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been recently suggested that the order Entomophthorales should be divided into two phylogenet- ically distinct groups: the genus Basidiobolus should be classified in a new order, the Basidiobolales, while Conidiobolus appears to be closely related to the Mucorales and should form a distinct group (5,8). The class of Zygomycetes is characterized in culture by broad, nonseptate or sparsely septated hyphae and by the presence of sporangiophores supporting sporangia, which contain sporangiospores (see Fig. 2.7, Chapter 2). During sexual reproduction in culture zygospores may be produced. The Zygomycetes are characterized in tissue by the formation of wide, ribbonlike, hyaline, aseptate or sparsely septated hyphae with wide- angle (approximately 90°) branching. The substantial differences among these and other structures allow mycology laboratories to diagnose organisms by genus and species (3). 3. EPIDEMIOLOGY The Zygomycetes are ubiquitous in soil and can be isolated from decaying organic matter including hay, decaying vegetation, and a variety of food items. Human infection is usually acquired through inhalation of sporangiospores from environmental sources. Acquisition via the cutaneous or percutaneous route is also common, either through traumatic disruption of skin barriers or with the use of catheters and injections. Less commonly, infection through the gastrointestinal route may occur (1,3,6). 12. Zygomycosis (Mucormycosis) 229 Zygomycosis is approximately 10- to 50- fold less common than invasive Candida or Aspergillus infections, with a prevalence of 1 to 5 cases per 10,000 autopsies and an estimated incidence of 1.7 cases per million per year in the United States (4,9). A clear male predisposition has been observed, as demonstrated by an approximate 2:1 male to female ratio among cases (1). Unlike other filamentous fungi, targeting mainly immunocompromised patients, the Zygomycetes cause disease in a wider and more heterogeneous population. While the Mucorales mainly affect patients with underlying immunosuppression or other medical conditions, the Entomophthorales largely afflict immunocompetent hosts in tropical and subtropical areas of developing countries. Lately, however, infections caused by the Entomophthorales in immunocompromised hosts have occasionally been documented (6,7,10). The most common underlying condition for development of zygomycosis is diabetes, both type I and type II. A significant proportion of these patients will present with concomitant ketoacidosis, while in others zygomycosis may even present as the diabetes-defining illness. Other significant underlying conditions include the presence of hematological malignancy, solid organ or bone marrow transplantation, deferox- amine therapy, and injection drug use (1,6). During the 1980s and 1990s, the percentage of patients with hematological malignancy, solid organ or bone marrow transplan- tation, and injection drug use among all cases of zygomycosis increased significantly (1,11,12). In the aforementioned groups of hematological patients and transplant recip- ients, factors associated with this infection have been reported to include prolonged neutropenia, corticosteroid use, and graft versus host disease (GvHD) (6,12). Less commonly, the Zygomycetes may cause invasive disease in the presence of renal failure, diarrhea, and malnutrition in low birth weight infants and in HIV patients. Occasionally zygomycosis has developed in patients with persistent metabolic acidosis secondary to causes other than diabetes (1,6). A significant proportion of zygomycosis cases have as well been observed in persons with no primary underlying disease at the time of infection. In many of these cases there was a history of penetrating trauma, surgery, or burn before the development of infection (1,6). 4. PATHOGENESIS AND IMMUNOLOGY The epidemiologic profile of zygomycosis cases (patients with diabetes, hemato- logical malignancies or on deferoxamine therapy, transplant recipients) may in part be explained by our current understanding of the pathogenesis of these infections. As with other filamentous fungi, an effective immune response following inoculation of sporangiospores requires the presence of adequate phagocytic activity of the host effector cells, including tissue macrophages and neutrophils. The macrophages ingest the sporangiospores to inhibit germination, while the neutrophils are involved in hyphal damage (13). Consequently, the host immune response against the Zygomycetes may be compromised if the phagocytic cells are insufficient in number as in the case of chemotherapy-induced neutropenia, or dysfunctional, as in the case of corticosteroid treatment or diabetes mellitus (3,13). Experimental evidence also suggests an important role of iron in the pathogenesis of infections caused by Rhizopus species, whose growth is promoted in the presence of 230 Charalampos Antachopoulos, Juan C. Gea-Banacloche, and Thomas J. Walsh increased iron uptake. Deferoxamine, an iron chelator, has siderophore activity for these fungi, allowing significant increase in iron uptake. Further, the availability of serum iron is increased in the presence of acidic pH, suggesting an additional mechanism for the development of zygomycosis in patients with diabetic ketoacidosis (4,13). An almost universal feature in infections caused by the Mucorales is the presence of extensive angioinvasion associated with thrombosis and ischemic necrosis (3). This is likely an important mechanism by which these organisms survive antifungal therapy because adequate blood supply is necessary for the delivery of antifungal agents. Recent data also have demonstrated the ability of R. oryzae sporangiospores or hyphae to adhere to subenthodelial matrix proteins and human endothelial cells (4,14). Pregerminated sporangiospores of R. oryzae were able to damage endothelial cells in vitro, following adherence to and phagocytocis by these cells. R. oryzae viability was not required for endothelial cell damage, suggesting that in the setting of established infection even fungicidal therapy may not prevent subsequent tissue injury (14). 5. CLINICAL MANIFESTATIONS The clinical presentations of zygomycosis in humans largely depend on the causative agent (Mucorales versus Entomophthorales) and the patient’s condition (intact immune response versus immunosuppresion or other underlying conditions). Thus, while the Mucorales cause rapidly progressive disease characterized by angioinvasion, throm- bosis, tissue necrosis, and dissemination in susceptible hosts, infections caused by the Entomophthorales usually lack these features, inducing a chronic inflammatory response and following an indolent course in immunocompetent patients (3,6,7). Conse- quently the patterns of human disease are described separately for these two orders of Zygomycetes, with more emphasis given to infections caused by the Mucorales, because organisms of this order are most likely to be encountered in developed countries. The clinical manifestations of human infection caused by the Mucorales can be classified as sinus disease, localized or extended to the orbit and/or brain, pulmonary, cutaneous, gastrointestinal, disseminated and miscellaneous infection. Some of these manifestations may occur with increased frequency in patients with certain underlying conditions (Table 12.2) (1,6). However, this is not always the case and zygomycosis in these patient groups may still present with any of the above patterns. 5.1. Sinus Infection Sinus disease may be confined to the paranasal sinuses or may infiltrate the orbit (sino-orbital) and/or the brain parenchyma (rhinocerebral). This form represents approximately two-thirds of all cases of zygomycosis in diabetic patients (1). The infection originates in the paranasal sinuses after inhalation of sporangiospores. Initial symptoms may suggest sinusitis and include sinus pain, discharge, soft tissue swelling, and perinasal cellulitis/paresthesia. Fever is variable and may be absent in up to half of cases (4,15,16). The tissues involved become red, violaceous, and finally black, as vascular thrombosis leads to tissue necrosis. A blood-tinged nasal discharge may be present. Extension of the infection to the mouth may produce painful necrotic ulcer- ations in the hard palate. Extension into the periorbital area and ultimately the orbit 12. Zygomycosis (Mucormycosis) 231 Table 12.2 Predominant site of Mucorales infection according to the patient’s underlying condition Underlying condition Type of infection, by site Diabetes Sinus Malignancy Pulmonary Solid organ transplantation Pulmonary Bone marrow transplantation Pulmonary Deferoxamine therapy Pulmonary, sinus, disseminateda Injection drug use Cerebral No underlying condition Cutaneous Data from ref. 1. aNo clear predominance among the three sites. may be manifested by periorbital edema, lacrimation, chemosis, and proptosis. Subse- quent ocular or optic nerve involvement may be suggested by pain, diplopia, blurring or loss of vision. Alteration of mental status and cranial nerve palsies may signify invasion of the central nervous system. Occasionally thrombosis of the cavernous sinus or the internal carotid artery may follow, with resultant neurological deficits, while dissemination of the infection also may occur (4,6,16,17). 5.2. Pulmonary Infection Pulmonary disease is most commonly observed in patients with hematological malignancies, solid organ or bone marrow transplant recipients, and in those receiving deferoxamine treatment (1). Not infrequently it may occur with concomitant sinus disease (sinopulmonary infection) (18). Lung involvement may be manifested as infil- trates, consolidation, and solitary nodular or cavitary lesions (Fig. 12.1) (19,20). Fungal invasion of the pulmonary vessels may result in thrombosis and subsequent infarcts in the lung parenchyma (Fig. 12.2). Angioinvasion may also lead to intra- parenchymal bleeding or even hemoptysis, which can be fatal if major vessels, such as the pulmonary artery, are involved. Extension of the infection to the chest wall, pericardium, myocardium, mediastinum, and diaphragm has been described (4,6,19). A predilection for the upper lobes has been reported; however, any part of the lung may be involved, and bilateral disease is not uncommon (19). Presenting signs and symptoms are nonspecific and include fever, cough, chest pain, dyspnea, hemoptysis, tachypnea, crackles, decreased breath sounds, and wheezing (4,19,20). 5.3. Cutaneous Infection Cutaneous zygomycosis is often observed in individuals with no underlying condition as a result of infection of a preexisting lesion, such as skin trauma or burn. Alternatively, it may occur in the context of disseminated disease or extensive local infection in immunocompromised hosts (1,3,21,22). In the case of primary cutaneous inoculation, the lesion appears acutely inflamed with redness, swelling, induration, and frequent progression to necrosis. Extensive local invasion may occur involving the 232 Charalampos Antachopoulos, Juan C. Gea-Banacloche, and Thomas J. Walsh Fig. 12.1. Thoracic CT scan of profoundly neutropenic patient with pulmonary zygomycosis demonstrates rapid evolution of pulmonary nodule to involve the pleural surface and to manifest a halo sign at the interface with radiologically normal lung. The two scans are separated by 5 days. [Figure in color on CD-ROM]. Fig. 12.2. Histopathology of pulmonary zygomycosis. Characteristic broad nonseptated ribbonlike hyphae with nondichotomous branching invading a pulmonary blood vessel. The specimen was obtained from the lung lesion seen on CT scan in Figure 12.1. GMS. [Figure in color on CD-ROM]. 12. Zygomycosis (Mucormycosis) 233 Fig. 12.3. Development of zygomycosis in the skin and subcutaneous tissues of the right lower extremity in a patient with cutaneous T-cell lymphoma. The top left panel depicts the lesions of cutaneous T-cell lymphoma, which were possibly infected by direct inoculation. The top right panel reveals the extensive necrosis and destruction of soft tissue caused by the rapidly invading zygomycete. The bottom panel demonstrates the soft tissues following extensive surgical debridement. [Figure in color on CD-ROM]. adjacent subcutaneous fat, muscle, bone tissues, and fascial layers (Fig. 12.3). When cutaneous disease is the result of disseminated infection, it usually presents as nodular subcutaneous lesions that may ulcerate (3,6,22). 5.4. Gastrointestinal Infection Gastrointestinal disease is rare, occurring mainly in malnourished patients and premature neonates, in whom it can present as necrotizing enterocolitis (1,23). After ingestion of the sporangiospores, fungal invasion of the mucosa, submucosa, and vascular structures of the gastrointestinal tract may occur, often resulting in necrotic ulcers, rupture of the intestinal wall, and peritonitis. Symptoms are nonspecific, including fever, abdominal pain, distention, vomiting, and gastrointestinal hemorrhage (3,4,23). 5.5. Disseminated Infection Disseminated infection refers to involvement of at least two noncontiguous sites and is commonly observed in patients receiving deferoxamine therapy (1). Dissemination occurs through the hematogenous route and may originate from any of the above sites of primary infection, although it seems to be more frequently associated with lung 234 Charalampos Antachopoulos, Juan C. Gea-Banacloche, and Thomas J. Walsh disease. The most common site of dissemination is the brain, but other organs may also be involved (4,25). 5.6. Other Infection Isolated cerebral zygomycosis is usually observed in injection drug users (1). Endocarditis is a potential complication of cardiac surgery. Isolated peritonitis is often associated with peritoneal dialysis. Renal infection and external otitis also have been reported (4,6). 5.7. Infection Due to the Entomophthorales In contrast to the Mucorales infections, human disease caused by the Entomophtho- rales usually follows an indolent course, as previously mentioned. A chronic subcuta- neous disease, characterized by slowly enlarging subcutaneous nodules that eventually ulcerate, is typically caused by B. ranarum. C. coronatus infections commonly present as chronic sinusitis that usually does not extend to the central nervous system (3,7). Less commonly, involvement of other body sites or even aggressive disseminated infection by members of the order Entomophthorales has been reported for immunocompromised and immunocompetent patients (3,7,10,23). 6. DIAGNOSIS As |
infections caused by the Zygomycetes, and particularly the Mucorales, in humans may be rapidly fatal, timely diagnosis is crucial to avoid treatment delay. While confirmation of the diagnosis and species identification of the causative organism should be pursued, treatment should be initiated as soon as the diagnosis is suspected, owing to the severity of these infections. Currently, the diagnosis of zygomycosis relies on a constellation of the following: high index of suspicion, assessment of presenting signs and symptoms, imaging studies, cultures of clinical specimens, and histopathology (Fig. 12.4). 6.1. Clinical Assessment The high index of suspicion should be based on the knowledge of the underlying conditions that predispose to zygomycosis and the usual presentation of the infection in each of these conditions (Table 12.2). Nevertheless, less common manifestations of the disease should not be excluded. A common scenario is the development of zygomycosis in oncological patients or transplant recipients who are receiving antifungal therapy for prophylaxis or treatment of other opportunistic fungal infections, such as invasive aspergillosis. If the antifungal agents that are being administered to the patient are not active against the Zygomycetes (including, fluconazole, voriconazole, and the echinocandins), then clinical deterioration or appearance of new signs and symptoms in these patients should alert the clinician to the possibility of zygomycosis (18). Most of the signs and symptoms that are associated with the clinical manifestations of zygomycosis are nonspecific. However, their diagnostic significance may increase if they are interpreted in relation to the patient’s underlying condition. For example, the development of sinusitis in a leukemic or diabetic patient should raise the suspicion of zygomycosis. Other findings have probably greater specificity for this infection, 12. Zygomycosis (Mucormycosis) 235 Host factors Relevant clinical findings Diabetes Sinusitis Malignancy Pneumonia Bone marrow transpl. Cutaneous ulcers Solid organ transpl. CNS lesions Deferoxamine therapy Injection drug use Rapid progression of infection Physician alertness, awareness of risk factors & presentation Presumed diagnosis of zygomycosis Diagnostic steps Treatment Imaging Rapid initiation Reversal of predisposing Cultures & condition Histopathology of Antifungal agents biopsy material Surgical debridement Final diagnosis Optimization of treatment Fig. 12.4. Diagnosis and management of zygomycosis. such as the presence of blood-tinged nasal discharge or necrotic eschars in the hard palate. In addition, the presence of hemoptysis in a susceptible host is consistent with angioinvasion and should raise the possibility of zygomycosis (19). An alarming sign should also be the rapid spread of the infection. Finally, even after the diagnosis has been made, careful periodic clinical assessment should be performed in order to detect progression of the disease. For example, in a patient with pulmonary zygomycosis, palpation of the skin for subcutaneous nodules and neurological evaluation for changes in mental status and focal neurological signs should be performed repeatedly to detect dissemination to the skin and brain, respectively. 6.2. Diagnostic Imaging Imaging studies are helpful in assessing the burden of the disease, involvement of adjacent tissues, and response to treatment. They are also helpful in guiding more invasive procedures to obtain biopsy specimens for histopathology and culture (26). Although imaging findings may be suggestive of zygomycosis in the appropriate clinical setting, they are not sufficiently specific to establish the diagnosis. In sinus disease, computerized tomography (CT) detects subtle mucosal thickening or bony erosions of the sinuses, but it is less sensitive than magnetic resonance imaging (MRI) for the detection of extension of the infection to the soft tissues of the orbit (15,27). 236 Charalampos Antachopoulos, Juan C. Gea-Banacloche, and Thomas J. Walsh In the case of pulmonary disease, high-resolution CT is more sensitive than chest radiograph for early diagnosis of the infection and can more accurately determine the extent of pulmonary involvement. Radiographic features may be consistent with infliltrate, cavity, or consolidation (Fig. 12.1). The air crescent and halo signs, which are recognized radiologic features of invasive aspergillosis, have been reported as well for zygomycosis (19,20,28). In patients with pulmonary zygomycosis the presence of an air crescent sign seems to be associated with increased risk for massive hemoptysis (19). Another suggestive finding could be expansion of a mass or consolidation across tissue planes, in particular toward the great vessels in the mediastinum (4,29). In the case of cutaneous disease, MRI is superior to CT scan for assessment of extension of the infection to the adjacent soft or bone tissues. 6.3. Culture Recovery of Zygomycetes from cultures of clinical specimens would allow not only establishment of diagnosis, but also identification of the causative organism to the species level. Although the Zygomycetes may contaminate laboratory material, their isolation from clinical specimens of susceptible hosts should not be disregarded as contamination. Despite the ability of these organisms to invade tissues, they are rarely isolated from cultures of blood, urine, cerebrospinal fluid, feces, sputum, paranasal sinuses secretions, bronchoalveolar lavage, or swabs from infected areas (3,15,19). The recovery of Zygomycetes from biopsy material may be compromised if processing of the specimens involves tissue grinding, a procedure that kills the nonseptate hyphae of these fungi. The recovery rate is enhanced, however, if thin slices of minimally manipulated tissue are placed onto the culture medium. Consequently, for proper handling of the specimens, the laboratory should be notified of the possibility of zygomycosis. In any case, negative cultures do not rule out the infection (3,6). 6.4. Endoscopic Findings and Histopathology Given the above limitations of cultures or imaging studies, diagnosis of zygomy- cosis is almost always based on histopathologic examination of appropriately collected samples. The latter should be pursued in the presence of strong suspicion for zygomy- cosis if the cultures or imaging studies are negative or nonspecific. Depending on the presentation of the disease, the samples may be collected by fiberoptic bronchoscopy, radiographically guided transthoracic needle aspiration, open lung biopsy, nasal endoscopy, paranasal sinus biopsy or débridement, and biopsies of skin or other infected tissues (4,6,19,26). In the case of lung disease, endobronchial findings include stenosis or airway obstruction, erythematous mucosa, fungating or polypoid mass, and, less often, granulation tissue or mucosal ulceration (19). In sinus disease, nasal endoscopy may show black necrotic crusts on the nasal septum and turbinates; in the early phases the mucosa may still look pink and viable (4). We refer to these necrotic ulcers along the nasal mucosa or turbinates as “sentinel eschars,” as they may represent an early phase of infection or may be more amenable to biopsy than a deep maxillary sinus infection. Because the hyphae of Zygomycetes in tissue specimens may stain poorly with hematoxylin and eosin (H&E), a second more fungus-specific tissue stain should 12. Zygomycosis (Mucormycosis) 237 also be used, such as Gomori methenamine silver (GMS) or periodic acid Schiff (PAS) (3). As previously mentioned, the hallmark of zygomycosis is the demonstration of wide, ribbonlike, aseptate (nonseptate or rarely septate) hyphae with wide-angle branching in biopsy specimens (Fig. 12.2) (see also Fig. 3.11, Chapter 3). For Mucorales infections, the hyphae are seen to invade the adjacent blood vessels. Mycotic emboli may thrombose small vessels in which they are lodged. Extensive tissue necrosis or hemorrhage may be observed (3). Although histopathology is sensitive and reliable for diagnosing zygomycosis, obtaining biopsy material from hematological patients may not always be feasible owing to concomitant thrombocytopenia. Finally, because fixing and staining of the biopsy specimens takes time, a promising method for accelerating diagnosis by use of frozen sections in tissue specimens has been proposed recently (30). 6.5. Other Non-Culture Diagnosis Unfortunately, no reliable molecular or antigen detection methods are available to date for primary diagnosis of zygomycosis. A number of molecular techniques are currently used by research laboratories for species identification of zygomycetes isolates, epidemiologic studies, or determination of taxonomic assignments (3,18). 7. TREATMENT There are four cornerstones of successful management of zygomycosis: (1) rapid initiation of therapy, (2) reversal of the patient’s underlying predisposing condition, (3) administration of appropriate antifungal agents, and (4) surgical débridement of infected tissues (Fig. 12.4) (4,6). If not treated or if diagnosed with delay, infections caused by the Mucorales in humans are typically fatal (1,11,12). Even if timely diagnosis is made, treatment is challenging due to a number of reasons such as the underlying condition of the patient, the rapid progression of the disease, and the high degree of angioinvasion and thrombosis that compromises the delivery of antifungal agents active against the causative organisms. As mentioned previously, in the presence of certain conditions such as diabetes, immunosuppression, and others (Table 12.2), treatment for zygomycosis should be initiated as soon as a strong suspicion for this infection is raised, without awaiting formal confirmation of the diagnosis, which may take time. Meanwhile of course, all the required actions to establish the diagnosis should be undertaken, as previously described (Fig. 12.4). Reversal of the underlying condition can be fairly quickly achieved in certain circumstances, such as diabetic ketoacidosis, which should be promptly corrected, or deferoxamine therapy, which should be discontinued. However, timely reversal of the disease- or treatment-related immunosuppression in patients with hematological malignancies or transplant recipients is challenging. In these patients, temporary discon- tinuation of corticosteroid treatment or myelotoxic chemotherapy should be strongly considered until the infection is brought under control. However, even with these measures, spontaneous restoration of phagocytic activity or recovery from neutropenia are likely to occur after several days, during which time the infection may progress. In vitro and in vivo studies, as well as case reports, have suggested that the administration of cytokines, such as granulocyte colony-stimulating factor (G-CSF), granulocyte- macrophage colony-stimulating factor (GM-CSF), and interferon-, may accelerate 238 Charalampos Antachopoulos, Juan C. Gea-Banacloche, and Thomas J. Walsh immune recovery (31,32). In support of these general recommendations for cytokine augmentation of host defense are recent in vitro studies demonstrating enhancement of neutrophil activity against R. oryzae and other species of Zygomycetes in the presence of interferon- and GM-CSF (33). An alternative approach has been the administration of granulocyte transfusions in neutropenic patients with invasive fungal infections (31,32). These immunomodulatory interventions may be considered on an individual patient basis as adjunctive therapy for zygomycosis in immunocompromised hosts. Nonetheless, as a caveat, there is a lack of adequately powered clinical trials to evaluate their clinical efficacy and potential complications (6,31,32). 7.1. Amphotericin B Amphotericin B is the drug of choice for the treatment of zygomycosis. This polyene agent exerts good in vitro and in vivo activity against the Zygomycetes. However, apparent in vitro resistance, with elevated minimal inhibitory concentrations (MICs) of amphotericin B, may be observed among clinical isolates and is relatively common among Cunninghamella species (34–36). The efficacy of amphotericin B in the treatment of zygomycosis was demonstrated in a recent review of 929 cases, where survival was 61% for patients treated with amphotericin B deoxycholate versus 3% for those who received no treatment (1). The lipid formulations of amphotericin B (mainly amphotericin B lipid complex and the liposomal formulation) also have been used in the treatment of zygomycosis. These formulations are associated with significantly less toxicity than amphotericin B deoxycholate and demonstrate at least equivalent clinical efficacy (1,37,38). However, no randomized controlled trials have been conducted to compare the efficacy of deoxycholate versus lipid formulations of amphotericin B in the treatment of zygomycosis. When treatment with amphotericin B is initiated for documented zygomycosis, full doses should be given from the onset, foregoing the past practice of dose escalation. The optimal dosage of amphotericin B formulations for the treatment of zygomycosis has not been systematically evaluated in clinical studies. A study of the safety, tolerance, and plasma pharmacokinetics of liposomal amphotericin B in patients with invasive fungal infections found no demonstrable dose-limiting nephrotoxicity or infusion- related toxicity over a dose range of 7.5 to 15 mg/kg per day (39). Plasma concentrations of liposomal amphotericin B achieved an upper limit at 10 mg/kg per day and were not increased by further dosage increases. Nevertheless, the efficacy of higher dosages of liposomal amphotericin B compared to the US Food and Drug Administration (FDA)- approved dosage of 3 to 5 mg/kg per day for aspergillosis has not been investigated through clinical trials in zygomycosis. In the absence of such studies, an increase of dosage of liposomal amphotericin B to 7.5 or 10 mg/kg per day could be considered on an individual basis for patients with zygomycosis progressing through liposomal amphotericin B at 5 mg/kg per day (39). Amphotericin B lipid complex has been used in a dosage of 5 mg/kg per |
day in salvage treatment of zygomycosis with complete or partial responses in 17 (71%) of 24 cases (38). 12. Zygomycosis (Mucormycosis) 239 7.2. Azoles Of the azole agents, fluconazole and voriconazole have little or no activity against the Zygomycetes (40). Itraconazole is active in vitro against some of these organisms, but has demonstrated poor efficacy in animal models (41,42). The investigational azole posaconazole is active in vitro and in vivo against many of the Zygomycetes (34,35,40–42). Results from two recent case series as well as a number of case reports on the use of posaconazole in patients with zygomycosis refractory to or intolerant of conventional antifungal treatment provide encouraging data regarding posaconazole as an alternative salvage therapy for zygomycosis (43–48). The use of this azole, however, as monotherapy or in combination with amphotericin B, for the treatment of zygomycosis awaits further evaluation in a randomized clinical trial versus deoxy- cholate amphotericin B or a lipid formulation of amphotericin B. 7.3. Surgery Appropriate and early surgical débridement is a critical intervention for the successful management of zygomycosis for a number of reasons: the infection progresses rapidly, vascular thrombosis compromises the delivery of antifungal agents to the site of infection, and there is massive tissue necrosis. Several retrospective studies have demonstrated that the survival of patients treated with antifungal therapy combined with surgical débridement was significantly higher than that of patients treated with antifungal therapy alone (1,4,6,19). Surgical treatment should aim in removing all necrotic tissues and should be considered for any of the clinical presenta- tions of zygomycosis (sinus disease, pulmonary or cutaneous). It should be performed early in the course of treatment and repeated if necessary. It may include excision of the infected sinuses, débridement of retroorbital space, or even enucleation in the case of sinus/sinoorbital disease, and wedge resection, lobectomy, or pneumonectomy in the case of pulmonary disease (15,16,19,22,29). If the patient survives the infection, plastic surgery is likely to be needed to correct disfiguring resulting from débridement (4). 7.4. Hyperbaric Oxygen Besides the aforementioned important aspects of management of zygomycosis, hyperbaric oxygen is a therapeutic modality that has been occasionally used as adjunctive treatment. Hyperbaric oxygen has a theoretical potential for being beneficial in the treatment of zygomycosis because it is known to inhibit fungal growth at high pressures, correct tissue hypoxia and lactic acidosis, promote healing, and enhance phagocytosis (49,50). In a number of case reports and small case series of zygomy- cosis, administration of hyperbaric oxygen was associated with a favorable outcome (49). Currently, however, the absence of randomized controlled clinical trials on the efficacy of hyperbaric oxygen in this setting does not allow firm recommendations regarding its use as adjunctive treatment of zygomycosis. 7.5. Prognosis The prognosis of zygomycosis largely depends on the patient’s underlying condition, the clinical presentation of the infection, the time of initiation of therapy, and the type of treatment provided. Mortality may range from less than 10% for localized sinus 240 Charalampos Antachopoulos, Juan C. Gea-Banacloche, and Thomas J. Walsh disease to approximately 100% for disseminated infection, with an overall percentage of 47% for zygomycosis cases reported in the 1990s (1,4). 8. PREVENTION Prevention may be feasible for a proportion of cases through adequate control of diabetes and judicious use of deferoxamine and corticosteroids. For severely immuno- compromised hosts, measures to reduce the risk of exposure to airborne sporan- giospores should be undertaken, including Hepafiltration of air supply, positive room air pressures, exclusion of plants from the wards, and wearing of masks when leaving the room. Owing to the relatively low incidence of zygomycosis, the cost-effectiveness of prophylactic treatment is questionable; the development of a preemptive therapy approach, however, based on validated early indicators of the disease and risk assumption, should be a target for research in the near future. In the meantime, physi- cians caring for susceptible patients should maintain a high level of suspicion and be alert to the early signs and symptoms of zygomycosis in order to achieve early diagnosis and timely initiation of treatment. REFERENCES 1. 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Zygomycosis in a tertiary-care cancer center in the era of Aspergillus-active antifungal therapy: a case-control observational study of 27 recent cases. J Infect Dis 2005;191:1350–1360. 19. Lee FY, Mossad SB, Adal KA. Pulmonary mucormycosis: the last 30 years. Arch Intern Med 1999;159:1301–1309. 20. McAdams HP, Rosado de Christenson M, Strollo DC, Patz EF, Jr. Pulmonary mucormy- cosis: radiologic findings in 32 cases. AJR Am J Roentgenol 1997;168:1541–1548. 21. Hay RJ. Mucormycosis: an infectious complication of traumatic injury. Lancet 2005;365:830–831. 22. Losee JE, Selber J, Vega S, Hall C, Scott G, Serletti JM. Primary cutaneous mucormycosis: guide to surgical management. Ann Plast Surg 2002;49:385–390. 23. Lyon GM, Smilack JD, Komatsu KK, et al. Gastrointestinal basidiobolomycosis in Arizona: clinical and epidemiological characteristics and review of the literature. Clin Infect Dis 2001;32:1448–1455. 24. Woodward A, McTigue C, Hogg G, Watkins A, Tan H. Mucormycosis of the neonatal gut: a “new” disease or a variant of necrotizing enterocolitis? J Pediatr Surg 1992;27:737–740. 25. Cuvelier I, Vogelaers D, Peleman R, et al. Two cases of disseminated mucormycosis in patients with hematological malignancies and literature review. Eur J Clin Microbiol Infect Dis 1998;17:859–863. 26. Maschmeyer G. Pneumonia in febrile neutropenic patients: radiologic diagnosis. Curr Opin Oncol 2001;13:229–235. 27. Fatterpekar G, Mukherji S, Arbealez A, Maheshwari S, Castillo M. Fungal diseases of the paranasal sinuses. Semin Ultrasound CT MR 1999;20:391–401. 28. Jamadar DA, Kazerooni EA, Daly BD, White CS, Gross BH. Pulmonary zygomycosis: CT appearance. J Comput Assist Tomogr 1995;19:733–738. 29. Reid VJ, Solnik DL, Daskalakis T, Sheka KP. Management of bronchovascular mucormy- cosis in a diabetic: a surgical success. Ann Thorac Surg 2004;78:1449–1451. 30. Hofman V, Castillo L, Betis F, Guevara N, Gari-Toussaint M, Hofman P. Usefulness of frozen section in rhinocerebral mucormycosis diagnosis and management. Pathology 2003;35:212–216. 31. Antachopoulos C, Roilides E. Cytokines and fungal infections. Br J Haematol 2005;129:583–596. 32. Pappas PG. Immunotherapy for invasive fungal infections: from bench to bedside. Drug Resist Updat 2004;7:3–10. 33. Gil-Lamaignere C, Simitsopoulou M, Roilides E, Maloukou A, Winn RM, Walsh TJ. Interferon-gamma and granulocyte-macrophage colony-stimulating factor augment the activity of polymorphonuclear leukocytes against medically important zygomycetes. J Infect Dis 2005;191:1180–1187. 34. Dannaoui E, Meletiadis J, Mouton JW, Meis JF, Verweij PE. In vitro susceptibilities of zygomycetes to conventional and new antifungals. J Antimicrob Chemother 2003;51:45–52. 242 Charalampos Antachopoulos, Juan C. Gea-Banacloche, and Thomas J. Walsh 35. Sun QN, Fothergill AW, McCarthy DI, Rinaldi MG, Graybill JR. In vitro activities of posaconazole, itraconazole, voriconazole, amphotericin B, and fluconazole against 37 clinical isolates of zygomycetes. Antimicrob Agents Chemother 2002;46:1581–1582. 36. Singh J, Rimek D, Kappe R. In vitro susceptibility of 15 strains of zygomycetes to nine antifungal agents as determined by the NCCLS M38-A microdilution method. Mycoses 2005;48:246–250. 37. Gleissner B, Schilling A, Anagnostopolous I, Siehl I, Thiel E. Improved outcome of zygomycosis in patients with hematological diseases? Leuk Lymphoma 2004;45: 1351–1360. 38. Walsh TJ, Hiemenz JW, Seibel NL, et al. Amphotericin B lipid complex for invasive fungal infections: analysis of safety and efficacy in 556 cases. Clin Infect Dis 1998;26:1383–1396. 39. Walsh TJ, Goodman JL, Pappas P, et al. Safety, tolerance, and pharmacokinetics of high- dose liposomal amphotericin B (AmBisome) in patients infected with Aspergillus species and other filamentous fungi: maximum tolerated dose study. Antimicrob Agents Chemother 2001;45:3487–3496. 40. Boucher HW, Groll AH, Chiou CC, Walsh TJ. Newer systemic antifungal agents: pharma- cokinetics, safety and efficacy. Drugs 2004;64:1997–2020. 41. Dannaoui E, Meis JF, Loebenberg D, Verweij PE. Activity of posaconazole in treatment of experimental disseminated zygomycosis. Antimicrob Agents Chemother 2003;47: 3647–3650. 42. Sun QN, Najvar LK, Bocanegra R, Loebenberg D, Graybill JR. In vivo activity of posaconazole against Mucor spp. in an immunosuppressed-mouse model. Antimicrob Agents Chemother 2002;46:2310–2312. 43. Greenberg RN, Scott LJ, Vaughn HH, Ribes JA. Zygomycosis (mucormycosis): emerging clinical importance and new treatments. Curr Opin Infect Dis 2004;17:517–525. 44. Greenberg RN, Mullane K, van Burik JA, et al. Posaconazole as salvage therapy for zygomycosis. Antimicrob Agents Chemother 2006;50:126–133. 45. van Burik JA, Hare RS, Solomon HF, Corrado ML, Kontoyiannis DP. Posaconazole is effective as salvage therapy in zygomycosis: a retrospective summary of 91 cases. Clin Infect Dis 2006;42:e61–65. 46. Tobon AM, Arango M, Fernandez D, Restrepo A. Mucormycosis (zygomycosis) in a heart-kidney transplant recipient: recovery after posaconazole therapy. Clin Infect Dis 2003;36:1488–1491. 47. Garbino J, Uckay I, Amini K, Puppo M, Richter M, Lew D. Absidia posttraumatic infection: successful treatment with posaconazole. J Infect 2005;51:e135–e138. 48. Dupont B. Pulmonary mucormycosis (zygomycosis) in a lung transplant recipient: recovery after posaconazole therapy. Transplantation 2005;80:544–545. 49. John BV, Chamilos G, Kontoyiannis DP. Hyperbaric oxygen as an adjunctive treatment for zygomycosis. Clin Microbiol Infect 2005;11:515–517. 50. Gill AL, Bell CN. Hyperbaric oxygen: its uses, mechanisms of action and outcomes. QJM 2004;97:385–395. SUGGESTED READINGS Gonzalez CE, Rinaldi MG, Sugar AM. Zygomycosis. Infect Dis Clin North Am 2002;16: 895–914. Greenberg RN, Scott LJ, Vaughn HH, Ribes JA. Zygomycosis (mucormycosis): emerging clinical importance and new treatments. Curr Opin Infect Dis 2004;17:517–525. Lee FY, Mossad SB, Adal KA. Pulmonary mucormycosis: the last 30 years. Arch Intern Med 1999;159:1301–1309. 12. Zygomycosis (Mucormycosis) 243 Prabhu RM, Patel R. Mucormycosis and entomophthoramycosis: a review of the clinical manifestations, diagnosis and treatment. Clin Microbiol Infect 2004;10 (Suppl 1):31–47. Ribes JA, Vanover-Sams CL, Baker DJ. Zygomycetes in human disease. Clin Microbiol Rev 2000;13:236–301. Roden MM, Zaoutis TE, Buchanan WL, et al. Epidemiology and outcome of zygomycosis: a review of 929 reported cases. Clin Infect Dis 2005;41:634–653. Spellberg B, Edwards Jr J, Ibrahim A. Novel perspectives on mucormycosis: pathophysiology, presentation, and management. Clin Microbiol Rev 2005;18:556–569. 13 Pneumocystosis Francis Gigliotti, MD and Terry W. Wright, PhD 1. INTRODUCTION Pneumocystis is the classic opportunistic pathogen in that it does not produce any recognizable disease in an immunologically intact host, yet infection of the at- risk immunocompromised host results in a pneumonitis that is universally fatal if untreated. The organism was first identified in the early 1900s but was not appreciated to be a significant human pathogen until |
after World War II, when outbreaks of Pneumocystis pneumonia (PCP) occurred in orphanages in Europe. These young infants who developed what was termed “interstitial plasma cell pneumonitis” were suspected to be immunosuppressed secondary to severe malnutrition. Two subsequent events firmly established Pneumocystis as a major opportunistic pathogen; the development of successful cancer chemotherapy in the late 1950s and 1960s and the start of the acquired immunodeficiency syndrome (AIDS) epidemic in the early 1980s. In fact it was the recognition of a cluster of this “rare” pneumonia, PCP, in gay men over a short period of time that led to the recognition that a new syndrome (AIDS) and infection (human immunodeficiency virus [HIV]) had emerged (1,2). At present the population of patients at risk to develop PCP is growing steadily as we develop new modalities of therapy and potent immunosuppressive drugs to treat malignancies, organ failure, and autoimmune and inflammatory diseases. For example, in solid organ transplant recipients, as survival improves so does the recognition that these patients are at risk of developing PCP if not on specific prophylaxis. Most recently, the addition of antitumor necrosis factor (TNF) therapy to the management of patients with Crohn’s disease has resulted in the occurrence of PCP in this population that had previously not been considered to be at risk for the development of PCP. 2. ETIOLOGIC AGENT All strains of Pneumocystis are extracellular organisms found in the lungs of mammals. The taxonomic placement of these organisms has not been unequivocally established, largely owing to the inability to adequately culture the organism. However, nucleic acid homologies indicate it is most closely related to the fungi, despite its From: Infectious Disease: Diagnosis and Treatment of Human Mycoses Edited by: D. R. Hospenthal and M. G. Rinaldi © Humana Press Inc., Totowa, NJ 245 246 Francis Gigliotti and Terry W. Wright morphologic features and susceptibility to drugs that are similar to those of protozoa. Both phenotypic and genotypic analysis demonstrates that each mammalian species is infected by a unique strain of Pneumocystis (3–5). A biological correlate for these differences is evidenced by animal experiments that have shown organisms are not transmissible from one mammalian species to another (6). This restricted host range is the one biologic characteristic of Pneumocystis that might achieve the level of uniqueness sufficient to define species of Pneumocystis. Two forms of Pneumocystis are found in the alveolar spaces, thick-walled cysts (Fig. 13.1) that are 5 to 8 μm in diameter and may contain up to eight pleomorphic intracystic sporozoites, and trophozoites, which are 2 to 5 μm diameter cells with a more typical cell membrane, thought to be derived from excysted sporozoites. The terminology sporozoites and trophozoites are based on the morphological similarities to protozoa, because there are not exact correlates for these forms of the organism among the fungi. Sporozoites are also called intracystic bodies and trophozoites are referred to as trophic forms. As noted in the preceding text, the host-species specificity of Pneumocystis has led some to propose the division of P. carinii into multiple unique species, with the nomenclature P. jirovecii being used to refer to human P. carinii (7). The proposal for a change in nomenclature is controversial because it also calls for species distinction Fig. 13.1. Silver-stained bronchoalveolar lavage specimen showing characteristic clusters of Pneumocystis cysts. [Figure in color on CD-ROM]. 13. Pneumocystosis 247 based on variation in gene sequences not known to result in a unique phenotype. Opposing opinions have also been published calling for the nomenclature P. carinii to be retained for all P. carinii or at least for P. carinii infecting humans (8). Until consensus is achieved, P. carinii can also be clearly defined using “special form” nomenclature (e.g., P. carinii f. sp. hominus for human P. carinii) or simply by identi- fying the mammalian source (e.g., mouse P. carinii to describe P. carinii isolated from mice). 3. EPIDEMIOLOGY PCP occurs only in patients who are significantly immunosuppressed, typically with abnormalities in CD4+ T lymphocytes or B cells. Serologic studies have demonstrated that a high proportion of the population has evidence of infection and that serocon- version typically occurs during childhood. A recent prospective longitudinal study demonstrated that seroconversion began in the first few months of life and by 20 months of age 85% of the infants in the study had seroconverted (9). Aside from the serologic data, Pneumocystis was not known to actually infect the immunologically normal host. However, animal studies have proved that Pneumocystis produces a typical pattern of infection, transmission, and resolution in the normal host (10). The other important biological feature of Pneumocystis infection is that the strain (or species) of Pneumocystis from any given mammalian host is transmissible only to members of the same host species. Cross-species transmission has never been convincingly demonstrated. Because of the finding of early seroconversion followed by disease later in life, PCP was postulated to be the result of reactivation of latent infection. However, no evidence for latency has ever been demonstrated, and mouse and rat models of PCP have shown that latency does not develop after infection. Considering all of these features it would seem most likely that PCP is the result of new infection rather than reactivation of a latent infection. Person-to-person trans- mission is likely, based on the cumulative experience in animal models, but difficult to prove. Without prophylaxis, PCP develops in approximately 70% of adults and 40% of infants and children with AIDS, and 10% of patients with organ transplants. It is often the sentinel event identifying infants with severe congenital immunodeficiencies such as severe combined immunodeficiency syndrome. PCP also is a frequent occurrence in patients being treated for malignancies, occurring with an overall frequency of 10% to 15%. The actual incidence for any given malignancy depends on the treatment regimen and is positively correlated with number of chemotherapeutic agents and intensity of treatment. 4. PATHOGENESIS AND IMMUNOLOGY Control of infection is dependant on normally functioning CD4+ T lymphocytes. Studies in patients with AIDS show an increase in the occurrence of Pneumocystis pneumonia as CD4+ T lymphocytes drop. For adults and children older than 6 years of age a CD4+ T cell count of 200 cells/μl or lower is a marker of very high risk for development of PCP. Based on the occurrence of PCP in some patients and mouse strains with various immunologic defects that result in defective antibody production, 248 Francis Gigliotti and Terry W. Wright a possible role for CD4+ T lymphocytes could be to provide help for the production of specific antibody. Passively administered antibody has been shown to aid in the clearance of Pneumocystis in mouse models. Thus antibody could be involved in the clearance of organisms through interaction with complement, phagocytes, and/or T lymphocytes. The mechanism by which Pneumocystis damages the lung is not yet fully defined. Animal models have been valuable in helping us understand the immunopathogenesis of PCP (11). Infection of severe combined immunodeficiency (SCID) mice with Pneumo- cystis produces very little alteration in lung histology or function until very late in the course of the disease. However, if Pneumocystis-infected SCID mice are immunolog- ically reconstituted with normal splenocytes there is a rapid onset of an inflammatory response that results in an intense cellular infiltrate, markedly reduced lung compliance, and significant hypoxia, all changes seen in humans with PCP. These inflammatory changes are associated with marked disruption of surfactant function. T-cell subset analysis has shown that CD4+ T lymphocytes produce an inflammatory response that clears the organisms but also results in lung injury. In contrast, CD8+ T lymphocytes are ineffective in the eradication of Pneumocystis, but do produce a marked injurious inflammatory response, especially in the absence of CD4+ T lymphocytes. Immune reconstitution inflammatory syndrome (IRIS), also called immune resti- tution disease or immune reconstitution syndrome, is a recently described manifestation of pulmonary infection in AIDS patients with Pneumocystis, Mycobacterium tuber- culosis, and other pulmonary pathogens who are experiencing rapid reconstitution of their immune system due to administration of effective antiretroviral therapy (12). In general, the severity of IRIS is directly related to the degree and rapidity of T-cell recovery. Mouse models of PCP suggest that CD8+ T lymphocytes help modulate the inflammation produced by CD4+T lymphocytes, but as mentioned in the preceding text, their ineffectual inflammatory response can also contribute significantly to lung injury. These various T-cell effects may be responsible for the variations in presentation and outcome of Pneumocystis pneumonia observed in different patient populations. The inflammatory processes taking place during PCP do not appear to result in major long-term damage to the lung in those who recover. A long-term follow up of 23 children with cancer and PCP showed a return to normal lung function by 6 months in all 18 survivors. Similar studies in adults are complicated by the fact that adult patients, especially those with AIDS, might have multiple pulmonary insults. While some studies, primarily of adult AIDS patients, suggest long-term pulmonary damage after PCP, other studies of renal transplant recipients have shown pulmonary function returned to nearly normal after recovery from PCP. 5. CLINICAL MANIFESTATIONS 5.1. Pneumocystis Pneumonia There are at least three distinct clinical presentations of PCP. In patients with profound immunodeficiency, such as young infants with congenital immunodefi- ciency, severe malnutrition, or in AIDS patients with very few CD4+ T lympho- cytes, the onset of hypoxia and symptoms is subtle, with cough, dyspnea on exertion, 13. Pneumocystosis 249 or tachypnea, often without fever. Infants may show progression to nasal flaring and intercostal, suprasternal, and infrasternal retractions. As the disease progresses, patients develop hypoxia, with cyanosis in severe cases. In the sporadic form of PCP, occurring in children and adults with underlying immunodeficiency, the onset of hypoxia and symptoms is usually more abrupt, with fever, tachypnea, dyspnea, and cough, progressing to severe respiratory compromise. This latter type accounts for the majority of cases, although the severity of clinical expression may vary. Rales are usually not detected on physical examination. The third pattern of disease is that associated with rapid restoration of immune function referred to as IRIS. It has been best described in newly diagnosed AIDS patients who are severely immunocompro- mised and present with PCP as their initial manifestation of AIDS (12). These patients appear to respond well to therapy for PCP but 3 to 6 weeks after beginning treatment they experience an unexpected recurrence of pulmonary symptoms and chest x-ray abnormalities that coincide with return of immune function. IRIS may also occur in bone marrow transplant patients who engraft while infected with Pneumocystis. 5.2. Extrapulmonary Infections Extrapulmonary infection with Pneumocystis is rare. The incidence is not well defined, but is estimated to be 1000-fold less likely than PCP itself (13). The most commonly reported sites of infection include the ear and eye. Why these two sites seem to predominate is unclear but may reflect the fact that infection at these sites may quickly produce readily apparent signs and symptoms. Other sites of involvement are the thyroid gland, liver, kidney, bone marrow, lymph nodes, spleen, muscle, and gastrointestinal tract. How the organism arrives at these sites is unknown. Response to treatment is usually good when extrapulmonary infections occur in the absence of pulmonary infection. 6. DIAGNOSIS Pulmonary symptoms in at risk patients should always raise the suspicion of PCP. The classic chest radiograph reveals bilateral diffuse alveolar disease with a granular pattern (see Fig. 5.7, Chapter 5). The earliest densities are perihilar, and progression proceeds peripherally, typically sparing the apical areas until last. Less common chest radiograph appearances in PCP include cystic lesions, pneumothorax, or isolated focal infiltrates. In patients receiving aerosolized pentamidine for prophylaxis there may be a predisposition for upper lobe infiltrates. The arterial oxygen tension (Pao2) is invariably decreased. A clinical pearl is that an elevated lactate dehydrogenase (LDH) may be a hint that one is dealing with PCP, because LDH is a useful marker of alveolar and inflam- matory cell damage. Because Pneumocystis is a diffuse alveolar infection it tends to result in higher and more often elevated levels of LDH than some other opportunistic pulmonary infections. For example, a recent analysis of LDH and pulmonary oppor- tunistic infections in AIDS patients showed that about 90% of those with definite PCP had elevated serum LDH (14). Thus while not specific for PCP, very high LDH levels should raise |
one’s suspicion for PCP, and normal levels make the diagnosis of PCP much less likely. 250 Francis Gigliotti and Terry W. Wright PCP can be definitively diagnosed only by demonstrating Pneumocystis in the lungs of a patient with compatible pulmonary signs and symptoms. Appropriate specimens for analysis include bronchoalveolar lavage, tracheal aspirate, transbronchial lung biopsy, bronchial brushings, percutaneous transthoracic needle aspiration, and open lung biopsy. Induced sputum samples are gaining popularity, but are helpful only if positive; the absence of Pneumocystis in an induced sputum sample does not exclude infection. The open lung biopsy is the most reliable method, although bronchoalveolar lavage is generally more practical. Estimates of the diagnostic yield of the various specimens are as follows: induced sputum 20% to 40%, tracheal aspirate 50% to 60%, bronchoalveolar lavage 75% to 95%, transbronchial biopsy 75% to 95%, and open lung biopsy 90% to 100%. Once obtained, the specimens are typically stained with one of four commonly used stains: Gomori methenamine silver (GMS) and toluidine blue stain only cyst forms; polychrome stains such as Giemsa stain for both tropho- zoites and sporozoites; and the fluorescein-labeled monoclonal antibody stains for both trophozoites and cysts. Pneumocystis can also be visualized by Papanicolaou stain. Polymerase chain reaction (PCR) analysis of respiratory specimens offers promise as a rapid diagnostic method, but a standardized system for clinical use has not been established. 7. TREATMENT The clear drug of choice for the treatment of PCP is trimethoprim-sulfamethoxazole (TMP-SMX) (Table 13.1). Generally TMP-SMX is administered intravenously, but it may be given orally if disease is mild and no malabsorption or diarrhea is present. The duration of treatment is generally 3 weeks for patients with AIDS and 2 weeks for other patients. Adverse reactions occur frequently, more so in adults than children, with TMP-SMX. These include rash, fever, and neutropenia in patients with AIDS. These side effects are less common in non-AIDS patients. For patients who cannot tolerate or fail to respond to TMP-SMX after 5 to 7 days, pentamidine isethionate may be used. Adverse reactions are frequent with pentamidine and include renal and hepatic dysfunction, hyperglycemia or hypoglycemia, rash, and thrombocytopenia. Atovaquone is an alternative treatment that has been used primarily in adults with mild to moderate disease. For adults and adolescents atovaquone is given twice a day with food. Less information is available for the treatment of younger children with this agent. Other effective therapies include trimetrexate glucuronate or combinations of trimethoprim plus dapsone and of clindamycin plus primaquine. Administration of corticosteroids in addition to anti-Pneumocystis drugs increases the chances for survival in moderate and severe cases of PCP (15). The recommended regimen of corticosteroids for adolescents older than 13 years of age and for adults is oral prednisone, 80 mg/day divided in two doses on days 1 to 5, 40 mg/day once daily on days 6 to 10, and 20 mg/day once daily on days 11 to 21. Although specific studies of adjunctive corticosteroid therapy in young children are not available, a reasonable regimen for children is oral prednisone, 2 mg/kg per day for the first 7 to 10 days, followed by a tapering regimen for the next 10 to 14 days. 13. Pneumocystosis 251 Table 13.1 Recommended treatment for Pneumocystis pneumoniaa Adults Children Treatment of first choice Trimethoprim- TMP 15–20 mg/kg/d with TMP 15–20 mg/kg per day sulfamethoxazole SMX 75–100 mg/kg with SMX 75–100 mg/kg per (TMP-SMX) per day IV divided into day IV divided into 4 doses; 3 or 4 doses; PO for PO for mild disease mild disease Alternate treatment regimens Pentamidine 4 mg/kg per day IV as 4 mg/kg per day as single dose single dose Atovaquone 750 mg PO bid 3–24 mo of age: 45 mg/kg per day PO divided into 2 doses; 1–3 mo and over 24 mo: 30 mg/kg per day in 2 divided doses (max. daily dose 1500 mg) Dapsone plus Dapsone 100 mg, PO Dapsone 2 mg/kg per day (100 trimethoprim once daily;TMP 15 mg max.) PO once daily; mg/kg per day PO in 3 TMP 15 mg/kg per day PO in divided doses 3 divided doses Primaquine plus Primaquine 15–30 Primaquine 0.3 mg/kg (max clindamycin mg, PO once 30 mg) PO once daily; daily;clindamycin 600 clindamycin 40 mg/kg per mg IV every 8 hours day IV in 4 divided doses (no pediatric data) Trimetrexate Trimetrexate plus <50 kg: 1.5 mg/kg per leucovorin day IV once daily 50–80 kg: 1.2 mg/kg per day IV once daily> 80 kg: 1.0 mg/kg per day 45 mg/m2 IV once daily IV once daily Leucovorin (continue 3 days beyond trimetrexate) <50 kg: 0.8 mg/kg per day 20 mg/m2 IV or PO every 6 IV or PO every 6 hours hours >50 kg: 0.5 mg/kg per day IV or PO every 6 hours IV, intravenous; PO, orally; mg/kg, milligrams/kilogram; mg/kg perday, milligrams/kilogram per day; mo, months of age; bid, twice daily. aDuration of therapy is typically 3 weeks in patients with AIDS and 2 weeks in other immunosup- pressed patients. 252 Francis Gigliotti and Terry W. Wright 8. PREVENTION PCP is effectively prevented by the use of antimicrobial prophylaxis; thus all patients at high risk for PCP should be placed on chemoprophylaxis. As noted in the preceding text, CD4+ T-cells are the key cell in determining susceptibility to PCP. However, defining the risk for PCP is not always clear. In AIDS patients there is clear-cut correlation between cell number and function so that firm cutoffs can be given. In adults with AIDS, prophylaxis is indicated at CD4+ T-cell counts of below 200 cells/μl. Because of rapid changes in CD4+ T-cell counts in young infants prophylaxis is recommended for all HIV-infected children during their first year of life. Thereafter prophylaxis is started at CD4+T-cell counts drop below 750 cells/μl for infants 12 to 23 months of age, 500 cells/μl for children from 2 to 6 years of age, and 200 cells/μl for those 6 years of age and older. Prophylaxis is also recommended for all ages if CD4+T- cell percentages drop below 15%. In other disease states in which patients are placed at risk of PCP from being on immunosuppressive drugs, both lymphocyte number and function will be affected. Thus while a patient may have a lymphocyte count above the threshold for susceptibility to develop PCP, suppressed function of remaining lymphocytes may place him or her at risk for PCP. Because of the demonstrated increased risk of PCP with increasing intensity of chemotherapy in patients with cancer, it would seem prudent, in our opinion, to consider prophylaxis for patients receiving prolonged (more than 6 to 8 weeks) therapy with two immunosuppressive agents and to give prophylaxis to all patients receiving three or more immunosuppressive agents. Table 13.2 Recommended antibiotic prophylaxis for Pneumocystis pneumonia Drug Adults Children Trimethoprim- 1 single or double TMP 5 mg/kg per day with sulfamethoxazole strength tablet daily SMX 25 mg/k per day (TMP-SMX) or 3 days/week given once daily or divided into 2 doses Dapsone 100 mg daily or twice 2 mg/kg per day as single weekly dose (max. 100 mg/dose) Atovaquone 1500 mg once daily 30 mg/kg per day as single dose for children aged 1–3 months and older than 24 mo; 45 mg/kg per day as single dose for children 4–23 mo Aerosolized 300 mg monthly given For children > 5 pentamidine by Respigard II years—same as for adults nebulizer IV, intravenous; PO, orally; mg/kg per day, milligrams/kilogram per day; mo, months of age. 13. Pneumocystosis 253 TMP-SMX is the drug of choice for Pneumocystis prophylaxis and may be given for 3 days each week, or, alternatively, each day (Table 13.2). The original study testing less than daily administration of TMP-SMX used a schedule of 3 consecutive days on TMP- SMX and 4 days off with the idea of reducing potential bone marrow suppression from the TMP-SMX. Subsequent studies have used alternate day schedules such as dosing on Monday, Wednesday, and Friday. The double strength tablet is preferred for adults receiving 3-days-a-week dosing. Alternatives for prophylaxis, all of which are inferior to TMP-SMX, include dapsone, atovaquone, and aerosolized pentamidine Prophylaxis must be continued as long as the patient remains immunocompromised. Studies in adult AIDS patients who reconstitute adequate immune response during antiretroviral therapy show that prophylaxis may be withdrawn without risk of developing PCP. Small studies in children have provided similar results. Criteria of maintaining the CD4+ T-cell count at or above 200 cells/μl for at least 3 months have been established for discontinuation of both primary and secondary prophylaxis (16). REFERENCES 1. Masur H, Michelis MA, Greene JB, et al. An outbreak of community-acquired Pneumocystis carinii pneumonia: initial manifestation of cellular immune dysfunction. N Engl J Med 1981;305:1431–1438. 2. Gottlieb MS, Schroff R, Schanker HM, et a. Pneumocystis carinii pneumonia and mucosal candidiasis in previously healthy homosexual men: evidence of a new acquired cellular immunodeficiency. N Engl J Med 1981;305:1425–1431. 3. Gigliotti F. Host species-specific antigenic variation of a mannosylated surface glycoprotein of Pneumocystis carini. J Infect Dis 1992;165:329–336. 4. Gigliotti F, Haidaris PJ, Haidaris CG, Wright TW, Van der Meid KR. Further evidence of host species-specific variation in antigens of Pneumocystis carinii using the polymerase chain reaction. J Infect Dis 1993;168:191–194. 5. Wakefield AE. Genetic heterogeneity in Pneumocystis carinii: an introduction. FEMS Immunol Med Microbiol 1998;22:5–13. 6. Gigliotti F, Harmsen AG, Haidaris CG, Haidaris PJ. Pneumocystis carinii is not universally transmissible between mammalian species. Infect Immun 1993;61:2886–2890. 7. Stringer JR, Cushion MT, Wakefield AE. New nomenclature for the genus Pneumocystis. J Eukaryot Microbiol 2001;Suppl:184S–189S. 8. Gigliotti F. Pneumocystis carinii: has the name really been changed? Clin Infect Dis 2005;41:1752–1755. 9. Vargas SL, Hughes WT, Santolaya ME, et al. Search for primary infection by Pneumocystis carinii in a cohort of normal, healthy infants. Clin Infect Dis 2001;32:855–861. 10. Gigliotti F, Harmsen AG, Wright TW. Characterization of transmission of Pneumo- cystis carinii f. sp. muris through immunocompetent BALB/c mice. Infect Immun 2003;71:3852–3856. 11. Wright TW, Gigliotti F, Finkelstein JW, McBride JT, An CL, Harmsen AG. Immune- mediated inflammation directly impairs pulmonary function, contributing to the patho- genesis of Pneumocystis carinii pneumonia. J Clin Invest 1999;104:1307–1317. 12. Cheng VC, Yuen KY, Chan WM, Wong SS, Ma ES, Chan RM. Immunorestitution disease involving the innate and adaptive response. Clin Infect Dis 2000;30:882–892. 13. Ng VL, Yajko DM, Hadley WK. Extrapulmonary pneumocystosis. Clin Microbiol Rev 1997;10:401–418. 254 Francis Gigliotti and Terry W. Wright 14. Butt AA, Michaels S, Kissinger P. The association of serum lactate dehydrogenase level with selected opportunistic infections and HIV progression. Int J Infect Dis 2002;6:178–181. 15. Briel M, Bucher HC, Boscacci R, Furrer H. Adjunctive corticosteroids for Pneumo- cystis jiroveci pneumonia in patients with HIV-infection. Cochrane Database Syst Rev 2006:3:CD006150. 16. Centers for Disease Control and Prevention. Guidelines for preventing opportunistic infec- tions among HIV-infected persons - 2002. Recommendations of the U.S. Public Health Service and the Infectious Diseases Society of America. MMWR 2002;51 (RR-8):1–52. SUGGESTED READINGS Gigliotti F, Wright TW. Immunopathogenesis of Pneumocystis carinii pneumonia. Exp Rev Molec Med 2005;7:1–16, www.expertreviews.org. Steele C, Shellito JE, Kolls JK. Immunity against the opportunistic fungal pathogen Pneumo- cystis. Med Mycol 2005;43:1–19. Thomas CF, Limper AH. Pneumocystis pneumonia. N Engl J Med 2004;350:2487–2498. Walzer PD, Cushion MT. Pneumocystis pneumonia. 3rd ed. New York: Marcel Dekker, 2005. 14 Cryptococcosis Methee Chayakulkeeree, MD and John R. Perfect, MD 1. INTRODUCTION Cryptococcosis is an infectious disease caused by pathogenic encapsulated yeasts in the genus Cryptococcus. Currently, two species of these fungi commonly cause disease in humans: C. neoformans which cause cryptococcosis in both immunocom- petent and immunocompromised hosts, and C. gattii , which is primarily a pathogen in apparently immunocompetent patients. C. neoformans was identified as a human pathogen in 1894 by two German physicians, Otto Busse and Abraham Buske, when they described a circular yeastlike microorganism in a lesion on the tibia of a woman; the microorganism was initially named Saccharomyces hominis (1). The name Crypto- coccus neoformans has been consistently adopted in both the mycology and medical literature since 1950 (2). In the mid-1970s, when Kwon-Chung discovered two mating types of C. neoformans that produced fertile basidiospores, the organisms were subse- quently separated into two varieties, var. neoformans (serotypes A and D) and var. gattii (serotypes B and C). These two varieties were recently separated into two species, C. neoformans and C. gattii, based on their genetic |
background and phylogenetic diversity, as proposed by Kwon-Chung in 2002 (3). It is possible, as more molecular information is gathered from genome sequencing, that C. neoformans var. neoformans (serotype D) and C. neoformans var. grubii (serotype A) will be divided into separate species as well. The incidence of cryptococcosis began to rise by the late 1970s. Early case reports of cryptococcal infections were primarily associated with cancer, autoimmune diseases, organ transplantation, and receipt of corticosteroids as these immunocompromised populations enlarged (4). A major surge in new cases of cryptococcosis occurred in the mid-1980s to 1990s. In the first two decades of the human immunodeficiency virus (HIV) infection pandemic, cryptococcal infection was an important opportunistic infection in all parts of the world. Further, C. gattii has recently caused a localized outbreak of cryptococcosis in apparently immunocompetent individuals on Vancouver Island (5). As a result, these fungi are not only major pathogens in immunocompromised patients such as those with acquired immune deficiency syndrome (AIDS), cancer, and From: Infectious Disease: Diagnosis and Treatment of Human Mycoses Edited by: D. R. Hospenthal and M. G. Rinaldi © Humana Press Inc., Totowa, NJ 255 256 Methee Chayakulkeeree and John R. Perfect immunosuppressive therapies, but also cause disease in apparently immunocompetent hosts. Despite the development of highly active antiretroviral therapy (HAART), which has decreased the rate of HIV-related cryptococcosis in developed countries, its preva- lence is still very high in developing countries and in individuals without access to healthcare. 2. ETIOLOGIC AGENTS Cryptococcus is a genus of basidiomycetous fungi containing more than 30 species. However, the common pathogenic organisms of cryptococcosis currently consist of two species that can be classified further into three varieties, five serotypes (based on capsular agglutination reactions), and eight molecular types (Table 14.1). C. neoformans has been classified into serotype A, D, and the hybrid strain, AD, whereas serotype B and C strains were classified as C. gattii. Serotype A strains have also been named C. neoformans var. grubii and serotype D strains were named C. neoformans var. neoformans. Recently, both C. neoformans and C. gattii were further classified into four molecular types for each species, VN I-IV and VG I-IV, respectively. The life cycle of C. neoformans and C. gattii involve asexual (yeast) and sexual (basidiospores/hyphae) forms. The asexual stage is the encapsulated yeast form found in clinical specimens, whereas the sexual stage, which exists in one of two mating types, “alpha” or “a,” is observed only under certain conditions resulting in meiosis to form basidiospores. Since the sexual stage of C. neoformans and C. gattii has been described, their teleomorphs were named Filobasidiella neoformans and Filoba- sidiella bacillospora, respectively. The majority of cryptococcal infections are caused by serotype A strains worldwide. However, cryptococcal diseases caused by serotype B and C strains, mostly VG II molecular type, are endemic in some subtropical areas, and serotype D is commonly found in Europe (6). C. neoformans and C. gattii usually appear as white-to-cream, opaque, and mucoid colonies that grow to several millimeters in diameter on most routine agar within 48 to 72 hours. With some strains, a few colonies occasionally develop sectors with different pigmentation. Both cryptococcal species can readily grow on most fungal culture media without cycloheximide at 30 to 37 C under aerobic conditions. However, C. neoformans is generally more thermotolerant to higher temperature than C. gattii, and serotype A is generally more tolerant than serotype D. Besides the ability to grow at 37 C, the yeast can produce a thick shedding polysaccharide capsule, melanin Table 14.1 Classification of Cryptococcus neoformans and Cryptococcus gattii Serotype Species and varieties Molecular types A C. neoformans var. grubii VN I, VN II B C. gattii VG I, VG II, VG III, VG IV C C. gattii VG I, VG II, VG III, VG IV D C. neoformans var. neoformans VN IV AD C. neoformans VN III 14. Cryptococcosis 257 pigments, and the enzymes urease and phospholipase, which are considered to be yeast virulence factors. Cryptococcus asexually reproduces by budding, but under specific conditions, it can have sexual reproduction or haploid fruiting in which the formation of the basidium and basidiospores occurs. The vast majority of infections and environmental isolates are caused by mating locus alpha strains. 3. EPIDEMIOLOGY Cryptococcosis was considered to be an uncommon infection before the AIDS epidemics, most associated with malignancies and immunosuppressive treatments. Since 1981, when HIV was rapidly becoming prevalent, the incidence of cryptococ- cosis in certain patients increased significantly and between 6% and 10% of AIDS patients developed cryptococcosis (7,8). In fact, HIV/AIDS was found to be associated with cryptococcosis in about 80% of cases worldwide. Cryptococcal infection became a major opportunistic infection in HIV-infected patients as their CD4+ cell count dropped below 100 cells/μl, especially those without access to HAART. Now that antiretro- viral treatment has been widely implemented, in many well developed countries the incidence of cryptococcosis has fallen significantly. The incidence of cryptococcal infection in persons not infected with HIV has not changed during this time. Never- theless, in developing countries with HIV epidemics and limited resources for HAART, cryptococcosis is still associated with a high incidence of disease and death. Besides HIV infection, other risk factors for acquiring cryptococcal infections include many conditions which result in an immunocompromised status (Table 14.2). Although both C. neoformans and C. gattii can cause cryptococcosis in apparently normal hosts, the Table 14.2 Predisposing factors of cryptococcosis HIV infection Malignanciesa (e.g., Hodgkin’s disease, other lymphomas and chronic lymphocytic leukemia) Lymphoproliferative disordersa Idiopathic CD4+ T cell lymphopenia Rheumatologic or immunologic diseasesa Sarcoidosis Systemic lupus erythematosus Rheumatoid arthritis Hyper-IgM syndrome or hyper-IgE syndrome Monoclonal antibodies (etanercept, infliximab, alemtuzumab) Corticosteroid and/or immunosuppressive therapies Diabetes mellitus Solid organ transplantationa Chronic pulmonary diseases Renal failure and/or peritoneal dialysis Chronic liver diseasesb aImmunosuppressive therapies add to the risk. bPoor prognosis. 258 Methee Chayakulkeeree and John R. Perfect Table 14.3 Distribution of C. neoformans and C. gattii Cryptococcus species Primary areas of distribution C. neoformans var. grubii serotype A Worldwide; pigeon guano, tree hollows C. gattii Tropical and subtropical regions: southern California, Hawaii, Brazil, Australia, Southeast Asia, and central Africa; Eucalyptus trees, firs, and oak trees C. neoformans var. neoformans serotype D Europe: Denmark, Germany, Italy, France, Switzerland; less common in the environment than serotype A percentage of C. gattii infections causing disease in such patients is significantly higher than for C. neoformans. C. neoformans is found throughout the world in association with excreta from certain birds such as pigeons and in tree hollows. C. gattii has been commonly associated with several species of eucalyptus trees and other trees (9). The link between environmental sources of infection and cryptococcosis cases is not well established although there is some evidence of cryptococcosis and positive serologies associated with intense bird exposures. Recently, there has been a strong link between the C. gattii outbreak in humans on Vancouver Island and common environmental yeast exposures. Although these fungi can be detected as endobroncheal colonization in humans without disease, clinicians should be alert for subclinical disease or potential for disease when these yeasts are isolated from any clinical specimens. Approximately 95% of cryptococcal infections are caused by serotype A strains (C. neoformans var. grubii) with the remaining 4% to 5% of infections caused by serotype D (C. neoformans var. neoformans) or serotype B and C strains (C. gattii). Whereas C. neoformans serotype A is found worldwide, serotypes B and C are found primarily in tropical and subtropical regions such as southern California, Hawaii, Brazil, Australia, Southeast Asia, and central Africa, and the serotype D is predominantly found in European countries (Table 14.3) (10). In Australia and New Zealand, serotypes B and C caused up to 15% of all cryptococcosis cases in one study, but the serotype A was still the predominant serotype (6). Only C. gattii strains have been reported to cause a widespread defined outbreak of disease (5). 4. PATHOGENESIS AND IMMUNOLOGY Cryptococcosis occurs primarily by inhalation of the infectious propagules, either dehydrated (poorly encapsulated) yeasts or basidiospores, into the alveoli within the lungs. Direct inoculation into tissue due to trauma can be a portal of entry in occasional cases, and potentially the yeast might enter through gastrointestinal tract. After the yeasts are inhaled into the lungs of a susceptible host, they come in contact with the alveolar macrophages, and other inflammatory cells are recruited through release of cytokines and chemokines such as interleukin-12 (IL-12), IL-18, 14. Cryptococcosis 259 monocyte chemotactic protein (MCP)-1, and macrophage inflammatory protein (MIP)- 1. Cryptococcal infection primarily involves granulomatous inflammation which is a result of a helper T-cell (Th1) response with cytokines including tumor necrosis factor, interferon-, and IL-2 (11). In many circumstances, the yeasts can remain dormant in hilar lymph nodes or pulmonary foci of an asymptomatic individual for years and then disseminate outside those complexes when the local immunity is suppressed (10). In a patient with a severely compromised cell-mediated immunity, the yeasts reactivate and proliferate at the site of infection and then disseminate to other sites, causing a progression in clinical manifestations. Recent advances in molecular biologic research into cryptococcal pathogenesis have confirmed several virulence factors in C. neoformans. The three classical virulence factors of C. neoformans include capsule formation, melanin pigment production, and ability to grow well at 37 C (9,11). The prominent antiphagocytic polysaccharide capsule, which is composed of glucuronoxylomannan (GXM), is unique to Crypto- coccus species and is considered an essential virulence factor that has multiple effects on host immunity. In addition, C. neoformans possesses an enzyme that catalyzes the conversion of diphenolic compounds to form melanin, which may have a biological role in protecting the yeasts from host oxidative stresses. Finally, its ability to grow at 37C is a basic part of the virulence composite for most of the human pathogenic fungi including Cryptococcus, as high-temperature growth has been shown to be linked with certain signaling pathways and enzymes through molecular studies. Other virulence factors include phospholipase and urease production and enzymes associated with protection against oxidative stresses. 5. CLINICAL MANIFESTATIONS Infections caused by C. neoformans and C. gattii have a predilection for establish- ing clinical disease in the lungs and central nervous system (CNS). Other organs that may be involved in cryptococcosis include skin, prostate, eyes, bone, and blood (2,8,10,12). In fact, this yeast may cause disease in any organ of the human body, and widely disseminated cryptococcal infection may affect multiple organs in severely immunosuppressed patients (Table 14.4). 5.1. Pulmonary Infection The respiratory tract serves as the most important portal of entry for this yeast and thus there are many clinical manifestations of pulmonary cryptococcosis, ranging from asymptomatic colonization of the airway or nodule on radiograph to a life-threatening fungal pneumonia (2,8). In a normal host with cryptococcal infection, asymptomatic pulmonary cryptococcosis can occur in about one third of patients with pulmonary infection, and patients may present to healthcare with only an abnormal chest radio- graph. The most common radiologic findings of cryptococcosis include well-defined single or multiple nodules (Fig. 14.1) and pulmonary infiltrates (Fig. 14.2), but other less frequent radiographic findings include pleural effusions, hilar lymphadenopathy, and lung cavitation. Patients with pulmonary cryptococcosis can present with symptoms of acute onset of fever, productive cough, respiratory distress, chest pain, and weight 260 Methee Chayakulkeeree and John R. Perfect Table 14.4 Clinical manifestations of cryptococcosis Organs Common clinical manifestations Central nervous system Acute/subacute/chronic meningoencephalitis Cryptococcomas Lung Asymptomatic with abnormal chest radiographs; pulmonary nodule(s), hilar lymphadenopathy, lobar/interstitial infiltrates, lung cavities, miliary infiltrates Acute/subacute pneumonia Acute respiratory distress syndrome Endobronchial lesions Pleural effusion/pneumothorax Skin Papules with central ulceration (molluscum contagiosum-like) Abscesses Nodules/papules Cellulitis Draining sinuses Ulcers Eyes Papilledema Endophthalmitis Optic nerve atrophy Chorioretinitis Genitourinary tract Prostatitis Cryptococcuria Renal abscess Bones and joints Osteolytic lesions Arthritis Cardiovascular system Cryptococcemia Endocarditis Mycotic aneurysm Other organs Peritonitis Myositis Hepatitis Thyroiditis Adrenal mass loss (13). The outbreak of C. gattii infections in Vancouver Island had involved several cases of severe symptomatic pulmonary cryptococcosis in apparently immunocom- petent individuals. In an immunocompromised patient, especially with HIV infection, cryptococcal pneumonia is usually symptomatic and can progress rapidly to an acute respiratory distress syndrome. However, most immunocompromised patients with cryptococcal infection usually present with CNS rather than pulmonary symptoms. In fact, more than 90% of HIV/AIDS patients with cryptococcal infection already have CNS cryptococcosis at the time of diagnosis. The findings in chest |
radiographs of immunocompromised patients with pulmonary cryptococcosis are the same as those in 14. Cryptococcosis 261 Fig. 14.1. Chest radiograph of pulmonary cryptococcosis presents as a single nodule in the lung at right lower lung field. (Reproduced with permission from A. Casadevall and J.R. Perfect, Cryptococcus neoformans, ASM Press, Washington, DC, 1998.). Fig. 14.2. Chest radiograph of pulmonary cryptococcosis presents as a left lobar infiltrates. (Reproduced with permission from A. Casadevall and J.R. Perfect, Cryptococcus neoformans, ASM Press, Washington, DC, 1998.). 262 Methee Chayakulkeeree and John R. Perfect immunocompetent patients, but alveolar and interstitial infiltrates tend to be more frequent and can mimic Pneumocystis pneumonia. In pulmonary cryptococcosis, if the infection is confined to the lung, serum cryptococcal polysaccharide antigen is usually negative and a positive serum polysaccharide antigen may indicate the dissemination of the yeast from the lung and therefore a lumbar puncture needs to be considered to rule out CNS cryptococcosis without symptoms. In immunocompromised individuals with pulmonary cryptococcosis, a lumbar puncture to rule out CNS disease is suggested regardless of the patient’s symptoms or serum polysaccharide antigen test results. The only setting in which a screening lumbar puncture may not necessarily need to be performed is in asymptomatic, immunocompetent patients with disease that appears limited to the lungs. 5.2. CNS Infection Clinical manifestations of CNS cryptococcosis include headache, fever, cranial neuropathy, alteration of consciousness, lethargy, memory loss, and meningeal irritation signs (2,8). These findings are usually present for several weeks and therefore cause a clinical syndrome of subacute meningitis or meningoencephalitis. However, on some occasions, patients can present with acute and/or intermittent headaches, or even with an altered mental status without headache. In HIV-infected patients with CNS crypto- coccosis, the burden of fungal organisms in the CNS is usually higher. Therefore, these patients may have a shorter onset of signs and symptoms, higher CSF polysac- charide antigen titers, higher intracranial pressures, and slower CSF sterilization after starting antifungal treatment. Further, patients who receive antiretroviral treatment can have a syndrome called immune reconstitution inflammatory syndrome (IRIS) with cryptococcal infections (14). This syndrome usually develops in the few months after HAART is introduced and when the CD4+ cells rise and the immune status improves. This syndrome can also occur in patients with solid organ transplants associated with allograft loss or altered immunosuppression (15). It has been hypothesized that crypto- coccal infections are made clinically apparent as inflammation is mobilized to interact with yeasts and/or their capsular antigen. Patients with cryptococcal IRIS can present with signs and symptoms that are indistinguishable from progressive cryptococcal meningitis/meningoencephalitis (i.e., worsening headaches, new inflammation noted on magnetic resonance imaging [MRI] scan, increased intracranial pressure). However, cultures from clinical specimens are always negative for cryptococci although yeasts may be present on a smear. Identification of IRIS has major implications for treatment strategies because it is not an antifungal treatment failure but a host immunity response issue. Different cryptococcal species may produce differences in clinical manifestations. For instance, one species may have a predilection to cause disease in brain parenchyma rather than the meninges. In certain areas of the world, C. gattii tend to cause cerebral cryptococcomas (Fig. 14.3) and/or hydrocephalus with or without large pulmonary mass lesions in immunocompetent hosts more frequently than C. neoformans. These patients with brain parenchymal involvement usually have high intracranial pressure, cranial neuropathies, and a poor response to antifungal therapy. 14. Cryptococcosis 263 Fig. 14.3. CT scan of the brain showing multiple cryptococcomas in an apparently normal host. (Reproduced with permission from A. Casadevall and J.R. Perfect, Cryptococcus neoformans, ASM Press, Washington, DC, 1998.). 5.3. Skin Infection Cutaneous infections are the third most common clinical manifestations of crypto- coccosis. Patients can manifest several types of skin lesions. One common skin lesion is a papule or maculopapular rash with central ulceration that may be described as “molluscum contagiosum-like” lesions. These lesions cannot be distinguished from those found in other fungal infections caused by Histoplasma capsulatum, Coccidioides, and Penicillium marneffei. Other cutaneous lesions of cryptococcosis include acneiform lesions, purpura, vesicles, nodules, abscesses, ulcers (Fig. 14.4), granulomas, pustules, plaques, draining sinus, and cellulitis. Because there are many skin manifestations in cryptococcosis that mimic other infections, skin biopsy with culture and histopathology are essential for definitive diagnosis. Skin lesions of cryptococcosis usually occur as a sign of disseminated cryptococcal infection. Primary cutaneous cryptococcosis is very rare and is usually associated with skin injury and direct inoculation of the yeasts. Patients with solid organ transplants receiving tacrolimus seem to be more likely to develop skin, soft tissue, and osteoarticular cryptococcal infections (16). Tacrolimus 264 Methee Chayakulkeeree and John R. Perfect Fig. 14.4. Skin ulceration and cellulitis as cutaneous cryptococcosis. [Figure in color on CD-ROM]. has anticryptococcal activity at high temperatures, but loses this activity as environ- mental temperatures decrease, which potentially explains the increased frequency of cutaneous cryptococcosis in these solid organ transplant recipients. Despite this series of patients, the most common site of disseminated infection in solid organ transplant recipients still remains the CNS, including in patients receiving tacrolimus. 5.4. Prostate Infection Prostatic cryptococcosis is usually asymptomatic and, in fact, the prostate gland is considered to be a sanctuary site for this yeast from the full impact of antifungal treatment. Itmaybean important reservoir for relapseofcryptococcosis inpatientswithahighburden of yeasts (17). A latent C. neoformans infection has even been recognized to spread into the blood during urological surgery on the prostate (18). Cultures of urine or seminal fluid may still be positive for Cryptococcus after initial antifungal treatment of cryptococcal meningoencephalitis in AIDS patients (19), strongly supporting a need for prolonged antifungal treatment to clear the prostate in these severely immunocompromised patients. 5.5. Eye Infection In the early reports of cryptococcal meningoencephalitis before the AIDS epidemic, ocular signs and symptoms were noted in approximately 45% of cases (20). The most common manifestations are ocular palsies and papilledema. However, in the present HIV era, several other manifestations of ocular cryptococcosis have been identified, including the presence of extensive retinal lesions with or without vitritis which can lead to blindness. Further, catastrophic loss of vision without evidence of endophthalmitis has also been reported (21). Visual loss may be due to one of two pathogenic processes. 14. Cryptococcosis 265 The first is caused by infiltration of the optic nerve with the yeasts, producing rapid visual loss with few effective treatments. The second is due to increased intracranial pressure. In this setting patients have slower visual loss, and treatment with serial lumbar punctures or ventricular shunts can prevent or slow down visual loss. 5.6. Infection at Other Body Sites In addition to lung, CNS, skin, prostate, and eye, C. neoformans can cause disease in many other organs (Table 14.4). Cryptococcemia can occur in severely immunosup- pressed patients but rarely causes endocarditis. Bone involvement of cryptococcosis typically presents as one or more circumscribed osteolytic lesions in any bone of the body and can be associated with sarcoidosis. Cryptococcal peritonitis (22) and crypto- coccuria are also reported in several case series. Any organ of the human body can be a site of cryptococcal infections. 6. DIAGNOSIS Several methods are used for diagnosis of cryptococcosis, including direct exami- nation of the fungus in body fluids, histopathology of infected tissues, serological studies, and culture of body fluids or tissues. 6.1. Direct Examination The most rapid method for diagnosis of cryptococcal meningitis is direct microscopic examination for encapsulated yeasts by an India ink preparation of cerebrospinal fluid (CSF). Cryptococcus can be visualized as a globular, encapsulated yeast cell with or without budding, ranging in size from 5 to 20 μm in diameter. It is easily distinguished in a colloidal medium of India ink when mixed with CSF (Fig. 14.5). Approximately 1 to 5 ml of specimen is recommended for use in the India ink preparation. India ink examination can detect encapsulated yeasts in a CSF specimen with a threshold between 103 and 104 colony-forming units of yeasts per milliliter of fluid. The sensitivity of India ink preparation technique is 30% to 50% in non-AIDS-related cryptococcal meningitis and up to 80% sensitive in AIDS-related cryptococcal meningitis. Some false-positive results can be found from intact lymphocytes, myelin globules, fat droplets, and other tissue cells. Also, dead yeast cells can remain in the CSF and be visualized via India ink preparation for varying periods of time during and after appropriate antifungal treatment. This is a limitation of direct microscopy of CSF during the management of cryptococcal meningitis (23). 6.2. Cytology and Histopathology Cryptococcus can be prominently identified by histological stains of tissues from lung, skin, bone marrow, brain, or other organs (24). Histopathological staining of a centrifuged CSF sediment has proven to be more sensitive for rapid diagnosis of cryptococcal meningitis than the India ink method (25). Peritoneal fluid from chronic ambulatory peritoneal dialysis (CAPD), seminal fluid, bronchial wash, or bronchoalveolar lavage fluid can also be used for cytology preparations in the diagnosis of cryptococcal infections (26,27). Fine-needle aspiration (FNA) for cytology of peripheral lymph nodes, adrenal glands; or vitreous aspiration, percutaneous transtho- racic biopsy under real-time ultrasound guidance; or video-assisted thorascopic lung 266 Methee Chayakulkeeree and John R. Perfect Fig. 14.5. India ink preparation showing budding encapsulated yeasts of C. neoformans. [Figure in color on CD-ROM]. biopsy on pulmonary nodules, masses, or infiltrative lesions can be used to obtain tissues for cytology/histopathology (28). A variety of positive staining methods have been described to demonstrate the yeast cells in tissue or fluids; ranging from the nonspecific Papanicolaou or hematoxylin and eosin stains, to the more specific fungal stains such as Calcofluor, which binds fungal chitin, or Gomori methenamine silver (GMS), which stains the fungal cell wall (2,26) (Fig. 14.6). Several stains can identify the polysaccharide capsular material surrounding the yeasts. These stains can be especially useful in presumptively identi- fying Cryptococcus when cultures do not grow or are not obtained. They include Mayer’s mucicarmine, periodic acid-Schiff (PAS), and alcian blue stains (2) (Fig. 14.6) (also see Fig. 3.4, Chapter 3). The Fontana-Masson stain appears to identify melanin in the yeast cell wall. The fungus is observed as a yeast that reproduces by formation of narrow-based budding with a prominent capsule. Gram stain is not optimal for identification of this yeast, but may show C. neoformans as a poorly stained gram positive budding yeast (Fig. 14.7) (2). The recognition of C. neoformans in Gram- stained smears of purulent exudates may be hampered by the presence of the large gelatinous capsule, which apparently prevents definitive staining of the yeast-like cells. 6.3. Serology Diagnosis of cryptococcosis has been significantly improved over the last several decades by the development of serological tests for cryptococcal polysaccharide antigen and/or antibody. Using serum cryptococcal antibodies as the only diagnostic tool for 14. Cryptococcosis 267 Fig. 14.6. Mouse tissues stained with various stains used to identify cryptococcal infection. Upper left panel is of brain stained with H&E showing meningoencephalitis with encapsulated yeast cells of C. neoformans. The upper right panel is of kidney stained with GMS. The middle left panel demonstrates lung stained with Mayer’s mucicarmine. Note orange-red staining of polysaccharide capsular material of C. neoformans. The middle right panel is liver tissue stained with PAS. Lung stained with Alcian blue stain is seen in the bottom left panel. Lung stained by Fontana-Masson method is seen in the bottom right. Melanin pigment in the cell wall of C. neoformans stains dark with this stain. (Courtesy of Dr. W.A. Schell.). [Figure in color on CD-ROM]. cryptococcosis has not been adopted for early diagnosis of cryptococcosis. In contrast, detection of cryptococcal capsular polysaccharide antigen in serum or body fluids via a latex agglutination technique has been robust in its performance and is the most useful diagnostic serological test available for cryptococcosis. This test uses latex particles coated with polyclonal cryptococcal capsular antibodies or antiglucuronoxylomannan monoclonal antibodies and has overall sensitivities and specificities of 93% to 100% 268 Methee Chayakulkeeree and John R. Perfect Fig. 14.7. Gram stain of sputum of a patient with pulmonary cryptococcosis. C. neoformans appears as poorly stained gram-positive budding yeasts. (Courtesy of Dr. W.A. Schell, Duke University Medical Center.). [Figure in color on CD-ROM]. and 93% to 98%, respectively (29,30). The false-positive rate of cryptococcal capsular polysaccharide antigen testing is only 0% to 0.4% (31). These can be caused by technical error, presence of rheumatoid factor or interference proteins, and infections with |
Trichosporon beigelii or some bacterial species. However, most of the false- positive results of latex agglutination testing for cryptococcal polysaccharide antigen have initial reciprocal titers of 8 or less (29). Therefore results of such low titers must be carefully interpreted within the clinical context. False-negative results of latex agglutination test for cryptococcal polysaccharide antigen in cryptococcal meningitis are unusual but can be due to prozone effect and therefore high-risk negative specimens may need to be diluted and retested (32). Low fungal burden, as in chronic low-grade cryptococcal meningitis or in a very early stage of cryptococcal infection, and improper storage of patient sera can also cause false-negative results in latex cryptococcal polysaccharide antigen agglutination tests (33). Enzyme immunoassay (EIA) for detection and quantification of cryptococcal polysaccharide antigen of all four serotypes of C. neoformans in sera and CSF has been developed to detect the major component of the polysaccharide capsule, glucuronoxy- lomannan (GXM), with sensitivities and specificities of 85.2% to 99% and 97%, respectively (29,34). Previous studies compared EIA and latex cryptococcal polysac- charide antigen agglutination tests and revealed that there was no significant difference between these tests. EIA for cryptococcal polysaccharide antigen did not give discrepant results with rheumatoid factor, syneresis fluid, or serum macroglobulins, and is not affected by prozone reactions. 14. Cryptococcosis 269 Although the presence of cryptococcal polysaccharide antigen in serum is undoubtedly suggestive for dissemination of cryptococcal infection outside the lung, the precise value of cryptococcal polysaccharide antigen for diagnosis of nondisseminated pulmonary cryptococcosis remains less certain. Generally, a positive or negative serum cryptococcal polysaccharide antigen will not prove or disprove limited pulmonary disease, but detectable antigen in serum should make clinicians consider that infection is now also located outside the lung. In a high-risk patient with meningitis, identifi- cation of cryptococcal antigen in CSF or serum is rapid, specific, noninvasive, and is virtually diagnostic of meningoencephalitic or disseminated cryptococcosis even when the India ink examination or culture is negative (35,36). The latex agglutination test for serum cryptococcal polysaccharide antigen is widely used for detecting crypto- coccal infection in patients with AIDS, as an initial screening test for those with fever of unclear etiologies or neurological symptoms. If financially feasible, this test has become a part of routine clinical practice for suspected cases of cryptococcal infec- tions in geographical areas where a high density of cryptococcal disease is present. In some patients, it may represent the only means of achieving an etiologic diagnosis of invasive cryptococcosis or an early diagnosis prior to CNS involvement. Likely because of its sensitivity, the detection of cryptococcal polysaccharide antigen in the serum may precede clinically obvious disseminated cryptococcal disease (“isolated cryptococcal polysaccharidemia”) in severely immunosuppressed patients (37,38). The management of these cases, in which there is a positive serum antigen and other nonspecific clinical findings in HIV-infected patients with negative fluid or tissue cultures, is uncertain. Isolated cryptococcal antigenemia in persons in high-risk groups probably do benefit from antifungal therapy given to prevent or delay the development of overt cryptococcosis (37). Generally, positive serum antigen tests at titers of 1:4 or more strongly suggest cryptococcal infections in these patients. Baseline cryptococcal polysaccharide antigen titers in serum and CSF have been shown to be factors that may be used to predict outcome of patients with crypto- coccal meningitis (39). A study in HIV-related acute cryptococcal meningitis indicated that a baseline titer of CSF cryptococcal polysaccharide antigen of 1:1024 or greater was a predictor of death during systemic antifungal treatment (40). After initiation of systemic antifungal therapy, patients may respond to treatment and titers of crypto- coccal polysaccharide antigen fall. Similarly, a rise in CSF cryptococcal polysaccharide antigen titers during suppressive therapy has been associated with relapse of crypto- coccal meningitis (41). However, it is important to emphasize that the use of changing antigen titers to make therapeutic decision is not precise and should be done with caution. The kinetics of polysaccharide elimination remains unclear and despite the accuracy of commercial kits for general diagnosis, the accuracy of specific titers can vary from kit to kit even with the same specimen. 6.4. Culture and Identification Cryptococcus can be easily grown from biologic samples such as CSF, sputum, and skin biopsy specimens on routine fungal and bacterial culture media. Colonies can usually be observed on solid agar plates after 48 to 72 hours incubation at 30 to 35 C in aerobic conditions. Antibacterial agents, preferably chloramphenicol, can be added 270 Methee Chayakulkeeree and John R. Perfect to the media when bacterial contamination is considered. The yeasts, however, do not grow in the presence of cycloheximide at the concentration used in selective fungal isolation media (25 μg/ml). Despite relatively rapid growth for most strains, cultures should be held for 3 to 4 weeks before discarding, particularly for patients already receiving antifungal treatment. On the other hand, there may be negative cultures despite positive microscopic examinations (India ink) due to nonviable yeast cells which may have prolonged persistence at the site of infection. Positive blood cultures are frequently reported in AIDS patients, and this may actually be the first positive test for this infection in a febrile high-risk patient. C. neoformans colonies appear on routine fungal media as opaque, white, creamy colonies that may turn orange-tan or brown after prolonged incubation. The mucoid appearance of the colony is related to the capsule size around the yeasts. Cryptococcus does not routinely produce hyphae or pseudohyphae, or ferment sugars, but is able to assimilate inositol and hydrolyze urea (42). C. neoformans and C. gattii do not assimilate nitrate but have the ability to use galactose, maltose, galactitol, and sucrose (42). There are special media such as canavanine–glycine–bromthymol blue (CGB) agar which can be used to differentiate C. gattii strains from C. neoformans strains (43). 7. TREATMENT The 2000 practice guidelines for the management of cryptococcal disease from the Infectious Diseases Society of America (summarized in Table 14.5) provide a good starting point for therapeutic decision making (44). Patients with pulmonary cryptococ- cosis with or without HIV infection can be treated with an oral regimen of fluconazole. However, in patients with severe symptoms, treatment similar to cryptococcal menin- gitis is recommended (44). In brief, amphotericin B deoxycholate remains the drug of choice for disseminated cryptococcosis. A standard induction dose for amphotericin B is 0.7 to 1 mg/kg per day. Liposomal amphotericin B (AmBisome) at 4 mg/kg per day can be used as an alternative treatment with a similar outcome to that of amphotericin B deoxycholate but with less nephrotoxicity (45). Flucytosine (5-FC) is primarily used in combination therapy with amphotericin B for first-line therapy in cryptococcal meningitis or severe pulmonary cryptococcosis at the dosage of 100 mg/kg per day in divided doses in patients with normal renal function (46,47). The combination of amphotericin B and 5-FC represents the most potent fungicidal regimen which produces consistently negative CSF cultures at 2 weeks of treatment in non-AIDS patients. 5-FC levels should be monitored to keep 2-hour postdose levels under 100 μg/ml to reduce its primary side effect of bone marrow suppression. A three-stage approach is employed in the treatment of cryptococcal meningitis in HIV-infected patients; this can be also followed in non-HIV patients (46). Initial induction treatment usually begins with amphotericin B plus 5-FC for 2 weeks, followed by clearance treatment with fluconazole 400 to 800 mg/day for a minimum of 10 weeks. Finally, a long-term suppressive/maintenance therapy (secondary prophylaxis) usually begins with oral fluconazole, 200 to 400 mg given once daily. Secondary prophylaxis can be discontinued after 2 years in patients who respond to HAART with rise in CD4+ cell counts to greater than 100 cells/μl and decline in viral load (HIV RNA) 14. Cryptococcosis 271 Table 14.5 Treatments for cryptococcal disease Cryptococcal disease in HIV-negative patients Pulmonary Mild-to-moderate symptoms: Fluconazole, 200–400 mg/day for 6–12 months Itraconazole, 200–400 mg/day for 6–12 months Amphotericin B, 0.5–1 mg/kg per day (total 1–2 g) Severe symptoms and immunocompromised hosts: Treat like CNS disease Central nervous system Induction/consolidation or clearance therapy: Amphotericin B, 0.7–1 mg/kg per day (preferably 0.7 mg/kg per day) plus flucytosine, 100 mg/kg per day (assuming normal renal function) for 2 weeks, then fluconazole, 400–800 mg/day for minimum 10 weeks Alternative regimens: Amphotericin B, 0.3 mg/kg per day plus flucytosine, 100 mg/kg per day for 6–10 weeks Amphotericin B, 0.4–1 mg/kg per day for 6–10 weeks Lipid formulation of amphotericin B, 4–6 mg/kg per day for 6–10 weeks with or without 2 weeks of flucytosine Suppressive therapy: Fluconazole 200–400 mg/day for completion of 1 year of therapy. Cryptococcal disease in HIV-infected patients Pulmonary Mild-to-moderate symptoms Fluconazole, 200–400 mg/day, for 1–2 years depending on response to HAART Alternative regimen: Itraconazole, 200–400 mg/day, for 1–2 years depending on response to HAART Severe symptoms: Treat like CNS disease Central nervous systema Induction/consolidation or clearance therapy: Amphotericin B, 0.7–1 mg/kg per day (preferably 0.7 mg/kg per day) plus flucytosine, 100 mg/kg per day for 2 weeks, then fluconazole, 400–800 mg/day for a minimum of 10 weeks Alternatives regimens: Fluconazole, 400–800 mg/d ay for 10–12 weeks Fluconazole, 400–800 mg/day plus flucytosine, 100–150 mg/kg per day for 6–10 weeks Lipid formulation of amphotericin B, 4–6 mg/kg per day for 6–10 weeks with or without flucytosine Maintenance or suppressive therapy: 1–2 years and may consider stopping if response to HAART Fluconazole, 200–400 mg/day Alternatives regimens: Itraconazole, 200 mg/day Amphotericin B, 1 mg/kg IV 1–3 times/week Adapted from the 2000 IDSA Practice Guideline for the Management of Cryptococcal Diseases with personal suggestions (44). aStart HAART 8–10 weeks after beginning antifungal regimen. Control IRIS with corticosteroids. 272 Methee Chayakulkeeree and John R. Perfect to undetectable levels for at least 3 months (48,49). Criteria for stopping treatment in non-AIDS patients with cryptococcal meningitis include resolution of symptoms, at least two negative CSF cultures, and a normal CSF glucose; generally following at least 1 year of suppressive therapy. Patients may have prolonged positive cryptococcal polysaccharide antigen tests and/or slightly abnormal CSF findings for months during successful therapy. Itraconazole can be used as an alternative azole treatment for cryptococcosis, but first-line therapy is with fluconazole. Itraconazole has poor CSF penetration and incon- sistent oral bioavailability but has been successfully used in the treatment of crypto- coccal meningitis (50). It has been shown to have efficacy inferior to fluconazole during suppressive therapy (51). In patients with HIV-associated cryptococcal diseases, HAART has a major impact on the patient’s long-term prognosis. However, treatment with HAART can cause cryptococcal IRIS. There are limited data for formal recommendations in prevention and treatment of cryptococcal IRIS in AIDS. However, at present, a delay in initiation of HAART for 8 to 10 weeks after starting antifungal therapy for cryptococcal diseases is generally recommended to reduce the complexity of dealing with IRIS and the increased intracranial pressure that may occur during induction therapy. Interruption of HAART and/or corticosteroids treatment may be used to control symptoms if severe cryptococcal IRIS occurs. IRIS in cryptococcosis is not limited to HIV and HAART, although it has been reported in up to 30% of these patients (14). It can occur in any patient in whom immune status changes rapidly; IRIS has been reported in 5% of solid organ transplantations with cryptococcosis (52). Along with the use of antifungal therapy, management of increased intracranial pressure is also important. An intracranial pressure of 250 mm H2O or more is considered elevated intracranial pressure (ICP). HIV-infected patients with crypto- coccal meningitis and high ICP after 2 weeks of treatment have been shown to have poorer clinical responses (44). Attempts to control increased ICP generally occur after development of symptoms (increasing headache, mental status changes, and new neurological findings). Treatment options recommended for managing acutely elevated ICP include repeated lumbar punctures, lumbar drain insertion, ventriculostomy, or ventriculoperitoneal shunt (Table 14.6). Medical treatments such as corticosteroids (unless there is a component of IRIS linked to the increased ICP), mannitol, or aceta- zolamide have some clinical data, but are generally not recommended for use in management of increased ICP pressure in cryptococcal meningitis (53). Some patients may develop symptoms of obstructive hydrocephalus requiring a permanent ventricu- loperitoneal shunt during the first 1 to 2 years of treatment, and sometimes at initial presentation. The shunt can be put in once a patient is receiving appropriate antifungal therapy; delaying placement until cultures are sterile is not required. 8. |
PREVENTION Prevention of cryptococcal diseases can be definitely achieved by use of HAART in HIV-infected patients to improve their immunity. Fluconazole prophylaxis has been shown to be effective for preventing cryptococcosis in AIDS patients who have 14. Cryptococcosis 273 Table 14.6 Management of elevated intracranial pressure in HIV-infected patients with cryptococcosis Initial lumbar puncture Positive focal neurological signs or altered conscious status Radiographic imaging before lumbar puncture is indicated Normal opening pressure Initiate medical therapy, with follow-up lumbar puncture at 2 weeks Opening pressure ≥ 250 mmH2O with signs or symptoms Lumbar drainage sufficient to achieve closing pressure <200 mm H2O or 50% of initial opening pressurea Follow-up for elevated pressure Repeated drainage daily until opening pressure <200 mm H2O and symptoms/signs are stable If elevated pressure persists, consider Lumbar drain Ventriculoperitoneal shunt This table based on the 2000 IDSA Practice Guideline for the Management of Cryptococcal Diseases (44). aRecommendations are not evidence-based and provided as a guide only. persistently low CD4+ cell counts (below 100 cells/μl), but this is not currently recom- mended, and HAART remains the best strategy for prevention in HIV infection. Although cryptococcal GXM-tetanus toxoid conjugate vaccine and specific monoclonal antibodies to cryptococci have been developed, clinical trials have not been initiated to determine their usefulness. REFERENCES 1. Knoke M, Schwesinger G. One hundred years ago: the history of cryptococcosis in Greif- swald. Medical mycology in the nineteenth century. Mycoses 1994;37:229–233. 2. Casadevall A, Perfect JR. Cryptococcus neoformans. Washington, DC: ASM Press, 1998. 3. Kwon-Chung KJ, Boekhout T, Fell JW, Diaz M. Proposal to conserve the name Crypto- coccus gattii against C. hondurianus and C. bacillisporus. Taxon 2002;51:804–806. 4. Mitchell TG, Perfect JR. Cryptococcosis in the era of AIDS—100 years after the discovery of Cryptococcus neoformans. Clin Microbiol Rev 1995;8:515–548. 5. Kidd SE, Hagen F, Tscharke RL, et al. A rare genotype of Cryptococcus gattii caused the cryptococcosis outbreak on Vancouver Island (British Columbia, Canada). Proc Natl Acad Sci USA 2004;101:17258–17263. 6. 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Cryptococcus neoformans peritonitis in a patient with alcoholic cirrhosis: case report and review of the literature. Infection 2005;33:282–288. 23. Diamond RD, Bennett JE. Prognostic factors in cryptococcal meningitis. A study in 111 cases. Ann Intern Med 1974;80:176–181. 24. Shibuya K, Coulson WF, Wollman JS, et al. Histopathology of cryptococcosis and other fungal infections in patients with acquired immunodeficiency syndrome. Int J Infect Dis 2001;5:78–85. 25. Sato Y, Osabe S, Kuno H, Kaji M, Oizumi K. Rapid diagnosis of cryptococcal meningitis by microscopic examination of centrifuged cerebrospinal fluid sediment. J Neurol Sci 1999;164:72–75. 26. Kanjanavirojkul N, Sripa C, Puapairoj A. Cytologic diagnosis of Cryptococcus neoformans in HIV-positive patients. Acta Cytol 1997;41:493–496. 27. Malabonga VM, Basti J, Kamholz SL. Utility of bronchoscopic sampling techniques for cryptococcal disease in AIDS. Chest 1991;99:370–372. 28. Lee LN, Yang PC, Kuo SH, Luh KT, Chang DB, Yu CJ. Diagnosis of pulmonary crypto- coccosis by ultrasound guided percutaneous aspiration. Thorax 1993;48:75–78. 14. Cryptococcosis 275 29. Tanner DC, Weinstein MP, Fedorciw B, Joho KL, Thorpe JJ, Reller L. Comparison of commercial kits for detection of cryptococcal antigen. J Clin Microbiol 1994;32: 1680–1684. 30. Wu TC, Koo SY. Comparison of three commercial cryptococcal latex kits for detection of cryptococcal antigen. J Clin Microbiol 1983;18:1127–1130. 31. Kauffman CA, Bergman AG, Severance PJ, McClatchey KD. Detection of crypto- coccal antigen. Comparison of two latex agglutination tests. Am J Clin Pathol 1981;75: 106–109. 32. Stamm AM, Polt SS. False-negative cryptococcal antigen test. JAMA 1980;244:1359. 33. Bloomfield N, Gordon MA, Elmendorf DF. Detection of Cryptococcus neoformans antigen in body fluids by latex particle agglutination. Proc Soc Exp Biol Med 1963;114:64–67. 34. Gade W, Hinnefeld SW, Babcock LS, et al. Comparison of the PREMIER cryptococcal antigen enzyme immunoassay and the latex agglutination assay for detection of crypto- coccal antigens. J Clin Microbiol 1991;29:1616–1619. 35. Chuck SL, Sande MA. Infections with Cryptococcus neoformans in the acquired immun- odeficiency syndrome. N Engl J Med 1989;321:794–799. 36. Shih CC, Chen YC, Chang SC, Luh KT, Hsieh WC. Cryptococcal meningitis in non-HIV- infected patients. QJM 2000;93:245–251. 37. Feldmesser M, Harris C, Reichberg S, Khan S, Casadevall A. Serum cryptococcal antigen in patients with AIDS. Clin Infect Dis 1996;23:827–830. 38. Tassie JM, Pepper L, Fogg C, et al. Systematic screening of cryptococcal antigenemia in HIV-positive adults in Uganda. J Acquir Immune Defic Syndr 2003;33:411–412. 39. Bindschadler DD, Bennett JE. Serology of human cryptococcosis. Ann Intern Med 1968;69:45–52. 40. Saag MS, Powderly WG, Cloud GA, et al. Comparison of amphotericin B with fluconazole in the treatment of acute AIDS-associated cryptococcal meningitis. The NIAID Mycoses Study Group and the AIDS Clinical Trials Group. N Engl J Med 1992;326:83–89. 41. Powderly WG, Cloud GA, Dismukes WE, Saag MS. Measurement of cryptococcal antigen in serum and cerebrospinal fluid: value in the management of AIDS-associated cryptococcal meningitis. Clin Infect Dis 1994;18:789–792. 42. Viviani MA, Tortorano AM, Ajello L. Cryptococcus. In: Anaissie EJ, McGinnis MR, Pfaller MA, eds. Clinical mycology. Philadelphia: Churchill Livingstone, 2003:240–259. 43. Min KH, Kwon-Chung KJ. The biochemical basis for the distinction between the two Cryptococcus neoformans varieties with CGB medium. Zentralbl Bakteriol Mikrobiol Hyg [A] 1986;261:471–480. 44. Saag MS, Graybill RJ, Larsen RA, et al. Practice guidelines for the management of cryptococcal disease. Infectious Diseases Society of America. Clin Infect Dis 2000;30: 710–718. 45. Leenders AC, Reiss P, Portegies P, et al. Liposomal amphotericin B (AmBisome) compared with amphotericin B both followed by oral fluconazole in the treatment of AIDS-associated cryptococcal meningitis. AIDS 1997;11:1463–1471. 46. van der Horst CM, Saag MS, Cloud GA, et al. Treatment of cryptococcal meningitis associated with the acquired immunodeficiency syndrome. National Institute of Allergy and Infectious Diseases Mycoses Study Group and AIDS Clinical Trials Group. N Engl J Med 1997;337:15–21. 47. Dismukes WE, Cloud G, Gallis HA, et al. Treatment of cryptococcal meningitis with combination amphotericin B and flucytosine for four as compared with six weeks. N Engl J Med 1987;317:334–341. 48. Vibhagool A, Sungkanuparph S, Mootsikapun P, et al. Discontinuation of secondary prophylaxis for cryptococcal meningitis in human immunodeficiency virus-infected patients 276 Methee Chayakulkeeree and John R. Perfect treated with highly active antiretroviral therapy: a prospective, multicenter, randomized study. Clin Infect Dis 2003;36:1329–1331. 49. Mussini C, Pezzotti P, Miro JM, et al. Discontinuation of maintenance therapy for crypto- coccal meningitis in patients with AIDS treated with highly active antiretroviral therapy: an international observational study. Clin Infect Dis 2004;38:565–571. 50. Denning DW, Tucker RM, Hanson LH, Hamilton JR, Stevens DA. Itraconazole therapy for cryptococcal meningitis and cryptococcosis. Arch Intern Med 1989;149:2301–2308. 51. Saag MS, Cloud GA, Graybill JR, et al. A comparison of itraconazole versus fluconazole as maintenance therapy for AIDS-associated cryptococcal meningitis. National Institute of Allergy and Infectious Diseases Mycoses Study Group. Clin Infect Dis 1999;28: 291–296. 52. Singh N, Lortholary O, Alexander BD, et al. An immune reconstitution syndrome-like illness associated with Cryptococcus neoformans infection in organ transplant recipients. Clin Infect Dis 2005;40:1756–1761. 53. Newton PN, Thai le H, Tip NQ, et al. A randomized, double-blind, placebo-controlled trial of acetazolamide for the treatment of elevated intracranial pressure in cryptococcal meningitis. Clin Infect Dis 2002;35:769–772. SUGGESTED READINGS Casadevall A, Perfect JR. Cryptococcus neoformans. Washington, DC: ASM Press, 1998. Kidd SE, Hagen F, Tscharke RL, et al. A rare genotype of Cryptococcus gattii caused the cryptococcosis outbreak on Vancouver Island (British Columbia, Canada). Proc Natl Acad Sci USA 2004;101:17258–17263. Mitchell TG, Perfect JR. Cryptococcosis in the era of AIDS–100 years after the discovery of Cryptococcus neoformans. Clin Microbiol Rev 1995;8:515–548. Mussini C, Pezzotti P, Miro JM, et al. Discontinuation of maintenance therapy for crypto- coccal meningitis in patients with AIDS treated with highly active antiretroviral therapy: an international observational study. Clin Infect Dis 2004;38:565–571. Perfect JR. Cryptococcus neoformans. In: Mandell GL, Bennett JE, Dolin R, eds. Mandell, Douglas, and Bennett’s principles and practice of infectious diseases. 6th Ed. Philadelphia, PA: Elsevier Churchill Livingstone, 2005:2997–3012. Perfect JR, Casadevall A. Cryptococcosis. Infect Dis Clin North Am 2002;16:837–874. Powderly WG, Cloud GA, Dismukes WE, Saag MS. Measurement of cryptococcal antigen in serum and cerebrospinal fluid: value in the management of AIDS-associated cryptococcal meningitis. Clin Infect Dis 1994;18:789–792. Saag MS, Graybill RJ, Larsen RA, et al. Practice guidelines for the management of cryptococcal disease. Infectious Diseases Society of America. Clin Infect Dis 2000;30:710–718. Saag MS, Powderly WG, Cloud GA, et al. Comparison of amphotericin B with fluconazole in the treatment of acute AIDS-associated cryptococcal meningitis. The NIAID Mycoses Study Group and the AIDS Clinical Trials Group. N Engl J Med 1992;326:83–89. 15 Blastomycosis Stanley W. Chapman, MD and Donna C. Sullivan, PhD 1. INTRODUCTION Blastomycosis is the systemic mycosis, primarily involving the lungs, caused by the thermally dimorphic fungus Blastomyces dermatitidis. First described by Gilchrist as a cutaneous disease (1), later analysis showed that the lung was the primary route of infection (2) and that skin disease or other organ involvement was secondary to hematogenous dissemination. Blastomycosis of the lung may be asymptomatic or manifest as acute or chronic pneumonia. Hematogenous spread of the organism frequently results in extrapulmonary disease. Blastomycosis has been reported in North America, Africa, India, and parts of Europe, but the majority of cases are from the endemic region around the Mississippi and Ohio Rivers and in areas of southern Canada near the Great Lakes (3–5). 2. ETIOLOGIC AGENT Blastomyces dermatitidis is the imperfect (asexual) stage of Ajellomyces dermati- tidis, which exhibits thermal dimorphism growing as a mould (mycelial) form at 25º to 30°C and as a yeast form at 37°C (Fig. 15.1). The mycelia produce terminal conidia, which, when disturbed in the environment, easily become airborne. Human and animal infections typically occur after the inhalation of conidia, which convert to large budding yeast cells inside the lungs associated with the temperature shift to 37°C (6). Primary isolation of B. dermatitidis from clinical specimens is most reliable when grown as the mycelial form at 30°C. Mycelial colonies, which are white to brown in color, grow on agar in 1 to 3 weeks. Positive identification of B. dermatitidis, however, generally requires conversion to the yeast form |
at 37°C or nucleic acid amplification methods that allow early identification of mycelial phase growth. Yeast- like colonies are wrinkled and cream to tan in color. Asexual reproduction is by budding of single, broad-based, thick-walled, multinucleated daughter cells (Fig. 15.1). The same morphologic characteristics are observed in tissue samples from infected individuals, and in the right clinical situation can be used to make a presumptive diagnosis of blastomycosis (6). From: Infectious Disease: Diagnosis and Treatment of Human Mycoses Edited by: D. R. Hospenthal and M. G. Rinaldi © Humana Press Inc., Totowa, NJ 277 278 Stanley W. Chapman and Donna C. Sullivan Fig. 15.1. The mycelial phase of B. dermatitidis (left) produces no unique character- istics that allow organism identification. As a yeast (right), B. dermatitidis has characteristic thick-walled, multinucleated cells with single broad-based daughter cells. [Figure in color on CD-ROM]. Two serotypes of B. dermatitidis have been identified based on the presence or absence of the A exoantigen (7); A antigen deficient serotypes are restricted to Africa (8). Recent serologic differences in B. dermatitidis isolates from different regions of the United States and Africa have been noted via an enzyme-linked immunoassay (9). Restriction fragment length polymorphism analysis of isolates from different geographic regions of North America reveals a high degree of genetic similarity among isolates (10,11). Using a polymerase chain reaction (PCR)-based typing system, three major groups have been identified. These results indicate that different genotypic groups may exist (10,11). These methods of analysis may be useful in future epidemiological studies. 3. EPIDEMIOLOGY The ecological niche of B. dermatitidis has been difficult to conclusively establish. Environmental isolations of the organism associated with disease outbreaks indicate that the organism grows as microfoci in warm, moist soil in wooded areas that is enriched in organic material (12,13). Analysis of sporadic cases in humans and dogs, point source outbreaks, and infrequent environmental isolations has provided the major basis for the definition of endemic regions in North America (5,6). B. dermatitidis is endemic to the eastern United States, the Mississippi and Ohio River valleys, extending northward to the Great Lakes and southern Canada. Most cases have been reported in Mississippi, Arkansas, Kentucky, Tennessee, and Wisconsin. Within these endemic regions are hyperendemic areas with exceptionally high attack rates (14–16). Dogs are the most commonly infected animal although infection in other mammals also occurs (16–18). Although early studies of endemic cases indicated middle-aged men with outdoor occupations were at greatest risk for blastomycosis, subsequent review of reported outbreaks indicate no predilection for sex, age, race, occupation, or 15. Blastomycosis 279 seasonal exposure (6). Exposure to soil at work and at play appears to be a common factor associated with both endemic and epidemic disease. 4. PATHOGENESIS AND IMMUNOLOGY Blastomycosis is initiated by the inhalation of the conidia of B. dermatitidis. After inhalation, the infectious conidia may be nonspecifically phagocytosed and killed by polymorphonuclear leukocytes (PMNs), monocytes, and alveolar macrophages. This phagocytic response represents natural or innate immunity and may in part explain the asymptomatic cases in analysis of outbreaks. Conidia that escape the initial phagocytic response rapidly convert to a yeast form that is more resistant to phagocytosis and killing. Several virulence factors have been associated with the pathogenicity of B. dermatitidis. The thick cell wall of the yeast has been proposed to have antiphagocytic properties while higher concentrations of lipids and phospholipids have been associated with increased virulence in some strains (19,20). Conversion of B. dermatitidis to the yeast form also induces the expression of a yeast phase specific gene designated BAD- 1 (formerly WI-1). BAD-1 (WI-1) is a 120-kDa glycoprotein adhesion and immune modulator that has a number of essential properties, including CR3 and CD14+ binding and an epidermal growth factor (EGF)-like domain (21–24). Cellular immunity in humans, as monitored by antigen-induced lymphocyte prolif- eration, has been documented using whole yeast phase organisms, an alkali-soluble, water-soluble yeast extract, and BAD-1 (25–27). As with other endemic fungi, B. dermatitidis seems to require type 1-dependent cell mediated immunity (CMI) (28–31). Recent vaccine studies in animal models have shown that CMI can be mediated by both vaccine-induced CD4+ (30) and CD8+ T cells (32) which produce type 1 cytokines such as interferon-gamma (IFN-) and tumor necrosis factor-alpha (TNF-). In addition, the CD28+ T cell receptor has been shown to be required for the induction of protective T cell responses to B. dermatitidis infection (33). CD4+ cells require interleukin-12 (IL-12) for the development of CMI while CD8+ cells were less dependent on IL-12 for this process (34). These animal studies are promising and indicate that the development of a vaccine to prevent disease in humans is possible. 5. CLINICAL MANIFESTATIONS The clinical manifestations of blastomycosis are varied and include asymptomatic infection, acute or chronic pneumonia, and extrapulmonary disease. Extrapulmonary disease results from the hematogenous spread of the fungus from a primary pulmonary infection. Although B. dermatitidis infection has been reported to involve almost every organ of the human body, the skin, bones, and genital urinary system are most common (Table 15.1). It is important to note that blastomycosis mimics many other disease processes whether acute or chronic (35). For example, acute pulmonary blastomycosis may be diagnosed as bacterial community-acquired pneumonia or influenza. Chronic pulmonary blastomycosis most commonly mimics a malignancy or tuberculosis. Skin lesions are often misdiagnosed as pyoderma gangrenosa or keratoacanthoma. Blasto- mycosis of the larynx is frequently misdiagnosed as carcinoma. Thus, a high index of suspicion and a careful histologic evaluation of secretions or biopsy material should be performed in difficult cases. 280 Stanley W. Chapman and Donna C. Sullivan Table 15.1 Organ involvement in blastomycosis Organ system involved No. involved/total patients (%) Pulmonary 369/534 (69) Cutaneous 306/534 (57) Osseous 116/534 (22) Genitourinary 92/534 (17) Central nervous system 29/534 (5) Modified from ref. 6. Data obtained from clinical and autopsy findings compiled from seven studies (35–41). 5.1. Pulmonary Blastomycosis 5.1.1. Acute Infection Initial infection occurs after inhalation of conidia into the lungs. Unless associated with an outbreak or group exposure, acute infection is frequently unrecognized. Clinical Fig. 15.2. Acute pulmonary blastomycosis. Chest radiograph reveals peripheral alveolar infiltrate that appear to be pleural based. Although this patient clinically improved with antibi- otics sputum culture grew B. dermatitidis. 15. Blastomycosis 281 studies involving point source outbreaks of infection indicate that symptomatic acute pulmonary disease occurs in only 50% of individuals, usually after an incubation period of 30 to 45 days (12–14). Signs and symptoms of acute pulmonary blastomycosis are similar to those of influenza or bacterial pneumonia. Fever, chills, pleuritic chest pain, arthralgias, and myalgias usually occur abruptly. Cough begins as nonproductive but frequently becomes purulent as disease progresses. Chest radiographs usually reveal alveolar infiltrates with consolidation (Fig. 15.2) (42,43). Pleural effusions are uncommon and, if present, are typically small in volume. Hilar adenopathy is uncommon and is a useful sign in distinguishing acute blastomycosis from acute histo- plasmosis. Spontaneous cures of symptomatic acute infection have been documented, but the exact frequency of these cures has not been clearly established (44,45). Fig. 15.3. Progressive pulmonary disease showing extensive left mid-lung alveolar infil- trate. This patient failed multiple courses of oral and intravenous antibiotics over a 2-month period before diagnosis of blastomycosis. 282 Stanley W. Chapman and Donna C. Sullivan 5.1.2. Chronic Infection Most patients diagnosed with pulmonary blastomycosis have a chronic pneumonia that is clinically similar to tuberculosis, other fungal infections, and cancer. Symptoms include fever, weight loss, chronic productive cough, and hemoptysis. The most frequent radiologic findings are alveolar infiltrates (Fig. 15.3) with or without cavitation, mass lesions that mimic bronchogenic carcinoma (Fig. 15.4), and fibron- odular infiltrates (46,47). Although small pleural effusions have been reported, large pleural effusions (Fig. 15.5) are distinctly uncommon and, when present, have been associated with poor outcome (48). 5.1.3. Acute Respiratory Distress Syndrome Patients may occasionally present with acute respiratory distress syndrome (ARDS) associated with miliary disease or diffuse pneumonitis (Fig. 15.6). Mortality exceeds 50% in these patients, and most deaths occur within the first few days of therapy (46). Diffuse pulmonary infiltrates and respiratory failure are more likely to occur in immunocompromised patients, especially those with late stage acquired immunodefi- ciency syndrome (AIDS) (45). 5.2. Extrapulmonary Blastomycosis Extrapulmonary disease has been reported in as many as two-thirds of patients with chronic blastomycosis. This high frequency probably reflects selection bias as these Fig. 15.4. Chest radiograph with right hilar infiltrate that mimics bronchogenic carcinoma. Bronchoscopy with biopsy and pulmonary cytology should be performed in these patients presenting with this radiographic finding to rule out concomitant disease. 15. Blastomycosis 283 Fig. 15.5. Patient with life-threatening pulmonary disease whose chest radiograph reveals bilateral alveolar infiltrates and large left-sided pleural effusion. figures were reported in earlier studies before effective therapy was available and were autopsy based (6). More recent studies have documented extrapulmonary disease in only 25 to 40 percent of patients with blastomycosis (15,47). Extrapulmonary disease is usually seen in conjunction with active pulmonary disease. 5.2.1. Skin Disease Skin disease is the most common extrapulmonary manifestation of blastomycosis. Two types of skin lesions occur, verrucous and ulcerative (Fig. 15.7). The verrucous lesion is most common, typically with well-demarcated borders and color from gray to violaceous hues. These lesions often mimic squamous cell carcinoma. Microabscesses develop at the periphery of these lesions, and specimen samples taken from the margins usually reveal the diagnostic yeast form on wet preparation (Fig. 15.8). The second type of lesion is ulcerative. These ulcers, which bleed easily, usually have well-demarcated, 284 Stanley W. Chapman and Donna C. Sullivan Fig. 15.6. Diffuse pulmonary infiltrates in a patient with ARDS. Patients presenting with this syndrome have a mortality rate greater than 50%. heaped up borders. The ulcers of blastomycosis develop from subcutaneous pustular lesions that spontaneously drain. Regional lymphadenopathy is usually not present in cases of pulmonary dissem- ination. Patients with inoculation blastomycosis occurring after dog bite or autopsy accidents often have lymphadenopathy/adenitis as a prominent feature (49,50). Lesions may also appear on the mucosa of the nose, mouth, and larynx. Laryngeal blastomycosis mimics well differentiated squamous cell carcinoma both clinically and histopatholog- Fig. 15.7. Cutaneous blastomycosis typically produces verrucous (left) or ulcerative (right) lesions. [Figure in color on CD-ROM]. 15. Blastomycosis 285 Fig. 15.8. Diagnosis of blastomycosis. This figure shows the characteristic yeast forms in a wet preparation of a skin scraping. Scraping of the edges of the verrucous and ulcerative lesions yield the best diagnostic results. [Figure in color on CD-ROM]. ically (35). Subcutaneous nodules or cold abscesses are usually seen in patients with multiorgan disease. 5.2.2. Osseous Osteomyelitis is associated with as many as one-fourth of B. dermatitidis infections (51). The vertebrae, pelvis, sacrum, skull, ribs, or long bones are most frequently involved, although any bone may be infected. Granuloma, suppuration, or necrosis may be observed in biopsy specimens. A well-circumscribed osteolytic lesion may be observed on x-ray examination, although such lesions cannot be distinguished from other fungal, bacterial, or neoplastic disease. Patients with B. dermatitidis osteomyelitis usually present with contiguous soft tissue abscesses or chronic draining sinuses. Although most bone lesions resolve with prolonged antifungal therapy, some may require surgical débridement for cure. 5.2.3. Genitourinary In men, 10% to 30% of blastomycosis involves the genitourinary tract, primarily the prostate and epididymis (52). Prostatic involvement is frequently associated with symptoms of obstruction, an enlarged tender prostate, and pyuria. Urine cultures obtained following prostate massage are frequently positive. 5.2.4. Central Nervous System Central nervous system (CNS) involvement normally occurs in fewer than 5% of cases in immunocompetent patients. Persons with AIDS have been reported to have 286 Stanley W. Chapman and Donna C. Sullivan Fig. 15.9. CNS blastomycosis in an AIDS patient. Diagnosis may require aspiration of the abscesses if no active pulmonary or cutaneous disease is present. rates of CNS involvement of 40% (53). CNS blastomycosis may present as an abscess (epidural, cranial, or spinal) or as meningitis (Fig. 15.9) (54–56). Surgical intervention may be necessary for both diagnosis and to prevent progression of neurologic deterio- ration (57). 6. DIAGNOSIS Definitive diagnosis of blastomycosis requires the growth of the organism from sputum, pus, or biopsy material. Mycelial phase cultures grown at 30oC are the most reliable method for isolation of B. dermatitidis from clinical specimens and usually become positive within 1 to 3 weeks of incubation (Fig. 15.1). Sputum cultures in pulmonary blastomycosis have a |
high positive yield (75% per single sample, 86% per patient), but bronchoscopy yields an even higher positive rate (92% of patients). A presumptive diagnosis is often made by visualization of the characteristic large broad-based budding yeast in sputum, pus, or histopathologic specimens (Fig. 15.8). Because colonization with B. dermatitidis does not occur, observation of yeast forms in clinical specimens may prompt empiric therapy in the appropriate clinical presentation. Although direct examination of wet preparations have been reported to have relatively 15. Blastomycosis 287 low diagnostic yield (58), the simplicity of the procedure, low cost, and potential for rapid diagnosis warrant the use of this method. Cytology has been shown to have a higher diagnostic yield (59). Serologic diagnosis of blastomycosis is of limited usefulness. Complement fixation antibodies have been used for epidemiologic purposes, but are severely limited in their specificity because of cross-reactivity to antigens of other fungi, particularly Histoplasma capsulatum and Coccidioides immitis. Immunodiffusion tests for precip- itating antibodies to B. dermatitidis are more specific than complement fixation but lack sensitivity in early disease (60). Commercial radioimmunoassays, enzyme immunoassays, and enzyme-linked immunosorbent assays have been developed which offer the promise of higher sensitivity but with specificities similar to complement fixation (61,62). Immunoassays employing the BAD-1 yeast phase specific protein discussed in the preceding text are not currently commercially available. A commercially available Blastomyces assay that detects antigen in urine and serum has been developed by MiraVista Diagnostics (Indianapolis, IN; www.miravistalabs.com). Antigenemia is detected in 70% to 80% of patients with disseminated disease. Antigen detection in the urine was higher than serum, approaching 100%. However, specificity is reduced by the presence of cross-reactive antigens present in specimens obtained from patients with other fungal infections, especially histoplasmosis. Antigen levels are reported to decline with successful treatment and increase in treatment failure or relapse of disease. Nucleic acid detection techniques, both target and signal amplification methods, have been developed (63–65), including the GEN-PROBE® AccuProbe® culture identi- fication test. These facilitate early identification of B. dermatitidis in mycelial cultures without the requirement of conversion to the yeast form. PCR amplification of the rRNA gene along with specific probe hybridization has been used to identify yeast phase organisms in tissue specimens. These molecular techniques offer great promise for the rapid diagnosis of blastomycosis, but have not yet been evaluated in large prospective studies. 7. TREATMENT Most patients who have blastomycosis require therapy. Before the availability of azoles, amphotericin B was the mainstay of treatment. However, a series of clinical trials performed by the NIAID Mycoses Study Group have shown ketoconazole, itraconazole, and fluconazole to be effective, relatively nontoxic agents when compared to amphotericin B for treatment of patients with mild to moderate non-CNS disease (66–68). Other than its use in diagnosis, the role of surgery is limited. Along with specific antifungal therapy, surgery may be helpful for the drainage of large abscesses, resection of cerebral blastomycomas, and débridement of devitalized bone. Selection of an appropriate therapeutic regimen must be based on three major considerations: the clinical form and severity of the disease, the immune status of the patient, and the toxicity of the antifungal agent. Specific recommendations of dose and duration of therapy in defined clinical settings are listed in Table 15.2 (69,70). For more detail concerning drug interactions, toxicities, adverse reactions and pharmacokinetics 288 Stanley W. Chapman and Donna C. Sullivan Table 15.2 Treatment guidelines for Blastomyces dermatitidis infections Type of disease Primary therapy Alternate therapy Pulmonary Severe Amphotericin B Switch to itraconazole 200–400 mg/day once 0.7–1.0 mg/kg patient stabilized per day;a total dose of 1.5–2.5 g Mild to moderate Itraconazole Ketoconazole 400–800 mg/day, or fluconazole 200–400 400–800 mg/day mg/day Disseminated CNS Amphotericin If intolerant to full course of amphotericin B, B, 0.7–1.0 fluconazole 800 mg/day mg/kg per day; total dose of at least 2 g Non-CNS disease Serious Amphotericin B Switch to itraconazole 200–400 mg/day once 0.5–0.7 mg/kg patient stabilized per day; total dose of 1.5–2.5 g Mild to moderate Itraconazole Ketoconazole 400–800 mg/day, or fluconazole 200–400 400–800 mg/day mg/day Immunocompromised Amphotericin B Selected patients with non-CNS disease may 0.3–0.6 mg/kg be switched to itraconazole, 200–400 per day;a total mg/day, once clinically improved. dose of Suppressive therapy with itraconazole 1.5–2.5 g should be considered in patients whose immunocompromised state continues. For patients with CNS disease or intolerant to itraconazole, consider fluconazole, 800 mg/day Modified from ref. 69. aA lipid formulation of amphotericin B (3.0–5.0 mg/kg per day) may be substituted for conventional amphotericin B. about individual antifungal agents, the reader is referred to the chapter discussing individual agents in this text (Chapter 6). Amphotericin B (including lipid-based formulations) is currently reserved for the initial treatment of patients with life-threatening disease, immunocompromised patients, and those with CNS disease. In selected patients initially presenting with 15. Blastomycosis 289 life-threatening disease, itraconazole has been successfully substituted following an induction course of amphotericin B (70). Patients with CNS disease and severely immunocompromised patients should be treated with a full course of amphotericin B. Most experts recommend a total dose of 1.5 to 2.5 grams. Fluconazole has been used in only a limited number of patients, but appears effica- cious at doses of 400 to 800 mg/day. Two factors may eventually lead to more extensive use of fluconazole: it has fewer side effects and adverse drug interactions and it has excellent penetration into the CNS, suggesting a role for this drug in the treatment of CNS blastomycosis. Voriconazole and posaconazole have activity against B. dermatitidis in vitro and have been shown to be effective in murine models of blastomycosis (71,72). The authors are aware of anecdotal cases of patients failing itraconazole therapy, including cases of cerebral blastomycosis, successfully treated with voriconazole as salvage therapy. Caspofungin and micafungin have shown variable activity against B. dermatitidis and have not been studied extensively in animal models or in human cases of blastomycosis (72). In some immunocompetent patients with acute pulmonary blastomycosis, the difficult and controversial decision to withhold therapy has been proposed following the description of self-limiting infections. When the decision to withhold therapy is made, patients should be evaluated for the presence of extrapulmonary disease and carefully monitored for progression of pulmonary infection. These patients should be followed for many years for evidence of reactivation of pulmonary or extrapulmonary disease. In contrast, all patients with progressive pulmonary infection or extrapulmonary disease and all immunocompromised patients should be treated (69,70). In immuno- logically normal patients with mild to moderate pulmonary or extrapulmonary disease that does not involve the CNS, the azole antifungal agents, itraconazole, ketoconazole, and fluconazole, administered for 6 months have proven to be effective, less toxic alternatives to amphotericin B. Although no randomized, blinded studies have been performed to compare different azoles, and only a few comparative trials for blasto- mycosis therapy have been reported, itraconazole appears to be the best tolerated and most effective azole. Itraconazole is considered the drug of choice for patients with non-life-threatening, non-CNS blastomycosis (69,70). Amphotericin B is the drug of choice for blastomycosis occurring during pregnancy. Azoles are contraindicated (69). The clinical spectrum of blastomycosis in pediatric patients is similar to that seen in adults. Recent reports indicate blastomycosis in children is more difficult to diagnosis and less likely to respond to oral therapy. Children with life-threatening disease should be treated with amphotericin B. Itraconazole has been used successfully at a dosage of 5 to 7 mg/kg per day in a small cohort of pediatric patients (69). CNS disease occurs in approximately 40% of patients with AIDS or other diseases or therapies associated with immunosuppression. Likewise, disseminated disease and life-threatening pulmonary disease also appear more common in the clinical setting of immunosuppression. Hence, the recommendation that amphotericin B is the drug of choice for treatment of immunocompromised patients. Frequent relapses have been reported in patients whose immunosuppression persists and chronic suppressive therapy with an oral azole has been recommended by some experts (69,70). 290 Stanley W. Chapman and Donna C. Sullivan Relapse rates of 10% to 14% have been reported in patients treated with ketoconazole. Patients should be followed for many years for evidence of relapse, especially in the CNS. Relapse rates of less than 5% are reported in patients treated with amphotericin B and itraconazole. Owing to the problems with bioavailability of oral itraconazole and ketoconazole, serum blood levels may be clinically useful in guiding treatment of patients whose disease progresses on either of these azoles (69). REFERENCES 1. Gilchrist TC. Protozoan dermatitis. J Cutan Gen Dis 1894;12:496–499. 2. Schawartz J, BaumGL. Blastomycosis. Am J Clin Pathol 1951;21:999–1029. 3. Bradsher RW. Clinical features of pulmonary blastomycosis. Semin Respir Infect 1997;12:229–234. 4. DiSalvo AF. The epidemiology of Blastomyces dermatitidis. In: Al-Doory Y, DiSalvo AF, eds. Blastomycosis. New York: Plenum Press, 1992:83–90. 5. DiSalvo AF. The ecology of Blastomyces dermatitidis. In: Al-Doory Y, DiSalvo AF, eds. Blastomycosis. New York: Plenum Press, 1992:43–69. 6. Chapman SW. Blastomyces dermatitidis. In: Mandell GL, Bennett JE, Dolin R, eds. Mandell, Douglas, and Bennett’s principles and practices of infectious diseases. 6th ed. Philadelphia: Elsevier Churchill Livingstone, 2005:3026–3040. 7. Kaufman L, Standard PG, Weeks RJ, et al. Detection of two Blastomyces dermatitidis serotypes by exoantigen analysis. J Clin Microbiol 1983;18:110–114. 8. Turner S, Kaufman L. Immunodiagnosis of blastomycosis. Semin Respir Infect 1986;1:22–28. 9. Axtell RC, Scalarone GM. Serological differences in two Blastomyces dermatitidis isolates from different geographic regions of North America. Mycopathologia 2002;153:141–144. 10. Yates-Ciilata KE, Sander DM, Keith EJ. Genetic diversity in clinical isolates of the dimorphic fungus Blastomyces dermatitidis detected by PCR based random amplified polymorphic DNA assay. J Clin Microbiol 1995;33:2171–2175. 11. McCullough MJ, DiSalvo AF, Clemons KV, Park P, Stevens DA. Molecular epidemiology of Blastomyces dermatitidis. Clin Infect Dis 2000;30:328–335. 12. Klein BS, Vergeront JM, Weeks RJ, et al. Isolation of Blastomyces dermatitidis in soil associated with a large outbreak of blastomycosis in Wisconsin. N Engl J Med 1986;314:529–534. 13. Klein BS, Vergeront JM, DiSalvo AF, et al. Two outbreaks of blastomycosis along rivers in Wisconsin: Isolation of Blastomyces dermatitidis from riverbank soil and evidence of its transmission along waterways. Am Rev Respir Dis 1987;136:1333–1338. 14. Klein BS, Vergeront JM, Davis JP. Epidemiologic aspects of blastomycosis, the enigmatic systemic mycosis. Semin Respir Infect 1986;1:29–39. 15. Proctor ME, Klein BS, Jones JM, Davis JP. Cluster of pulmonary blastomycosis in a rural community: evidence for multiple high-risk environmental foci following a sustained period of diminished precipitation. Mycopathologia 2002;153:113–120. 16. Bumgardner DM, Buggy BP, Mattson BJ, Ludwig D. Epidemiology of blastomycosis in a region of high endemicity of north central Wisconsin. Clin Infect Dis 1992;15:629–635. 17. Baumgardner DJ, Paretsky DP, Yopp AC. The epidemiology of blastomycosis in dogs: north central Wisconsin, USA. J Med Vet Mycol 1995;33:171–176. 18. Legendre AM. Blastomycosis in animals. 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29. Cozad GC, Change CT. Cell mediated immunoprotection in blastomycosis. Infect Immun 1980;28:398–403. 30. Wuthrich M, Filutowicz HI, Klein BS. Mutation of the WI-1 gene yields an atten- uated Blastomyces dermatitidis strain that induces host resistance. J Clin Invest 2000;106: 1381–1389. 31. Wuthrich M, Filutowicz Warner T, Klein BS. Requisite elements in vaccine immunity to Blastomyces dermatitidis: plasticity uncovers vaccine potential in immune-deficient hosts. J Immunol 2002;169:6969–6976. 32. Wuthrich M, Filutowicz HI, Warner T, Deepe GS, Klein BS. Vaccine immunity to pathogenic fungi overcomes the requirements for CD4 help in exogenous antigen presen- tation to CD8+ T cells: implications for vaccine development in immune-deficient hosts. J Exp Med 2003;197:1405–1416. 33. Wuthrich M, Warner T, Klein BS. CD28 is required for optimal induction, but not maintenance, of vaccine-induced immunity to Blastomyces dermatitidis. Infect Immun 2005;73:7436–7441. 34. Wuthrich M, Warner T, Klein BS. IL-12 is required for induction but not maintenance of protective, memory responses to Blastomyces dermatitidis: implications for vaccine development in immune-deficient hosts. J Immunol 2005;175:5288–5297. 35. Bradsher RW, Chapman SW, Pappas PG. Blastomycosis. Infect Dis Clin N Am 2003;17:21–40. 36. Cherniss EI, Waisbren BA. North American blastomycosis: a clinical study of 40 cases. Ann Intern Med 1956;44:169–123. 37. Abernathy RS. Clinical manifestations of pulmonary blastomycosis. Ann Intern Med 1959;51:707–727. 292 Stanley W. Chapman and Donna C. Sullivan 38. Blastomycosis Cooperative Study of the Veterans Administration. Blastomycosis I: a review of 198 collected cases in Veterans Administration hospitals. Am Rev Respir Dis 1964;89:659–672. 39. Witorsch P, Utz JP. North American blastomycosis: a study of 40 patients. Medicine (Baltimore) 1968;47:169–200. 40. Lockwood WR, Allison F, Batson BE, et al. The treatment of North American blastomy- cosis: ten years experience. Am Rev Respir Dis 1969;100:314–320. 41. Duttera MJ, Osterhout S. North American blastomycosis: a survey of 63 cases. South Med J 1969;62:295–301. 42. Sheflin JR, Campbell JA, Thompson GP. Pulmonary blastomycosis: findings on chest radiographs in 63 patients. AJR Am J Roentgenol 1990;154:1177–1180. 43. Brown LR, Sweasen SJ, VanScovy RE, et al. Roentgenologic features of pulmonary blastomycosis. Mayo Clin Proc 1991;66:29–38. 44. Sarosi GA, Davies SF, Phillips JR. Self-limited blastomycosis: a report of 39 cases. Semin Respir Infect 1986;1:40–44. 45. Sarosi GA, Hammerman KJ, Tosh FE, et al. Clinical features of acute pulmonary blasto- mycosis. N Engl J Med 1974;290:540–543. 46. Meyer KC, McManus EJ, Maki DG. Overwhelming pulmonary blastomycosis associated with the adult respiratory distress syndrome. J Engl J Med 1993;329:1231–1236. 47. Chapman SW, Lin AC, Hendricks DA, et al. Endemic blastomycosis in Mississippi: epidemiological and clinical studies. Semin Respir Infect 1997;12:219–228. 48. Kinasewitz GT, Penn RL, George RB. The spectrum and significance of pleural disease in blastomycosis. Chest 1984;86:580–584. 49. Larson DM, Eckman MR, Alber RL, et al. Primary cutaneous (inoculation) blastomycosis: an occupational hazard to pathologists. Am J Clin Pathol 1983;79:253–255. 50. Gnann JW, Bressler GS, Bodet CA, et al. Human blastomycosis after a dog bit. Ann Intern Med 1983;98:48–49. 51. Moore RM, Green NE. Blastomycosis of bone. J Bone Joint Surg 1982;64:1094–1101. 52. Sarosi GA, Davies SF. Blastomycosis: state of the art. Am Rev Respir Dis 1979;120:911–938. 53. Pappas PG, Pottage JC, Powderly WG, et al. Blastomycosis in patients with acquired immunodeficiency syndrome. Ann Intern Med 1992;116:847–853. 54. Gonyea EF. The spectrum of primary blastomycotic meningitis: a review of central nervous system blastomycosis. Ann Neurol 1978;3:26–39. 55. Kravitz GR, Davies SF, Eckman MR, et al. Chronic blastomycotic meningitis. Am J Med 1981;71:501–505. 56. Roos KL, Bryan JP, Maggio WW, et al. Intracranial blastomycoma. Medicine (Baltimore) 1987;66:224–235. 57. Ward BA, Parent AD, Raila F. Indications for the surgical management of central nervous system blastomycosis. Surg Neurol 1995;43:379–388. 58. Martynowicz MA, Prakash UBS. Pulmonary blastomycosis: an appraisal of diagnostic techniques. Chest 2002;121:768–773. 59. Lemos LB, Guo M, Baliga M. Blastomycosis: organ involvement and etiologic diagnosis. A review of 123 patients from Mississippi. Ann Diagn Pathol 2000;4:391–406. 60. Klein BS, Vergeront JM, Kaufman L, et al. Serological test for blastomycosis: assess- ments during a large point-source outbreak in Wisconsin. J Infect Dis 1987;155: 262–268. 61. Bradsher RW, Pappas PG. Detection of specific antibodies in human blastomycosis by enzyme immunoassay. South Med J 1995;88:1256–1259. 15. Blastomycosis 293 62. Sekhorn AS, Kaufman L, Kobayashi AS, et al. The value of the Premier enzyme immunoassay for diagnosing Blastomyces dermatitidis infections. J Med Vet Mycol 1995;33:123–125. 63. Yeo SF, Wong B. Current status of nonculture methods for diagnosis of invasive fungal infections. Clin Microbiol Rev 2002;15:465–484. 64. Walsh TJ, Larone DH, Schell WA, Mitchell TG. Histoplasma, Blastomyces, Coccidioides, and other dimorphic fungi causing systemic mycoses. In: Murray PR, Barron EJ, Jorgensen JH, Pfaller MA, Yolken RH, eds. Manual of clinical microbiology. 8th Ed. Washington, DC: ASM Press, 2003:1781–1797. 65. Lindsey MD, Hurst SF, Iqbal NJ, Morrison CJ. Rapid identification of dimorphic and yeast- like fungal pathogens using specific DNA probes. J Clin Microbiol 2001;39:3505–3511. 66. Treatment of blastomycosis and histoplasmosis with ketoconazole. Results of a prospective randomized clinical trial. National Institute of Allergy and Infectious Diseases Mycoses Study Group. Ann Intern Med 1985;103:861–872. 67. Dismukes WE, Bradsher RW, Cloud GC, et al. Itraconazole therapy for blastomycosis and histoplasmosis. Am J Med 1992;93:489–497. 68. Pappas PG, Bradsher RW, Kauffman CA, et al. Treatment of blastomycosis with higher dose fluconazole. Clin Infect Dis 1997;25:200–205. 69. Chapman SW, Bradsher RW, Campbell GD, Pappas PG, Kauffman CA. Practice guidelines for the management of patients with blastomycosis. Clin Infect Dis 2000;30:679–683. 70. Bradsher RW. Histoplasmosis and blastomycosis. Clin Infect Dis 1996;22:S102–S111. 71. Sugar AM, Liu XP. Efficacy of voriconazole in treatment of murine pulmonary blastomy- cosis. Antimicrob Agents Chemother 2001;45:601–604. 72. Espinel-Ingroff A. Comparison of in vitro activities of the new triazole SCH56592 and the echinocandins MK-0991 (L-743,872) and LY303366 against opportunistic filamentous dimorphic fungi and yeasts. J Clin Microbiol 1998;36:2950–2956. SUGGESTED READINGS Bradsher RW. Blastomycosis. In: Dismukes WE, Pappas PG, Sobel JD, eds. Clinical mycology. New York: Oxford University Press, 2003:299–310. Bradsher RW, Chapman SW, Pappas PG. Blastomycosis. Infect Dis Clin N Am 2003;17:21–40. Chapman SW. Blastomyces dermatitidis. In: Mandell GL, Bennett JE, Dolin R, eds. Mandell, Douglas, and Bennett’s principles and practices of infectious diseases. 6th Ed. Philadelphia: Elsevier Churchill Livingstone, 2005:3026–3040. 16 Coccidioidomycosis Royce H. Johnson, MD and Shehla Baqi, MD 1. INTRODUCTION Coccidioidomycosis was first described in 1892, in Buenos Aires by Posadas and Wernicke (1,2). They thought that the individual in their case report suffered from a malignant disease with a likely infectious cause. Organisms seen microscopically were mistakenly thought to be parasites. The disease was next described in San Francisco in 1896 by Rixford and Gilchrist, whose paper was the first extensive study of coccid- ioidomycosis (3). They better understood that this was an infectious illness and were the first to appreciate the importance of the parasite as the agent of a new and distinctive disease. In 1900, Williams Ophuls began his work on coccidioidal disease. Although he noted the “protozoa” of Rixford and Gilchrist in pathological sections, he discovered that culture of the organism always produced colonies of a mould, what we now know to be the mycelial (saprobic) growth of Coccidioides. The life cycle was roughly outlined in a preliminary report and the fungus given the name of Coccidioides immitis (4). During 1925–1936, the early pathologic, epidemiologic, and mycologic studies were completed. Montenegro reported the first recovery of Coccidioides immitis from blood (5). Coccidioidal infection in farm animals was described by Beck (6). Menin- gitis was described first pathologically and subsequently clinically in the early part of the 20th century (7,8). Two important observations were also made during this period; that the lung is the portal of entry and that Coccidioides immitis can be isolated from soil (9,10). Coccidioidomycosis was considered to be a rare and fatal infection until an accidental laboratory exposure of a medical student at Stanford University resulted in only a transient pulmonary infection. This led to a reassessment of the natural history of coccidioidal infection. The work in Kern County by Dr. Myrnie Gifford on a local respi- ratory illness in the San Joaquin Valley of California, known as Valley Fever, eventually elucidated the primary infection as being predominantly pulmonary (11). During the latter part of the 1930s and 1940s, the natural history of the primary illness and the utility of the skin test and serology were developed by Charles E. Smith and co-workers. William Winn and Hans Einstein made further contributions to disease description and therapy with amphotericin B deoxycholate, both intravenously and intrathecally From: Infectious Disease: Diagnosis and Treatment of Human Mycoses Edited by: D. R. Hospenthal and M. G. Rinaldi © Humana Press Inc., Totowa, NJ 295 296 Royce H. Johnson and Shehla Baqi for meningitis. By the 1950s, the clinical spectrum of coccidioidal infection was well described, with the publication of an excellent monograph by Fiese (12). 2. ETIOLOGIC AGENTS Kingdom: Fungi Phylum: Ascomycota Class: Euascomycetes Order: Onygenales Family: Onygenaceae Genus: Coccidioides Coccidioides was originally described as noted above as one species, C. immitis. More recently, two genetically distinct populations of Coccidioides have been described, Coccidioides immitis and Coccidioides posadasii, correlating to separate endemic regions. Currently the C. immitis is maintained as the name of isolates that are Fig. 16.1. Life cycle of Coccidioides immitis depicting saprophytic (soil) and parasitic (host) phases. 16. Coccidioidomycosis 297 predominately found in California. The new species, C. posadasii, is predominately found in Texas, Mexico, Central America, and South America. Both species are found in Arizona (13). Coccidioides of both species, however, show few phenotypic differences and are mycologically and clinically indistinguishable. Coccidioides is a thermal dimorphic fungus that exists either as a mycelium or a spherule. The fungus is found as far North as the northern central valley in California and as far South as Argentina, the place of its original description. The fungus grows in conditions where the soil has a relatively high salinity, and in a climate that has mild winters with few freezes and hot dry summers (14). Under these ideal conditions, the fungus grows in isolated pockets as a mycelium by apical extension. These mycelia produce specialized aerial hyphae that segment and form arthroconidia. The connecting links between arthroconidia are quite fragile and separate easily with minimal mechanical force or air turbulence. The arthroconidia become airborne in a form capable of deposition in the lungs if inhaled, and can travel substantial distances, perhaps as far as 75 miles or more. These arthroconidia, if they find an appropriate soil niche, can reestablish the saprophytic phase. However, if they are inhaled by an appro- priate host, they undergo transformation from arthroconidia into spherules. Spherules reproduce by endosporulation, a process whereby the growing spherule is subdivided into numerous subcompartments, each of which become viable daughter cells or endospores. The spherule eventually ruptures, releasing endospores, each of which may continue to propagate in tissue or revert to mycelial growth in soil or on growth media (Fig. 16.1). 3. EPIDEMIOLOGY The disease was first described in Argentina, but other foci of infection in South America and Central America also exist. Coccidioides species are found solely in the Western Hemisphere, in the “lower Sonoran life zone” (15). The majority of the soils that support the organism are found in North America, particularly in the southwestern United States and northwestern Mexico. The areas of greatest endemicity are in the Southern San Joaquin Valley and south central Arizona. The disease extends to the Northern Central Valley in California and as far as Utah in the Great Plains (16). The total number of infections per year is not known, but prevalence surveys in the 1950s of school-age children in California’s central valley suggested an annual risk of infection of 15%. More recent estimates from California and Arizona have indicated that the risk has declined to 3% or less (17,18). It has been estimated that in the United States there are approximately 100,000 infections annually (19). There has been a recent increase in the reported number of patients per year that is believed to be due to changes in demographics and medicine. Regions in which coccidioidomycosis is endemic have experienced a tremendous increase in their population. In 2005, it was estimated the populations of southern Arizona and southern California increased by greater than 7 million inhabitants. In addition, there is greater recognition of the disease in patients with compromised cellular immunity and in the elderly, who are more likely to have more |
severe disease. It appears, however, that the absolute incidence of disease has also increased, particularly during the epidemics described in the 1990s and in the first 2 years of the new millennium (20–23). 298 Royce H. Johnson and Shehla Baqi 4. PATHOGENESIS AND IMMUNOLOGY Virtually all infections result from inhalation of arthroconidia. Inhaled arthroconidia transform into spherules, an inflammatory response ensues, and a local pulmonary lesion develops. In some infections, the Coccidioides species gain access to the vascular space, leaving the lungs and disseminating to other parts of the body. Control of coccidioidomycosis is predominantly cell mediated, with more severe infections seen in T-cell deficient patients (24–26). In addition, in vitro observations have shown that innate cellular responses, mediated by mononuclear cells or natural killer cells, may slow fungal proliferation after infection (27). It is conceivable that interleukin (IL)-12, IL-23, and interferon gamma may play an important role in protective immunity in coccidioidomycosis as recently demonstrated in paracoccidioidomycosis and histoplas- mosis (28,29). 5. CLINICAL MANIFESTATIONS 5.1. Pulmonary Coccidioidomycosis most commonly presents as primary pulmonary disease. The first symptoms of primary infection usually appear 7 to 21 days after infection, although infection is asymptomatic 60% of the time. In patients presenting with symptomatic disease, the majority present with an influenza-like syndrome. Of the 40% of total infections with symptoms, only one out of four is diagnosed; the majority of these present with pneumonic or pleural disease. There are a number of pulmonary compli- cations of primary coccidioidomycosis. The most common is severe and persistent pneumonia, with radiographic and clinical findings of pneumonic disease for greater than 6 weeks. Progressive primary coccidioidomycosis is a syndrome in which the patient has resolution of his or her pulmonary parenchymal disease with persistence of his or her hilar and mediastinal lymphadenitis. Rare cases of progressive fibro- cavitary coccidioidomycosis, which often resembles pulmonary tuberculosis, are also described. Solitary thin-walled pulmonary cavities are a frequent complication (see Fig. 5.11, Chapter 5). Residual nodules are often confused with a neoplasm, partic- ularly when individuals with unrecognized primary coccidioidomycosis present with a residual nodule on routine chest radiograph long after the time of infection (30). Modest amounts of pleural fibrosis, a residual of the primary infection, may also be seen. Cavitary disease may rupture into the pleural space causing coccidioidal empyema, not to be confused with simple pleural effusions which may occur as part of the primary disease process. Symptoms prevalent in primary coccidioidomy- cosis include fever (76%), cough (73%), chest pain (44%), fatigue (38%), erythema nodosum (26 %), myalgias (23%), shortness of breath (22%), sputum production (22%), chills (21%), headache (21%), night sweats (21%), and other rashes (14%) (31). Radiographic findings in primary coccidioidomycosis typically include infiltrate only (70%), infiltrate with hilar adenopathy (10%), or infiltrate with effusion (10%) (Figs. 16.2 through 16.5). Lung cavities are present in about 8% of adults, but are less common in children. Approximately 10% of individuals will have a negative chest radiograph at diagnosis. Skin manifestations develop as part of the primary illness, most often as a transient nonpruritic fine papular rash. Erythema nodosum is 16. Coccidioidomycosis 299 Fig. 16.2. Right upper lobe dense consolidation with associated right hilar adenopathy. fairly common in primary coccidioidal infection, with a strong predilection for women (Fig. 16.6). Less commonly, erythema multiforme and erythema sweetobullosum are seen (32,33). Migratory arthralgias are common; the triad of fever, erythema nodosum, and arthralgias has been referred to as “desert rheumatism.” Rarely, pulmonary coccid- ioidomycosis may present as a bronchial mass found on bronchoscopy. Chronic fibrocavitary pneumonia can occur, commonly in association with diabetes or preex- isting pulmonary fibrosis (34). Miliary disease with coccidioidomycosis is seen with significant frequency in the endemic area, where miliary coccidioidomycosis may be 10 times as frequent as miliary tuberculosis. Overwhelming miliary and/or alveolar coccidioidomycosis can result in respiratory failure. Human immunodeficiency virus (HIV)-infected patients often have a fulminant presentation, particularly when CD4+ T lymphocyte counts are less than 100 cells/μl. Probably the most common cause of death in the endemic area from pulmonary coccidioidomycosis is respiratory failure, although most coccidioidal respiratory infections resolve within several weeks to months without complications. 5.2. Disseminated Disease The rate of dissemination of coccidioidomycosis is highly dependent on the infected host. The majority of disseminated disease occurs in individuals with antecedent symptomatic pulmonary infection. However, in a minority of patients disseminated disease presents without obvious primary pulmonary infection. Risk factors for dissemination include the extremes of age, male sex, African American or Filipino ancestry, tobacco smoking, and low social economic status (35,36). Persons with 300 Royce H. Johnson and Shehla Baqi Fig. 16.3. Fibrocavitary changes involving both upper lobes. Right upper lobe cavity with airfluid level. Fig. 16.4. Right upper lobe pleural-based mass with surrounding infiltrate. 16. Coccidioidomycosis 301 Fig. 16.5. Right middle lobe giant cavity with air-fluid level and associated infiltrate. immunodeficiency, including that seen with advanced HIV infection, high-dose corti- costeroid therapy, Hodgkin’s lymphoma and solid organ or bone marrow transplan- tation, are at greater risk of dissemination (26,37–41). Pregnancy also predisposes to individuals to disseminated disease (42,43). The majority of dissemination is to skin, subcutaneous tissue, bone and joints. These sites taken together represent more than 50% of disseminated disease. Unfortunately, the single most common site of dissem- ination is the meninges. Cutaneous dissemination has a variable clinical appearance; perhaps the most characteristic is one or more verrucous lesions, which may vary in size from a few millimeters to a few centimeters (Fig. 16.7). Subcutaneous tissue infection, which usually presents as a cold abscess, is also seen with some frequency. Infections of virtually all joints have been described. Infections of the knee, elbow, wrist, and ankle are seen, with the knee most commonly involved (44–46). Dissem- ination similarly has been described in almost every bone. Particularly common are infections of the axial skeleton, the pelvic bones, tibia, and femur. It is not unusual to see osteomyelitis and joint involvement in the same patient. Single bone or joint infections are most common but multiple sites may be involved, particularly in African American males. The most severe disseminated manifestation of coccidioidomycosis is meningitis. This is the single most common dissemination site in Caucasian and Latino males. Untreated, meningitis is fatal within a few months, although there are rare reports of survival for 2 or more years (47). Meningitis usually develops within 302 Royce H. Johnson and Shehla Baqi Fig. 16.6. Erythema nodosum affecting the lower extremities. [Figure in color on CD- ROM]. Fig. 16.7. Characteristic lesions of cutaneous coccidioidomycosis. [Figure in color on CD-ROM]. 16. Coccidioidomycosis 303 6 months of the initial infection (48). The cerebrospinal fluid has an elevated white blood cell count and protein, with depressed glucose. Eosinophils are not common but when present are highly suggestive of the diagnosis of coccidioidal meningitis (49). Finally, Coccidioides may disseminate to virtually any site in the body. Coccidioidal endophthalmitis, peritonitis, and prostatitis have all been described. 6. DIAGNOSIS The diagnosis of coccidioidomycosis is dependent on a compatible clinical illness with positive laboratory confirmation by culture, histopathology, or serology. It is essential to obtain a detailed travel history for exposure to an endemic area. Exposure does not need to be over a prolonged period of time and infection has occurred after only briefly passing through an endemic area. 6.1. Culture Suitable material for culture is sputum, tissue aspirates, or biopsy specimens. Coccid- ioides species grow well on most culture media after 5–7 days of incubation in aerobic conditions at 25º, 30º, or 35°C. Typically these fungi produce a white mould, although more pigmented strains have been observed. Laboratory cultures are highly infectious when mature and arthroconidia have formed (see Fig. 2.14, Chapter 2). Typically it takes about 10 to 20 days for Coccidioides to mature and produce arthro- conidia. Because of their size, the arthroconidia are easily dispersed in the air and inhaled; therefore Coccidioides presents an extreme hazardous when cultured in the laboratory. At a minimum, Biosafety Level 2 practices and facilities are recommended for handling and processing of clinical specimens. When working with known Coccid- ioides, Biosafety Level 3 is required (50). Accidental percutaneous inoculation of the spherule form may result in local granuloma formation. Clinical specimens, prior to culture, however, are not infectious to personnel. The much larger size spherules are considerably less effective as airborne pathogens. Coccidioides will grow in most of the media used in the microbiology laboratories including 5% sheep blood agar, chocolate agar, Sabouraud dextrose agar, Mycosel agar, and brain heart infusion agar with or without blood. Growth on 5% sheep blood agar and chocolate agar incubated at 35°C can be seen in as little as 24 hours. Growth on Sabouraud dextrose agar and Mycosel agar incubated at 25°C (room temperature) can be seen after 3 to 4 days. Accidental recovery of Coccidioides on 5% sheep blood agar and chocolate agar from respiratory, tissue, aspirates and biopsy specimens can be extremely hazardous to laboratory personnel. Specimens from known or suspected cases should not be cultured on unsealed plated media. Tubed media must be used (Fig. 16.8). Presumptive identi- fication may be made based on colony morphology, growth rate, and the production of alternating arthroconidia. Care must be taken when attempting to identify Coccidioides because other mycoses may have similar macroscopic and microscopic morphologies, especially if arthroconidia are not abundant (Fig. 16.9). Laboratories that are not experienced with working with Coccidioides should refer these suspected isolates to qualified reference laboratories. Coccidioides species are dimorphic fungi that have the ability to grow vegetatively at 25°C as moulds, and at 37°C in tissue or in special medium (Converse liquid medium) in 10% CO2 incubator as spherules. Confirmation 304 Royce H. Johnson and Shehla Baqi Fig. 16.8. Growth of Coccidioides in tubed fungal medium. [Figure in color on CD-ROM]. 16. Coccidioidomycosis 305 Fig. 16.9. Mycelial form of Coccidioides immitis. [Figure in color on CD-ROM]. traditionally was performed by animal inoculation with identification of endosporu- lating spherules on histopathology. Exoantigen tests and the production of spherules in Converse liquid medium could also be used. These methods have now been supplanted by molecular testing; a DNA probe is available commercially. 6.2. Histopathology Diagnosis may also be confirmed histopathologically with the demonstration of endosporulating spherules usually in the setting of granulomatous inflammation (see Fig. 3–9, Chapter 3). 6.3. Serologic Testing The most common method of diagnosis is serologic testing in individuals who have typical clinical features or based on suspicion. Correctly performed serologic tests are both sensitive and specific for the disease. A negative serologic test, however, does not exclude the presence of infection, especially if recently acquired, and should be repeated over the course of several weeks to months. Serologic tests for Coccidioides are many. The most commonly used currently are enzyme immunoassay (EIA), immunodiffusion (ID), and complement fixation (CF) antibody tests. The EIA allows the detection of immunoglobulin M (IgM) antibodies for the determination of recent infection. Although this test suffers from many false positives, it is probably the most sensitive test for early infection. The ID IgM test has somewhat less sensitivity but a better specificity than the EIA test. The EIA IgG test appears to have a significant number of false negatives, which limit its utility in the diagnosis of severe and advanced disease. The ID IgG test, while somewhat less sensitive, has a high degree of specificity and fewer false negatives in individuals with significant or disseminated disease. Complement fixation tests are both sensitive and specific in the diagnosis of coccidioidal infections. The quantitative CF test is expressed as a titer and has the additional advantage of being not only diagnostic, but prognostic. There is an inverse relationship of the IgG antibody titer to prognosis. Individuals with low amounts of IgG antibodies tend to have modest primary infections. Individuals with high amounts of IgG antibodies are more likely 306 Royce H. Johnson and Shehla Baqi to have extensive primary infection or disseminated disease. It must be understood, however, that this holds true for a population of patients. In a given individual, the extent and severity of disease cannot be accurately predicted solely on the measurement of IgG antibodies. The majority of patients with disseminated disease will eventually have a titer greater than or equal to 1:16. At present, EIA results should be confirmed with the more established immunodiffusion or complement fixation tests |
(51,52). Immunodiffusion and complement fixation IgG tests will have a false-negative rate of approximately 1% in disseminated disease. The majority of individuals with disseminated disease who have falsely negative Coccidioides serology are HIV infected. The complement fixation titer in individuals with meningitis is higher than in individuals with primary disease but lower than in those with other forms of disseminated disease (53). 6.4. Skin Testing Skin test antigens derived from both mycelia and spherules have been marketed in the past. No skin testing reagents are currently commercially available. Skin testing detects the delayed-type hypersensitivity reaction to Coccidioides (54). Because skin tests commonly remain positive for life in most people, a positive result may not be related to current illness, analogous to tuberculin skin testing. The diagnosis of coccidioidomycosis can be made by demonstrating the conversion of the skin test from negative to positive. False-negative skin tests can occur in immunocompromised individuals and in the setting of overwhelming infection. Thus a negative skin test cannot exclude a diagnosis of current or past coccidioidal infection. Skin testing therefore is limited as a screening procedure for recent infection, but may be useful in epidemiologic studies. 6.5. Other Laboratory Findings Nonspecific laboratory tests such as the complete blood count and chemistry tests occasionally offer clues to coccidioidal infection. In an endemic area, an individual presenting with what appears to be community-acquired pneumonia who has an absolute eosinophilia (greater than 350 cells/μl) is more likely to have primary coccidioidal infection. It has also been noted that individuals who present with coccidioidomycosis and elevated alkaline phosphatase may also have liver involvement. 6.6. Radiologic Imaging Radiographic imaging may be of great help in defining the extent and severity of both pleural pulmonary and disseminated disease. Chest radiograph is mandatory in evaluation of primary disease. Computed tomography of the chest may be helpful in selected cases, especially with cavitary disease. Bone scan is the most frequently utilized test for osteomyelitis and plain radiograph is frequently utilized (see Figs. 5.13 and 5.14, Chapter 5). Magnetic resonance imaging (MRI) of bone and joint will help define problematic cases. Approximately 50% of patients with meningitis will have a normal study, but neuroimaging of the brain and spinal cord with MRI may reveal meningeal enhancement, hydrocephalus, or vasculitic infarction (55) (see Fig. 5.3, Chapter 5). 16. Coccidioidomycosis 307 7. TREATMENT 7.1. General Approach The treatment of coccidioidomycosis is both complex and at times controversial. A treatment guideline published by the Infectious Disease Society of America (IDSA) in 2005 gives a consensus framework (56). It is clear that many individuals infected with Coccidioides species recover. This is especially true when one notes that 60% of infections are asymptomatic and, by definition, go undiagnosed and untreated. Of symptomatic individuals, large numbers also go undiagnosed and most recover unevent- fully. There is some controversy as to whether individuals who are diagnosed with symptomatic primary disease need to be treated. Some experts believe that the majority of these persons will recover without treatment and therefore treatment ought not to be offered. Other authorities note that a small but significant percentage of individuals with primary disease will have either pulmonary or disseminated complications and it is difficult to predict with certainty who these individuals might be; thus the majority should be treated. Unfortunately, there is no evidence-based study of primary disease that has examined whether improvement in the primary symptom complex, rate of pulmonary complications, or frequency of dissemination, is affected by treating or not treating. What is generally agreed upon is that individuals with significant risk factors for dissemination or poor outcome should receive treatment for primary disease (Table 16.1). Thus advanced age; male sex; vulnerable race; presence of associated comorbid diseases such as diabetes, liver disease, and underlying lung disease; and elevated complement fixation titers should favor treatment of primary disease. HIV infection and other conditions associated with immunodeficiency, such as lymphoma, cancer chemotherapy, and organ transplantation, mandate early and aggressive therapy of primary disease. Pregnancy represents a special circumstance. The development of primary disease during the middle trimester through the early postpartum period puts an otherwise low-risk group of individuals at much higher risk. Coccidioidomycosis had been the leading cause of maternal mortality in Kern County, California for more than 50 years (57). Despite the risk, many women will have favorable outcomes without drug treatment, and abortions or early delivery in subjects with active infection is rare (58). 7.2. Primary Coccidioidomycosis Most individuals with uncomplicated acute coccidioidal pneumonia, if treated, are initiated on oral azole therapy (Fig. 16.10). Fluconazole 400 mg daily has been prescribed most often. Alternatively, itraconazole 200 mg twice daily is also commonly prescribed (59). Some institutions are initiating higher doses as primary therapy. This has especially been true with the recent availability of generic fluconazole. It should be noted that fluconazole and itraconazole are not approved for coccidioidomycosis by the Food and Drug Administration (FDA), nor are doses greater than 400 mg daily, in any disease. Despite lack of an FDA indication, these two drugs have become the mainstay in treatment of primary disease. Duration of recommended treatment ranges from 3 to 6 months, although longer courses may be prescribed in diabetics, persons 308 Royce H. Johnson and Shehla Baqi Table 16.1 Antifungal therapy of coccidioidomycosis Disease Primary therapy Alternate therapy Duration Acute pulmonary infection Uncomplicated 3–6 months Low risk Observation Oral azolea High riskb Oral azolec Amphotericin B Diffuse/severe Amphotericin Bd High dose fluconazolee ≥12 months Chronic infection Chronic infection Oral azole Surgical resection ≥12 months Pneumonia (cavitary disease only) Disseminated Oral azole Amphotericin B Years Nonmeningeal Meningeal Oral fluconazoleef Voriconazole, or Life-long itraconazole, or intrathecal amphotericin B Other Nodule Observation Cavity Asymptomatic Observation Symptomatic Oral azole Ruptured Surgery and antifungal >12 months therapy Modified from Infectious Diseases Society of America guidelines (56). aOral azoles include fluconazole and itraconazole, typically 400 mg or more daily. bSee text for description of patients at high risk. cAmphotericin B should be used in pregnant patients due to the teratogenicity of azoles. dTypically replaced with azole therapy once patient is clinically improving and stable. eHigh-dose fluconazole (800–1200 mg daily) is often used in severe disease, at least initially. f Consider early shunting if hydrocephalus is present and corticosteroids if vasculitis is present. of African American or Filipino descent, and in immunocompromised patients. In individuals presenting with severe, diffuse pulmonary coccidioidomycosis or miliary disease with respiratory failure, azoles are not the initial drugs of choice. In this circum- stance, amphotericin B deoxycholate, liposomal amphotericin B, or amphotericin B lipid complex continue to be preferred. It appears that there is a more rapid response to the amphotericin than to the azole drugs. There does not seem to be a difference in efficacy between amphotericin B compounds, albeit in other diseases there has been a demonstrable difference in toxicity (60). Several weeks of therapy with amphotericin B are often required for improvement, after which oral azole therapy is employed. A brief initial course of corticosteroids is considered beneficial by some in case of fulminant diffuse pneumonia with hypoxia (61). 16. Coccidioidomycosis 309 Hypoxia (PaO2 < 70 mmHg) yes Amphotericin no Nonpregnant female Pregnant female or male Minimal disease, Moderate to severe CF titer > 1:8 CF titer < 1:8, < 1 risk factor* disease, large mild disease infiltrate, multiple risk factors Observation Azole Amphotericin Observation Fig. 16.10. Approach to the patient with acute pulmonary coccidioidomycosis. CF, complement fixation antibody. See text for listing of the risk factors associated with poorer prognosis. These include age greater than 40, African or Asian ancestry, and immunodeficiency. 7.3. Pulmonary Nodule Antifungal therapy or resection is unnecessary for stable pulmonary nodules with an established diagnosis. If enlargement of the nodule occurs, reevaluation with sputum cultures and measurement of serum coccidioidal antibodies should be done to determine if the infection is active and warrants treatment. 7.4. Pulmonary Cavity Asymptomatic, cavitary disease caused by Coccidioides seldom requires inter- vention. Symptomatic solitary cavity coccidioidomycosis may benefit from azole therapy. A course of varying duration until symptoms are resolved is appropriate. Resolution of fever, cough and hemoptysis, improvement in appetite, and decrease in complement fixation titers, if any, may be used to guide therapy. Approximately one-half of cavities smaller than 3 cm will resolve in 6 to 12 months. If the cavity persists but the symptoms abate, a trial of withdrawal of azole therapy can be under- taken. If symptoms recrudesce, reinstitution of therapy for a longer period of time is suggested. Indications for resection of the cavity include recurrent bacterial superin- fections and recurrent or life-threatening hemoptysis. Rupture of the cavity into the pleural space, with development of empyema often requires surgical as well as medical therapy. 7.5. Chronic Progressive Fibrocavitary Pneumonia Fibrocavitary pneumonia of coccidioidomycosis in the pre-azole era often resulted in death from respiratory failure and pulmonary hypertension. Since the advent of azoles, death is less common. Fluconazole or itraconazole at 400 mg or more per day is the most common therapy. At this time, amphotericin has little role in the management of this subacute illness. 310 Royce H. Johnson and Shehla Baqi 7.6. Coccidioidomycosis in Pregnancy Because of demonstrated concerns of teratogenicity of azole compounds, ampho- tericin is the drug of choice in pregnancy for those requiring therapy. Pregnant females with mild disease of limited extent and with low complement fixation titers are usually followed very closely, as often as weekly, without initiation of any therapy. Those with greater extent of disease or with high complement fixation titers are immediately placed on amphotericin. This should be done in concert with an obstetrician who deals with high-risk pregnancies. 7.7. Disseminated Disease (Extrapulmonary) Therapy of disseminated disease requires more expertise and judgment than does uncomplicated pulmonary disease (Fig. 16.11). It has been noted that minimal cutaneous disseminated disease may remit without specific antifungal therapy. At this time however, no expert recommends that treatment of disseminated disease not include antifungal therapy. Disseminated disease of the skin, soft tissue, joints and bones that is limited and not life or limb threatening is usually treated with azole therapy. Some experts prefer itraconazole for disseminated disease, particularly bony dissemination, because of a trend toward superior resolution at 1 year with itraconazole 200 mg every 12 hours when compared with fluconazole 400 mg daily (59). However, both drugs are used, albeit fluconazole is now commonly used at doses greater than 400 mg per day. A duration of therapy substantially longer than a year is frequently recommended. Some experts are recommending therapy for 3 years for significant disseminated disease. Severe multifocal osseous disease that affects the axial skeleton or a major long bone may be treated with azole therapy, though many experts prefer to use amphotericin initially in this circumstance. If the disease is amenable to surgical debridement, this may be a valuable adjunct. After individuals undergo treatment with amphotericin, secondary therapy with azoles is undertaken for protracted periods. Doses higher than 400 mg of fluconazole or itraconazole are frequently administered. In coccidioidal meningitis, fluconazole is the preferred drug, given at doses of 800–1200 mg daily as a single dose (62,63). Itraconazole is not as commonly used Meningitis Nonmeningeal disease Minimal extent and serverity Fluconazole 800- 1200 mg daily* yes no Azole† Amphotericin Fig. 16.11. Approach to the patient with disseminated coccidioidomycosis. ∗ Alternatives for treatment failure include voriconazole, itraconazole, and intrathecal amphotericin B. † Fluconazole 400 to 1000 mg daily (as one dose) or itraconazole 400 to 800 mg daily (in divided doses). Voriconazole and posaconazole may also have a role. 16. Coccidioidomycosis 311 as fluconazole but has had reported success. In patients failing high dose fluconazole therapy, voriconazole has been used and has significant theoretical appeal as rescue therapy at a dose of 4 mg/kg every 12 hours (64,65). Intrathecal amphotericin B was the primary therapy of CNS coccidioidomycosis until supplanted by azole therapy. This therapy can and has been given by direct cisternal injection, via ventricular or cisternal reservoir, or via intrathecal lumbar injection or reservoir. It is now used primarily in those failing other initial or secondary therapies. Coccidioidal meningitis is often complicated by hydrocephalus (Fig. 16.12), which is treated by ventriculoperitoneal shunting (66). Therapy for coccidioidal meningitis is usually life-long. 7.8. Monitoring Therapy Patients with primary coccidioidomycosis should be monitored at 1- to 3-month intervals, both with laboratory and radiologic studies. If |
there is suspicion for dissemi- nation by history or on examination, biopsy and culture of suspected sites of infection should be performed. Lumbar puncture should be performed in patients who develop headaches after the initial primary infection or other neurologic signs at any time. Bone scan is indicated to evaluate bony or joint involvement. Fig. 16.12. Hydrocephalus in coccidioidal meningitis. 312 Royce H. Johnson and Shehla Baqi 8. PREVENTION Developing a vaccine has been a goal for many years. A formalin-killed, whole-cell spherule vaccine was used in a human field trial but was not found to be protective (67). New research on a subcellular vaccine has been initiated and is ongoing. ACKNOWLEDGMENT The authors wish to thank D. Caldwell, H. Einstein, J. Pusavat, and C. Burke for their help and/or advice in the preparation of this chapter. REFERENCES 1. Posadas A. Un nuevo caso de micosis fungoidea con psorospermias. Annales del Circulo Medico Argentina 1892;5:585–597. 2. Wernicke R. Ueber einen Protozoenbefund bei Mycosis fungoides. Zentralb Bakt 1892;12:859–861. 3. Rixford E, Gilchrist TC. Two cases of protozoan (coccidioidal) infection of the skin and other organs. John Hopkins Hosp Rep 1896;1:209–268. 4. Ophuls W, Moffitt HC. A new pathogenic mould (formerly described as a protozoan: Coccidioides immitis): preliminary report. Phila Med J 1900;5:1471–1472. 5. Montenegro J. Septicemia por Coccidioides immitis. Brasil-med 1925;1:69–70. 6. Beck MD. Occurrence of Coccidioides immitis in lesions of slaughtered animals. Proc Soc Exper Biol Med 1929;26:534–536. 7. Rand CW. Coccidioidal granuloma: report of two cases simulating tumor of the spinal cord. Arch Neurol Psychiat 1930;23:502–511. 8. Abbott KH, Cutler OI. Chronic coccidioidal meningitis: review of the literature and report of seven cases. Arch Path 1936;21:320–330. 9. Pulford DS, Larson EE. Coccidioidal granuloma; report of a case treated by intravenous dye, colloidal lead, and colloidal copper, with autopsy observations. JAMA 1929;93:1049–1056. (Discussed by William Ophuls) 10. Stewart RA, Meyer KF. Isolation of Coccidioides immitis (Stiles) from the soil. Proc Soc Exper Biol Med 1932;29:937–938. 11. Gifford MA. San Joaquin Fever. Kern County Dept Public Health Annu Rep 1936:22–23. 12. Fiese MJ. Coccidioidomycosis. Springfield, IL: Charles C Thomas, 1958. 13. Fisher MC, Koenig GL, White TJ, Taylor JW. Molecular and phenotypic description of Coccidioides posadasii sp. nov., previously recognized as the non-California population of Coccidioides immitis. Mycologia 2002;94:73–84. 14. Centers for Disease Control and Prevention. Coccidioidomycosis in workers at an arche- ologic site–Dinosaur National Monument, Utah, June–July 2001. MMWR Morb Mortal Wkly Rep 2001;50:1005–1008. 15. Abuodeh RO, Orbach MJ, Mandel MA, Das A, Galgiani JN. 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Paracoccidioides brasiliensis disseminated disease in a patient with inherited deficiency in the beta 1 subunit of the interleukin (IL)-12/IL-23 receptor. Clin Infect Dis 2005;41:e31–e37. 29. Zerbe CS, Holland SM. Disseminated histoplasmosis in persons with interferon gamma receptor 1 deficiency. Clin Infect Dis 2005;41:e38–e41. 30. Forseth J, Rohwedder JJ, Levine BE, Saubolle MA. Experience with needle biopsy for coccidioidal lung nodules. Arch Intern Med 1986;146:319–320. 31. Johnson RH, Caldwell JW, Welch G, et al. The great coccidioidomycosis epidemic: clinical features. In: Einstein HE, Catanzaro A, eds. Coccidioidomycosis. Proceedings of the Fifth International Conference. Washington DC: National Foundation for Infectious Diseases; 1996;77–87. 32. Quimby SR, Connolly SM, Winkelmann RK, Smilack JD. Clinicopathologic spectrum of specific cutaneous lesions of disseminated coccidioidomycosis. J Am Acad Dermatol 1992;26:79–85. 33. Elbaum DJ. 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Am J Med Sci 1978;275:283–295. 40. Blair JE, Logan JL. Coccidioidomycosis in solid organ transplantation. Clin Infect Dis 2001;33:1536–1544. 314 Royce H. Johnson and Shehla Baqi 41. Logan JL, Blair JE, Galgiani JN. Coccidioidomycosis complicating solid organ transplan- tation. Semin Respir Infect 2001;16:251–256. 42. Walker MP, Brody CZ, Resnik R. Reactivation of coccidioidomycosis in pregnancy. Obstet Gynecol 1992;79:815–817. 43. Peterson CM, Schuppert K, Kelly PC, Pappagianis D. Coccidioidomycosis and pregnancy. Obstet Gynecol Surv 1993;48:149–156. 44. Bisla RS, Taber TH. Coccidioidomycosis of bone and joints. Clin Orthop 1976;121: 196–204. 45. Bried JH, Galgiani JN. Coccidioides immitis infections in bones and joints. Clin Orthop 1986;211:235–243. 46. Lund PJ, Chan KM, Unger EC, Galgiani JN, Pitt MJ. Magnetic resonance imaging in coccidioidal arthritis. Skeletal Radiol 1996;25:661–665. 47. Rosen E, Belber JP. Coccidioidal meningitis of long duration: report of a case of four years and eight months duration, with necropsy findings. Ann Intern Med 1951;34:796–808. 48. Vincent T, Galgiani JN, Huppert M, Salkin D. The natural history of coccidioidal meningitis: VA-Armed Forces cooperative studies, 1955–1958. Clin Infect Dis 1993;16:247–254. 49. Ragland AS, Arsura E, Ismail Y, Johnson RH. Eosinophilic pleocytosis in coccidioidal meningitis: frequency and significance. Am J Med 1993;95:254–257. 50. Biosafety in Microbiological and Biomedical Laboratories. CDC. Fourth ed, May 1999. 51. Kaufman L, Sekhon AS, Moledina N, Jalbert M, Pappagianis D. Comparative evaluation of commercial Premier EIA and microimmunodiffusion and complement fixation tests for Coccidioides immitis antibodies. J Clin Microbiol 1995;33:618–619. 52. Wieden MA, Lundergan LL, Blum J, Jalbert M, Pappagianis D. Detection of coccidioidal antibodies by 33-kDa spherule antigen, Coccidioides EIA, and standard serologic tests in sera from patients evaluated for coccidioidomycosis. J Infect Dis 1996;173:1273–1277. 53. Smith CE, Saito MT, Beard RR, Keep RM, Clark RW, Eddie BU. Serological tests in the diagnosis and prognosis of coccidioidomycosis. Am J Hyg 1950;52:1–21. 54. Drutz DJ, Catanzaro A. Coccidioidomycosis: Part 1. Am Rev Respir Dis 1978;117:559–585. 55. Penrose J, Johnson R, Einstein H, et al. Neuroimaging in coccidioidal meningitis. Proceedings of the 39th Annual Coccidioidomycosis Study Group Meeting, Bakersfield, CA, April 1, 1995. 56. Galgiani JN, Ampel NM, Blair JE, et al. Coccidioidomycoses. Clin Infect Dis 2005;41: 1217–1223. 57. Vaughn JE, Ramirez H. Coccidioidomycosis as a complication of pregnancy. Calif Med J 1951;74:121–125. 58. Caldwell JW, Arsura EL, Kilgore WB, Garcia AL, Reddy V, Johnson RH. Coccidioidomy- cosis in pregnancy during an epidemic in California. Obstet Gynecol 2000;95:236–239. 59. Galgiani JN, Catanzaro A, Cloud GA, et al. Comparison of oral fluconazole and itraconazole for progressive, nonmeningeal coccidioidomycosis: a randomized, double-blind trial. Mycoses Study Group. Ann Intern Med 2000;133:676–686. 60. Walsh TJ, Finberg RW, Arndt C, et al. Liposomal amphotericin B for empirical therapy in patients with persistent fever and neutropenia. National Institute of Allergy and Infectious Diseases Mycoses Study Group. N Engl J Med 1999;340:764–771. 61. Shibli M, Ghassibi J, Hajal R, O’Sullivan M. Adjunctive corticosteroids therapy in acute respiratory distress syndrome owing to disseminated coccidioidomycosis. Crit Care Med 2002;30:1896–1898. 62. Classen DC, Burke JP, Smith CB. Treatment of coccidioidal meningitis with Fluconazole. J Infect Dis 1988;158:903–904. 63. Galgiani JN, Catanzaro A, Cloud GA, et al. Fluconazole therapy for coccidioidal meningitis. Ann Intern Med 1993;119:28–35. 16. Coccidioidomycosis 315 64. Proia LA, Tenorio AR. Successful use of voriconazole for treatment of Coccidioides menin- gitis. Antimicrobial Agents Chemother 2004;48:2341. 65. Cortez KJ, Walsh TJ, Bennett JE. Successful treatment of coccidioidal meningitis with voriconazole. Clin Infect Dis 2003;36:1619–1622. 66. Johnson RH, Einstein HE. Coccidioidal Meningitis. Clin Infect Dis 2006;42:103–107. 67. Pappagianis D. Valley Fever Vaccine Study Group: Evaluation of the protective efficacy of the killed Coccidioides immitis spherule vaccine in humans. Am Rev Respir Dis 1993;148:656–660. SUGGESTED READINGS Galgiani JN. Coccidioides species. In: Mandell GL, Bennett JE, Dolin R, eds. Mandell, Douglas, and Bennett’s principles and practice of infectious diseases. 6th ed. Philadelphia: Elsevier Churchill Livingstone, 2005:3040–3051. Galgiani JN, Ampel NM, Blair JE, et al. Coccidioidomycosis. Clin Infect Dis 2005; 41: 1217–1223. Johnson RH, Einstein HE. Coccidioidal Meningitis. Clin Infect Dis 2006;43:103–107. Stevens DA. Current concepts: coccidioidomycosis. N Engl J Med 1995;332:1077–1082. 17 Histoplasmosis L. Joseph Wheat, MD and Nicholas G. Conger, MD 1. INTRODUCTION Histoplasma capsulatum is a dimorphic fungus primarily found in the Americas, Africa, and Asia, but may be found worldwide, particularly in travelers and immigrants from the endemic areas (1). Among the endemic mycoses, histoplasmosis is the leading cause of hospitalization and death in the United States (2). Darling first described the organism in 1906, believing it to be Leishmania. First thought to cause a progressive and fatal disseminated disease, subsequently histoplasmosis was shown to be very common and usually asymptomatic or clinically self-limited. 2. ETIOLOGIC AGENT Histoplasma is a dimorphic fungus, defined by its ability to grow as a mould in the environment and yeast at 37ºC. Clinical specimens viewed via potassium hydroxide (KOH) preparation, calcofluor white, Giemsa, or hematoxylin and eosin (H&E) stains may demonstrate the 2 to 4 μm budding yeast cells (Fig. 17.1). Histoplasma capsu- latum var. capsulatum causes the vast majority of clinical disease, while its closely related variant Histoplasma capsulatum var. duboisii is the etiologic agent of African histoplasmosis. 3. EPIDEMIOLOGY Histoplasmosis is most commonly reported to occur in and around the Mississippi and Ohio River valleys of the United States, South and Central America, and less so in parts of Africa and Asia. This is thought to be due to factors such as the climate, humidity, and soil acidity. Large amounts of bird and bat excreta enrich the soil in which the fungi are found, facilitating growth and accelerating sporulation. When disturbed, Dr. Wheat is the president of MiraVista Diagnostics, the company that developed and performs Histoplasma antigen testing. The views expressed herein are those of the authors and do not reflect the official policy of the Department of the US Air Force, Department of Defense, or other departments of the US Government. From: Infectious Disease: Diagnosis and Treatment of Human Mycoses Edited by: D. R. Hospenthal and M. G. Rinaldi © Humana Press Inc., Totowa, NJ 317 318 L. Joseph Wheat and Nicholas G. Conger Fig. 17.1. Hematoxylin and eosin stain of yeast in macrophages. [Figure in color on CD-ROM]. microfoci or niches harboring a large number of infective particles may lead to high infectivity rates or large outbreaks. In most cases, however, the exposure is small and usually unrecognized. Cases outside the endemic area usually occur in individuals who have traveled or previously lived in endemic area (3). However, microfoci containing the organism can sometimes be found outside the endemic area and may be the source for exposures. 4. PATHOGENESIS AND IMMUNOLOGY Infection occurs when aerosolized microconidia (spores) are inhaled (Fig. 17.2). Infection usually is |
asymptomatic in healthy individuals after low-level exposure. Further, infection usually is self-limited except after heavy exposure or in patients with Fig. 17.2. Lactophenol cotton blue stain of the mould grown at 25ºC. Note microconidia and tuberculate macroconidia. [Figure in color on CD-ROM]. 17. Histoplasmosis 319 underlying diseases that impair immunity. In addition, pulmonary infection may be progressive in patients with underlying obstructive lung disease. Also, for unknown reasons, pulmonary infection may rarely elicit exuberant mediastinal fibrosis. In the alveoli, the conidia attract phagocytic cells including macrophages, neutrophils, and dendritic cells (4). Within a few days the conidia transform to yeast, which multiply within the nonactivated macrophages and disseminate via the blood- stream to extrapulmonary organs. Neutrophils and dendritic cells inhibit proliferation of the organism, and dendritic cells present antigen to T lymphocytes as the initial step in development of specific cell-mediated immunity. Consequently, tumor necrosis factor- and interferon- are induced, which activate macrophages to inhibit the growth of the organism, leading to spontaneous recovery and immunity against reinfection in most individuals (5). Traditionally, humoral immunity is not believed to be important, but recent studies using monoclonal antibodies suggest otherwise. Reactivation of latent infection has been proposed as the mechanism for progressive disseminated histoplasmosis (PDH) in immunocompromised patients. However, the rarity of PDH in immunosuppressed patients (0.1% to 1%) argues against reactivation. Among more than 600 patients undergoing solid organ or bone marrow transplantation in Indianapolis, a hyperendemic area in which three outbreaks occurred between 1978 and 1993, none developed PDH during the year after transplantation (6). More likely, PDH in this population occurs because low-grade histoplasmosis was present at the time immunosuppression was initiated, or infection was acquired exogenously. In endemic areas, repeated exposure to Histoplasma spores probably occurs, permitting reinfection in immunosuppressed individuals whose immunity to H. capsulatum has waned. 5. CLINICAL MANIFESTATIONS 5.1. Asymptomatic Infection The infection is asymptomatic in most otherwise healthy individuals who experience low inoculum exposure, as indicated by skin test positivity rates above 50% to 80% in the endemic areas (7). Asymptomatic infection also may be identified via radiographic findings of pulmonary nodules or mediastinal lymphadenopathy, which eventually calcify, by splenic calcifications, or via positive serologic tests performed during screening for organ or bone marrow transplantation or epidemiologic investigation. In endemic areas about 5% of healthy subjects have positive complement fixation tests for anti-Histoplasma antibodies. 5.2. Acute Pulmonary Histoplasmosis Healthy individuals who experience a heavy exposure usually present with acute diffuse pulmonary disease 1 to 2 weeks after exposure. Fever, dyspnea, and weight loss are common, and physical examination may demonstrate hepatomegaly or splenomegaly as evidence of extrapulmonary dissemination. In most cases after heavy exposure the illness is sufficiently severe to require hospitalization, with some individuals experiencing respiratory failure. Chest radiographs usually show diffuse infiltrates, which may be described as reticulonodular or miliary (Fig. 17.3). 320 L. Joseph Wheat and Nicholas G. Conger Fig. 17.3. Chest radiograph showing diffuse infiltrate seen in acute pulmonary or dissemi- nated histoplasmosis. 5.3. Subacute Pulmonary Histoplasmosis In symptomatic cases the most common syndrome is a subacute pulmonary infection manifested by respiratory complaints and fever lasting for several weeks, then resolving spontaneously over 1 or 2 months. The chest radiograph or computed tomography (CT) scan usually shows focal infiltrates with mediastinal or hilar lymphadenopathy (Fig. 17.4). Symptoms caused by a mediastinal adenopathy may prevail, and in some cases persist for months to years (granulomatous mediastinitis). In some cases respiratory symptoms may be mild or absent, and findings of pericarditis or arthritis/arthralgia may be prominent. Pericarditis and this rheumatologic syndrome represents inflammatory reactions to the acute infection, rather than infection of the pericardium or joints. 5.4. Chronic Pulmonary Histoplasmosis Patients with underlying obstructive pulmonary disease develop chronic pulmonary disease after infection with H. capsulatum. The underlying lung disease prevents spontaneous resolution of the infection. Chest radiographs reveal upper lobe infil- trates with cavitation, often misdiagnosed as tuberculosis (Fig. 17.5). The course is chronic and gradually progressive, highlighted by systemic complaints of fever and sweats, associated with shortness of breath, chest pain, cough, sputum production, and 17. Histoplasmosis 321 Fig. 17.4. Chest radiograph showing hilar lymphadenopathy seen in subacute pulmonary histoplasmosis. occasional hemoptysis. Patients often experience repeated bacterial respiratory tract infections, and occasionally superinfection with mycobacteria or Aspergillus. 5.5. Progressive Disseminated Histoplasmosis (PDH) Hematogenous dissemination is common during acute pulmonary histoplasmosis, but is nonprogressive (8). With the development of specific cell-mediated immunity, the infection resolves in the lung and extrapulmonary tissues. In contrast, disease is progressive in individuals with defective cell-mediated immunity. In many cases the cause for immune deficiency is easily identified, and often includes the extremes of age, solid organ transplantation (9), treatment with immunosuppressive medica- tions (10), acquired immunodeficiency syndrome (AIDS) (11), idiopathic CD4+ T-lymphocytopenia (12), deficiency in the interferon-/interleukin-12 pathway (13), or malignancy (14). In other cases the cause for immunodeficiency remains unknown, awaiting a more complete understanding of immunity in histoplasmosis and devel- opment of better tests for diagnosis of immunodeficiency. Clinical findings in PDH include progressive fever and weight loss, often associated with hepatomegaly or splenomegaly, and laboratory abnormalities including anemia, leukopenia, from the cytopenia, liver enzyme elevation, and ferritin elevation (8). An elevation in serum lactate dehydrogenase has been associated with disseminated 322 L. Joseph Wheat and Nicholas G. Conger Fig. 17.5. Chest radiograph showing upper lobe infiltrates with cavitation seen in chronic pulmonary histoplasmosis. histoplasmosis, particularly levels greater than 600 IU/liter. Other less frequent sites of involvement include the central nervous system, gastrointestinal tract, skin, and adrenal glands. 5.6. Fibrosing Mediastinitis and Mediastinal Granuloma Fibrosing mediastinitis is a rare complication of pulmonary histoplasmosis (7). The mechanism for this manifestation appears to be an excessive fibrotic response to antigens released into the mediastinal tissues rather than progressive infection. It is unclear why the immune response from subclinical histoplasmosis can lead to either mediastinal granuloma formation (with inflammation and caseating necrosis) or this fibrotic process. Genetic influences, inoculum size, and host immunity are all likely factors. The clinical findings of fibrosing mediastinitis are caused by obstruction of mediastinal structures and may include involvement of the superior vena cava, airways, or pulmonary vessels. Infiltrative inflammatory mediastinal masses that do not respect fat or fascial planes are characteristic CT findings of fibrosing medias- tinitis. Fibrosing mediastinitis most commonly involves the right hemithorax, although 17. Histoplasmosis 323 bilateral involvement may occur. In most patients obstruction does not progress, but mild to moderate symptoms persist indefinitely. In fewer than one quarter of patients the illness is progressive, highlighted by repeated bouts of pneumonia, hemoptysis, respiratory failure, or pulmonary hypertension. No proven medical therapy exists for fibrosing mediastinitis due to histoplasmosis, including antifungal and corticos- teroid therapy. Because the pathogenesis involves fibrosis rather than inflammation or infection, antifungal or anti-inflammatory therapy is not effective. Because fibrosis infiltrates adjacent mediastinal structures, surgical therapy has been of little benefit and is associated with a high risk for surgical morbidity and mortality. Surgical therapy is rarely indicated, and should be considered only after careful consideration of the risks and benefits by experts in the management of patients with fibrosing medias- tinitis. Various procedures to relieve compression of vascular structures, and airway and esophageal compression are often employed with mixed results. Distinguishing fibrosing mediastinitis from mediastinal granuloma is key. Mediastinal granuloma represents persistent inflammation in mediastinal or hilar lymph nodes. Enlarged, inflamed nodes may cause chest pain with or without impingement upon soft mediastinal structures, such as the esophagus or superior vena cava. Fistulae may develop between the lymph nodes, airways, or esophagus. Improvement may occur spontaneously or following antifungal therapy. The enlarged lymph nodes are usually encased in a discrete capsule, which can be dissected free from the adjacent tissues with a low risk for surgical morbidity or mortality. Thus, surgery may be appropriate in patients with persistent symptoms despite antifungal therapy, weighing the risk of surgery with the severity of the clinical findings. However, studies establishing the effectiveness of surgery for mediastinal granuloma are scant, and surgical therapy is rarely necessary in such cases. Often mediastinal lymphadenopathy is asymptomatic, identified on chest radiographs or CT scans performed for other reasons. In such cases concern arises if the mass may represent malignancy. Differentiation of mediastinal lymphadenopathy caused by histoplasmosis or other granulomatous infection from that caused by a malignancy is best deferred to pulmonary disease consultants (7). The presence of calcification strongly suggests that lymphadenopathy is caused by granulomatous infection, but cannot rule out concomitant neoplasm. Conversely, the absence of calcification does not exclude granulomatous infection, and is quite typical of histoplasmosis during the first year or two after infection. Positron emission tomography (PET) scan has been suggested as a method to distinguish malignancy from nonmalignant causes for mediastinal or pulmonary masses, but PET scan is often positive in patients with histoplasmosis. For most patients lacking risk factors for malignancy, follow-up CT scan at 3- to 6-month intervals for 1 year is appropriate; lack of progressive enlargement supports the diagnosis of histoplasmosis. In others, especially those with risk factors for malignancy, biopsy may be necessary for definitive diagnosis. Choices include surgical excision or mediastinoscopy, although many recommend avoiding the latter because of risk of excessive bleeding. 324 L. Joseph Wheat and Nicholas G. Conger 5.7. African Histoplasmosis In addition to infection with Histoplasma capsulatum var. capsulatum, Histoplasma capsulatum var. duboisii causes disease in Africa. Bony abscesses, more commonly involving the axillary skeleton, and skin lesions are much more common with African histoplasmosis. Pulmonary disease is rare, although infection with this variant also likely occurs via inhalation of spores. Disseminated African histoplasmosis resembling PDH as described in the preceding text has been reported, with fever, multi-organ involvement, and a progressive course. The yeasts of H. capsulatum var. duboisii are 10 to 15 m in diameter (much larger than var. capsulatum) and can be seen within giant cells. This can be easily confused with Blastomyces dermatitis or Coccidioides immitis on histopathological examination. DNA and antigen tests with the African variant should react similarly to H. capsulatum var. capsulatum. Likewise, therapy is similar with the exception that isolated cutaneous disease, or “cold abscesses” may heal spontaneously or with excisional surgery. Disseminated African histoplasmosis, especially with HIV coinfection, has a poor prognosis. 6. DIAGNOSIS 6.1. Culture The only test specific for histoplasmosis is culture, but the sensitivity is low, and delays up to 1 month may be required to isolate the organism (15). Identification as H. capsulatum can be determined by conversion from the mould to the yeast, exoantigen detection, or through use of the commercially available DNA probe. Despite these limitations, culture should be performed unless the patient is already improving at the time the diagnosis is suspected. In patients with pulmonary disease, bronchoscopy may be required if patients are unable to produce sputum. In those with suspected PDH, fungal blood cultures should be obtained, but cultures of tissues requiring invasive procedures may be deferred if tests for antigen are positive. In such cases, failure to improve within 2 weeks after initiation of therapy would raise question about the diagnosis and support additional testing. 6.2. Antigen Detection Antigen detection is most useful method for rapid diagnosis of the more severe histoplasmosis syndromes, including acute diffuse pulmonary histoplasmosis and PDH (15). The Histoplasma antigen assay has been revised to reduce false-positive (16) and false-negative results (17). Antigen may be detected in any body fluid, but urine and serum testing is recommended in all cases. In some patients with pulmonary histoplasmosis, antigen may be detected in the bronchoalveolar lavage fluid but not in urine or serum (18). Antigen may also be detected in the cerebrospinal fluid in patients with central nervous system histoplasmosis (19), in pleural fluid, synovial fluid, peritoneal fluid, or in pericardial fluid in patients with infection localized to those tissues. Antigen levels decline with effective therapy, persist with ineffective therapy, and rebound in patients who relapse during or following treatment (15). 17. Histoplasmosis 325 Fig. 17.6. Gomori methenamine silver (GMS) stain of yeast showing narrow neck budding. [Figure in color on CD-ROM]. 6.3. Histopathology Demonstration of yeast-like structures in body fluids or tissues may provide a rapid diagnosis in some cases (7) (Fig. 17.6) (see also Fig. 3.6, Chapter 3). Limitations of histopathology include the need for performance of an invasive procedure and low sensitivity. Histopathology may be falsely negative or falsely positive |
when performed by pathologists inexperienced with recognition of fungal pathogens. If histopathology is inconsistent with clinical findings or other laboratory tests, the specimens should be reviewed by a pathologist experienced with recognition of fungal pathogens. 6.4. Serologic Tests Serologic tests for antibodies are most useful in patients with subacute pulmonary histoplasmosis, chronic pulmonary histoplasmosis, granulomatous mediastinitis, pericarditis, or rheumatologic syndromes (15). Immunodiffusion and complement fixation tests should be performed in all cases of suspected histoplasmosis. Serologic tests may be falsely negative during the first 2 months after acute exposure, limiting their usefulness in acute pulmonary histoplasmosis. Also, these tests may be falsely negative in immunosuppressed patients, limiting their role in PDH. Further, positive serologic tests persist for several years after recovery from histoplasmosis, and thus may provide misleading information in patients with other diseases. 6.5. Conclusions Accurate diagnosis of histoplasmosis requires skilled use of all of these laboratory methods. Except for culture, none of the tests are specific, and physicians must consider that the positive result may be falsely positive. Further, none of the tests are positive in all cases, and the physician must consider the possibility that the test is falsely negative. 326 L. Joseph Wheat and Nicholas G. Conger In cases in which the diagnosis is uncertain, or the laboratory tests are inconsistent, additional testing should be performed, including repetition of the antigen test or serology, and in some cases, invasive procedures to obtain tissues for histopathology and culture; expert advise should be sought. 7. TREATMENT Practice guidelines, published in 2000 (20), are currently being updated. Treatment is indicated in patients with acute diffuse pulmonary histoplasmosis, chronic pulmonary histoplasmosis, and PDH (Table 17.1). Adjunctive treatment with methyl prednisolone or prednisone may hasten recovery in otherwise healthy individuals with respiratory distress caused by acute pulmonary histoplasmosis, and can be administered safely if patients also receive antifungal therapy. Treatment also should be considered in patients with subacute pulmonary histo- plasmosis or granulomatous mediastinitis who are not improving within a month Table 17.1 Primary and alternate therapy for treatment for histoplasmosis Presentation Primary therapy Alternate therapy Acute pulmonary Itraconazole 200 mg qd or bid Posaconazole 400 mg bid, or Mild for 6–12 weeks voriconazole 200 mg bid, or fluconazole 800 mg qd Moderately Liposomal amphotericin B Amphotericin B lipid complex severe or severe 3–5 mg/kg per day for 3–5 mg/kg per day, or 1–2 weeks followed by amphotericin B deoxycholate itraconazole 200 mg bid for 12 0.7–1.0 mg/kg per day weeks;a methyl prednisolone or prednisone 0.5–1 mg/kg per day for 1–2 weeksb Chronic Itraconazole 200 mg bid for at Posaconazole 400 mg bid, or pulmonary least 12 months voriconazole 200 mg bid, or fluconazole 800 mg qd Disseminated Liposomal amphotericin B Amphotericin B lipid complex 3–5 mg/kg per day for 3–5 mg/kg per day, or 1–2 weeks followed by amphotericin B deoxycholate itraconazole 200 mg bid at 0.7–1.0 mg/kg per day least 12 monthsa qd, once daily; bid, twice daily; tid, three times daily; mg/kg per day, milligram/kilogram per day. aItraconazole may be given 200 mg tid × 3 days as a loading dose to achieve steady-state levels more quickly. The capsule formulation should be administered with food whereas the solution should be administered on an empty stomach. Measurement of drug concentrations is recommended because itraconazole drug exposure is highly variable. The intravenous formulation of itraconazole would be an alternative in a hospitalized patient with moderately severe or severe disease who cannot be treated with any amphotericin formulation. bAdjunctive treatment with methyl prednisolone or prednisone may hasten recovery in otherwise healthy individuals with respiratory distress caused by acute pulmonary histoplasmosis. 17. Histoplasmosis 327 or two of the onset of symptoms; however, the effectiveness of therapy for these manifestations remains uncertain. Treatment for fibrosing mediastinitis is ineffective, and is not indicated except in cases in which granulomatous mediastinitis cannot be reasonably excluded. Treatment is not indicated in patients with calcified or noncal- cified pulmonary nodules or as prophylaxis before immunosuppression in patients without evidence of active histoplasmosis within the last 2 or 3 years. Liposomal amphotericin B is the treatment of choice for patients with severe manifestations of histoplasmosis requiring hospitalization (21). In some cases, however, because of intolerance or cost, other lipid formulations may be used. In children, deoxycholate amphotericin B is well tolerated and preferred over the lipid formu- lations because of cost. Itraconazole is recommended for mild cases not requiring hospitalization and for continued therapy after response to liposomal amphotericin B. Treatment should be continued for 6 to 12 weeks in patients with acute pulmonary histoplasmosis and 1 year or longer in patients with chronic pulmonary histoplas- mosis or PDH. In patients with AIDS who achieve a good immunologic response to antiretroviral therapy, itraconazole may be stopped after 1 year if the CD4+ T lymphocyte count is above 200 cells/μl and the antigen concentration in urine and serum are below 4 ng/ml (22). However, in those with persistent immune deficiency, or who relapse after stopping an appropriate course of therapy, lifelong mainte- nance therapy may be required. Itraconazole blood levels should be monitored to ensure adequate drug exposure, and the dosage should be increased or the capsule formulation should be replaced with the solution if random concentrations are below 2 μg/ml. If the antigen test is positive, treatment should be continued until antigen levels become undetectable or below 4 ng/ml. Further, antigen levels should be monitored during the first year following discontinuation of therapy, and at the time of recurrence of symptoms suggesting relapse of histoplasmosis. The best alternative oral therapy in patients unable to take itraconazole is posaconazole, which is highly active in vitro (23), in animal models (23), and in patients (24). Fluconazole is less active in histoplasmosis, and relapse associated with development of resistance has been observed in patients with AIDS (25). Voriconazole is more active in vitro than fluconazole, but less active than itraconazole or posaconazole. Although minimum inhibitory concentrations (MICs) are lower to voriconazole than fluconazole, higher drug exposure with fluconazole offsets the lower MICs. Further, prior exposure to fluconazole or voriconazole may induce resistance to voriconazole (26). Voriconazole has not been studied in animal models or patients with histoplasmosis, and offers no clear advantage over fluconazole. Measurement of voriconazole or posaconazole blood levels is recommended because of the wide variation in drug levels. Voriconazole exhibits a short half-life (approximately 6 hours) and concentrations decline in at least twofold from the peak to the trough time after administration. Accordingly, trough concentrations of voriconazole of at least 0.5 μg/ml are recommended. Posaconazole exhibits a long half-life, similar to that of itraconazole (approximately 24 hours), supporting a similar target random concen- tration of 2 μg/ml. The echinocandins are not active in histoplasmosis and should not be used (27). 328 L. Joseph Wheat and Nicholas G. Conger REFERENCES 1. Panackal AA, Hajjeh RA, Cetron MS, Warnock DW. Fungal infections among returning travelers. Clin Infect Dis 2002;35:1088–1095. 2. Chu JH, Feudtner C, Heydon K, Walsh TJ, Zaoutis TE. Hospitalizations for endemic mycoses: a population-based national study. Clin Infect Dis 2006;42:822–825. 3. Antinori S, Magni C, Nebuloni M et al. Histoplasmosis among human immunodeficiency virus-infected people in Europe: report of 4 cases and review of the literature. Medicine (Baltimore) 2006;85:22–36. 4. Newman SL. Interaction of Histoplasma capsulatum with human macrophages, dendritic cells, and neutrophils. Methods Mol Med 2005;118:181–191. 5. Deepe GS. Modulation of infection with Histoplasma capsulatum by inhibition of tumor necrosis factor-alpha activity. Clin Infect Dis 2005;41 (Suppl 3):S204–S207. 6. Vail GM, Young RS, Wheat LJ, Filo RS, Cornetta K, Goldman M. Incidence of histo- plasmosis following allogeneic bone marrow transplant or solid organ transplant in a hyperendemic area. Transpl Infect Dis 2002;4:148–151. 7. Wheat LJ, Conces DJ, Allen S, Blue-Hnidy D, Loyd J. Pulmonary histoplasmosis syndromes: recognition, diagnosis, and management. Semin Respir Crit Care Med 2004;25:129–144. 8. Wheat LJ, Kauffman CA. Histoplasmosis. Infect Dis Clin North Am 2003;17:1–19. 9. Freifeld AG, Iwen PC, Lesiak BL, Gilroy RK, Stevens RB, Kalil AC. Histoplasmosis in solid organ transplant recipients at a large Midwestern university transplant center. Transpl Infect Dis 2005;7:109–115. 10. Wood KL, Hage CA, Knox KS, et al. Histoplasmosis after treatment with anti-TNF-alpha therapy. Am J Respir Crit Care Med 2003;167:1279–1282. 11. Wheat J. Histoplasmosis in the acquired immunodeficiency syndrome. Curr Top Med Mycol 1996;7:7–18. 12. Duncan RA, Von Reyn CF, Alliegro GM, Toossi Z, Sugar AM, Levitz SM. Idiopathic CD4+ T-lymphocytopenia—four patients with opportunistic infections and no evidence of HIV infection. N Engl J Med 1993;328:393–398. 13. Zerbe CS, Holland SM. Disseminated histoplasmosis in persons with interferon-gamma receptor 1 deficiency. Clin Infect Dis 2005;41:e38–e41. 14. Adderson EE. Histoplasmosis in a pediatric oncology center. J Pediatr 2004;144:100–106. 15. Wheat LJ. Current diagnosis of histoplasmosis. Trends Microbiol 2003;11:488–494. 16. Wheat LJ, Connolly P, Durkin M, et al. False-positive Histoplasma antigenemia caused by antithymocyte globulin antibodies. Transpl Infect Dis 2004;6:23–27. 17. Wheat LJ, Connolly P, Durkin M, Book BK, Pescovitz MD. Elimination of false-positive Histoplasma antigenemia caused by human anti-rabbit antibodies in the second generation Histoplasma antigen test. Transpl Infect Dis 2006;8:219-221. 18. Hage CA, Davis TE, Egan L, et al. Diagnosis of pulmonary histoplasmosis and blastomy- cosis by detection of antigen in bronchoalveolar lavage fluid using an improved second- generation enzyme-linked immunoassay. Respir Med 2007;101:43–47. 19. Wheat LJ, Musial CE, Jenny-Avital E. Diagnosis and management of central nervous system histoplasmosis. Clin Infect Dis 2005;40:844–852. 20. Wheat J, Sarosi G, McKinsey D, et al. Practice guidelines for the management of patients with histoplasmosis. Infectious Diseases Society of America. Clin Infect Dis 2000;30: 688–695. 21. Johnson PC, Wheat LJ, Cloud GA, et al. Safety and efficacy of liposomal amphotericin B compared with conventional amphotericin B for induction therapy of histoplasmosis in patients with AIDS. Ann Intern Med 2002;137:105–109. 17. Histoplasmosis 329 22. Goldman M, Zackin R, Fichtenbaum CJ, et al. Safety of discontinuation of mainte- nance therapy for disseminated histoplasmosis after immunologic response to antiretroviral therapy. Clin Infect Dis 2004;38:1485–1489. 23. Connolly P, Wheat LJ, Schnizlein-Bick C, et al. Comparison of a new triazole, posaconazole, with itraconazole and amphotericin B for treatment of histoplasmosis following pulmonary challenge in immunocompromised mice. Antimicrob Agents Chemother 2000;44: 2604–2608. 24. Restrepo A, Tobon A, Clark B, et al. Salvage treatment of histoplasmosis with posaconazole. J Infect 2007;54:319–327. 25. Wheat LJ, Connolly P, Smedema M, Brizendine E, Hafner R. Emergence of resistance to fluconazole as a cause of failure during treatment of histoplasmosis in patients with acquired immunodeficiency disease syndrome. Clin Infect Dis 2001;33:1910–1913. 26. Wheat LJ, Connolly P, Smedema M, et al. Activity of newer triazoles against Histoplasma capsulatum from patients with AIDS who failed fluconazole. J Antimicrob Chemother 2006;57:1235–1239. 27. Kohler S, Wheat LJ, Connolly P, et al. Comparison of the echinocandin caspofungin with amphotericin B for treatment of histoplasmosis following pulmonary challenge in a murine model. Antimicrob Agents Chemother 2000;44:1850–1854. SUGGESTED READINGS Goldman M, Zackin R, Fichtenbaum CJ, et al. Safety of discontinuation of maintenance therapy for disseminated histoplasmosis after immunologic response to antiretroviral therapy. Clin Infect Dis 2004;38:1485–1489. Johnson PC, Wheat LJ, Cloud GA, et al. Safety and efficacy of liposomal amphotericin B compared with conventional amphotericin B for induction therapy of histoplasmosis in patients with AIDS. Ann Intern Med 2002;137:105–109. Lee JH, Slifman NR, Gershon SK, et al. Life-threatening histoplasmosis complicating immunotherapy with tumor necrosis factor alpha antagonists infliximab and etanercept. Arthritis Rheum 2002;46:2565–2570. Limaye AP, Connolly PA, Sagar M, et al. Transmission of Histoplasma capsulatum by organ transplantation. N Engl J Med 2000;343:1163–1166. Vail GM, Young RS, Wheat LJ, Filo RS, Cornetta K, Goldman M. Incidence of histoplasmosis following allogeneic bone marrow transplant or solid organ transplant in a hyperendemic area. Transpl Infect Dis 2002;4:148–151. Wheat LJ. Histoplasmosis: a review for clinicians from non-endemic areas. Mycoses 2006;49:274–282. Wheat LJ, Conces DJ, Jr., Allen S, Blue-Hnidy D, Loyd J. Pulmonary histoplasmosis syndromes: recognition, diagnosis, and management. Semin Respir Crit Care Med 2004;25:129–144. Wheat LJ, Musial CE, Jenny-Avital E. Diagnosis and management of central nervous system histoplasmosis. Clin Infect Dis 2005;40:844–852. Woods JP. Histoplasma capsulatum molecular genetics, pathogenesis, and responsiveness to its environment. Fungal Genet Biol 2002;35:81—97. 18 Paracoccidioidomycosis Angela Restrepo, PhD, Angela M. Tobón, MD, and Carlos A. Agudelo, MD 1. INTRODUCTION Paracoccidioidomycosis (PCM), formerly known as South American blastomycosis, was first described by Lutz in Brazil in 1906. Lutz and Splendore described the etiologic agent but considered it |
to be a strain of Coccidioides immitis. It was not until 1930 when the de Almeida properly differentiated its etiologic agent and designated it Paracoccidioides brasiliensis. The Brazilian disease was soon diagnosed in other Latin American countries and its peculiar geographic limitation to Latin American countries recognized (1). PCM is an endemic and systemic disease caused by the thermally dimorphic fungus, Paracoccidioides brasiliensis, a microorganism exogenous to humans that has an as yet undiscovered habitat. The infection is acquired by the inhalation of mycelial form structures (conidia, mycelial fragments) that settle into the lungs where they convert into the tissue yeast form structure that characteristically reproduces by multiple budding. PCM exists in two forms, subclinical infection and the clinically manifested disease. The latter is usually chronic with involvement of the primary target, the lungs, and of other organs and systems (including the mucosa, skin, adrenal glands, and lymph nodes). The mycosis afflicts men more frequently than women and is more common in adults. Latency is known to exist and is frequently prolonged (2). 2. ETIOLOGIC AGENT P. brasiliensis is a thermally dimorphic fungus that at temperatures between 4ºC and 25°C grows as a white mould, microscopically composed of thin septated hyphae, occasional chlamydospores, and rare conidia; the latter are infectious. At 35º to 37ºC the colony is soft and wrinkled and microscopically is comprised of oval to round yeast cells of varying sizes (4 to 40 μm) that reproduce by budding. The key distinguishing feature is that of multiple budding yeast cells with a larger mother cell surrounded by multiple daughter cells (blastoconidia), a structure thought to resemble the pilot wheel of a ship. A thick refractive cell wall and prominent intracytoplasmic vacuoles From: Infectious Disease: Diagnosis and Treatment of Human Mycoses Edited by: D. R. Hospenthal and M. G. Rinaldi © Humana Press Inc., Totowa, NJ 331 332 Angela Restrepo, Angela M. Tobón, and Carlos A. Agudelo Fig. 18.1. P. brasiliensis yeast cell surrounded by multiple budding daughter blastoconidia (“pilot’s wheel”). [Figure in color on CD-ROM]. Fig. 18.2. P. brasiliensis in sputum sample. Note multiple budding yeast cell and single yeasts with prominent lipid vacuoles. KOH and blue ink. [Figure in color on CD-ROM]. 18. Paracoccidioidomycosis 333 further characterize the organism at these temperatures (Fig. 18.1). The aforementioned morphologic characteristics are also observed in clinical specimens (Fig. 18.2) (1,3). Despite the absence of a sexual stage or teleomorph, P. brasiliensis has been classified in the phylum Ascomycota, order Onygenales, family Onygenaceae on the basis of phylogenetic studies and on its sharing certain characteristics with other dimorphic fungi (e.g., Blastomyces dermatitidis, Histoplasma capsulatum) exhibiting teleomorphic stages, genus Ajellomyces (4). 3. EPIDEMIOLOGY Demographic data indicate that clinically manifested PCM predominates in adults, 80% to 95% of cases. In highly endemic areas, skin testing with paracoccidioidin has indicated that among healthy populations the prevalence of infection is close to 10%. The prevalence of disease in other areas of Latin America has been estimated to be much lower, 0.33 to 3 cases per 100,000 inhabitants (1,3). The disease is more often diagnosed in males than in females (ratio of 15:1), even though similar infection rates are demonstrated by paracoccidioidin skin testing. Most patients (73%) have or have had agriculture-related occupations (2,5–8). One of the most relevant characteristics of PCM is its restricted geographic limitation to Central and South America from Mexico (23º North) to Argentina (34º South), sparing certain countries within these latitudes (Chile, Surinam, the Guyana, Nicaragua, Belize, most of the Caribbean Islands) (3). Also of note is the fact that within endemic countries the mycosis is not diagnosed everywhere, but in areas with relatively well- defined ecologic characteristics. These characteristics include the presence of tropical and subtropical forests, abundant watercourses, mild temperatures (<27ºC), high rainfall (2000 to 2999 mm), and coffee/tobacco crops (9). Risk factors include living and working in these areas, as well as malnutrition. Alcoholism and smoking may also play a role. A direct relationship with underlying immunosuppressive conditions, including human immunodeficiency virus (HIV) infection, has not been clearly demonstrated although sporadic cases have been reported (1,3,10). In the case of the PCM–HIV coinfection, the total number of reported cases in the endemic areas appears to not have exceeded 200 patients (1,3,11). Approximately 60 cases of PCM have been reported outside of the endemic areas (North America, Europe, Asia); however all these patients had previously resided in recognized endemic countries (3,12,13). These cases demonstrate that P. brasiliensis can remain latent for long periods (mean 13 years) from the time of infection to disease manifestations (3). Latency may explain why the microniche of P. brasiliensis has not been precisely demonstrated, as with delay in presentation, the site and type of activities that led to infection are likely forgotten. No outbreaks have been reported and the isolation of the fungus from nature (e.g., soils) has seldom been successful (14). 4. PATHOGENESIS AND IMMUNOLOGY Lack of data on P. brasiliensis habitat and on the characteristics of the primary infection has hindered full understanding of the pathogenesis of PCM. Based on experimental animal studies it is accepted that infection is acquired by inhalation of the conidia produced by the mycelial form, structures that are sufficiently small to reach 334 Angela Restrepo, Angela M. Tobón, and Carlos A. Agudelo Infection (subclinical) Dissemination via lymphatics/ blood Distant foci Host – parasite interaction Adequate Inadequate - Disease Symptomatic Progressive Regressive form Chronic adult Acute/subacute juvenile form Treatment Viable, latent fungi→ Relapse Fungi destroyed→ Cure Response Death Residual form (sequelae) Fig. 18.3. Natural history of paracoccidioidomycosis. the alveoli, where they soon transform into yeast cells (15,16). The organism then multiplies in the lung parenchyma and disseminates by the venous/lymphatic routes to extrapulmonary organs (Fig. 18.3). Infection gives rise to an intense host response with alveolitis and abundant neutrophils engulfing the fungus, later on replaced by migrating mononuclear cells that convert into epithelial cells initiating granuloma formation and attracting CD8+ T lymphocytes (1,2,17). The host immune response determines the course of the infection, with subclinical infection predominating in immunocompetent individuals and clinically manifested disease appearing in those whose cellular immunity is deficient. Thus, children and some HIV-infected individuals develop disseminated disease with predominant involvement of the reticuloendothelial system, the acute/subacute juvenile type disease (18–20). Adult patients tend to develop a chronic, progressive disease, marked by more severe pulmonary damage accompanied in most cases by dissemination to the lymphatic system, the mucosa, the adrenals, skin, and other organs (1–3). Lung damage progresses to fibrosis, leaving behind important sequelae (1–3). An efficient T helper 1 (Th1) cellular immune response is required to control fungus invasion while a T helper 2 (Th2) type response is reflected by abundant fungal multiplication and extra- pulmonary dissemination with progression of disease (21,22). Antibodies are detected in most patients with either the acute or the chronic type of disease but their role in protection or dissemination is still unclear (1,23). 5. CLINICAL MANIFESTATIONS PCM is a disorder characterized by protean manifestations; usually it is a chronic progressive disease involving various organs and systems. In untreated and patients with chronic, advanced disease, mortality rates may be high; 1.4 per one million inhabitants in Brazil (24). Most patients present with constitutional symptoms such as general malaise, asthenia, weight loss and fever, as well as symptoms specific to the organs infected by the fungus. The lungs are the site of primary infection, but often neither the patient nor the clinical examiner will note abnormalities at this site. 18. Paracoccidioidomycosis 335 On the basis of the clinical presentation and the host immune response to PCM, the disease is categorized as (1) subclinical infection or (2) symptomatic infection, which is divided into two forms, the acute/subacute juvenile and the chronic adult type. The latter may involve one (unifocal) or various organs (multifocal) with the severity of the symptoms varying with the patient. The acute/subacute juvenile type is always disseminated. A third, residual form characterized by fibrotic sequelae has also been suggested (1–3). 5.1. Subclinical Infection Subclinical infection has no special characteristics and is detected mostly by a reactive paracoccidioidin skin testing, and sometimes by chest radiograph abnormalities (25). However, P. brasiliensis may remain latent in the infected host and give rise to symptomatic paracoccidioidomycosis years after the initial contact, as demonstrated by the cases diagnosed outside of the endemic areas for the mycosis (2,12,13). 5.2. Symptomatic Infection The clinically manifested disease varies with patient age. 5.2.1. Juvenile Type Disease The juvenile type disease is a serious disorder that afflicts children and immuno- compromised individuals of either sex; it represents fewer that 10% of all cases. Involvement of the reticuloendothelial system with lymphadenopathy, hepatomegaly, and splenomegaly characterize this form. Skin lesions, often multiple, may also be present, along with fever, marked weight loss, and general malaise. Bone involvement is frequent in the subacute severe cases. Respiratory symptoms are minimal but the fungus can be seen in respiratory secretions. High-resolution computed tomography (CT) studies reveal lung abnormalities in a significant proportion of these patients (8,18–20). 5.2.2. Chronic Adult Type Disease The chronic, adult type disease predominates (80% to 90%) in all case series, typically occurring in patients older 30 years of age with agriculture-related occupa- tions; males are more frequently afflicted than females (15:1). The disease course is characterized by protracted pulmonary and extrapulmonary organ damage, mainly of the mucous membranes and the skin, where the lesions tend to be ulcerative, granulo- matous, and infiltrated (Fig. 18.4). Sialorrhea, dysphagia, and dysphonia are common. Regional lymph nodes are hypertrophied and may spontaneously drain forming fistulae. The adrenals glands may also become involved, with associated symptoms of adrenal deficiency. Central nervous system (CNS) involvement is also considered important. Chest radiographs reveal infiltrates, mostly interstitial but at times also alveolar; these are located in the central and lower fields and are bilateral. Sequelae repre- sented mainly by pulmonary fibrosis can be observed in half of the cases (Fig. 18.5) (1,2,6,7,26–29). Usually, at time of the initial consultation, more than one organ is involved. 336 Angela Restrepo, Angela M. Tobón, and Carlos A. Agudelo Fig. 18.4. Cutaneous and mucosal lesions in a patient with paracoccidioidomycosis. Note lip edema, ulceration, and scarring. [Figure in color on CD-ROM]. Fig. 18.5. Pulmonary paracoccidioidomycosis. Note abundant fibronodular infiltrates in central fields and basal bullae. [Figure in color on CD-ROM]. 18. Paracoccidioidomycosis 337 6. DIAGNOSIS Diagnosis is based on directed diagnostic techniques in persons with the relevant symptoms and risk factors/endemic exposures (Table 18.1). Direct potassium hydroxide (KOH) examination of clinical material including sputum, bronchoalveolar lavage (BAL) fluid, exudates, lymph node drainage, and biopsies offer a diagnosis in more than 90% of the cases; the rather large yeast cells with their translucent wall and multiple budding allow prompt identification. Most cases can be diagnosed by the histopathological study of biopsies; although Gomori methenamine silver (GMS) stain is more reliable, hematoxylin and eosin (H&E), may reveal these fungi, especially in and around granulomas (Fig. 18.6). Isolation of P. brasiliensis in culture is evidence of active disease, but mould cultures are positive in only of 80% of the cases and require between 20 to 30 days for growth adequate for microscopic diagnosis. Room temperature incubation is preferred for nonsterile site specimens, but requires a transfer to 36–37C for transformation of the fungus into its yeast form (with its multiple budding cells) once growth is noted for confirmation. Primary isolation often requires multiple samples (e.g., sputum) and a variety of culture media (1,3). Antibody detection is useful not only for diagnosis but also for disease monitoring; antibodies of the immunoglobulin classes G, M, and E are regularly detected. Because of its simplicity, the gel immunodiffusion (ID) test is typically used in endemic countries. This test demonstrates circulating antibodies in 95% of the cases, and is very specific for PCM. Complement fixation (CF) testing allows quantification of antibody levels, and indirectly aids in determination of the severity of illness and response to treatment. CF is reactive in more than 85% of the cases, but cross-reactivity with H. capsulatum antigens is seen. Other tests, such as immunofluorescence, counterimmu- noelectrophoresis, dot-blot, enzyme-linked immunosorbent assay, and immunoblotting, are also currently used by some centers (30–32). Improvements in serodiagnosis include detection in more than 60% of the cases of circulating fungal antigens in patients’ Table 18.1 Key factors in the consideration of the diagnosis of paracoccidioidomycosis History |
of residence in an endemic country (even if many years previous to symptoms/lesions) History of working in agriculture or related occupation Being an adult male with a chronic, progressive illness Complaints related to external manifestations (mucous membrane, skin, and or lymph node lesions) Pulmonary involvement is often ignored by the patient but is almost always demonstrated by radiographic or other imaging studies Reticuloendothelial system (RES) involvement is commons in children and young adults. Presence of multiple skin lesions or bone damage may also be observed in these age groups Coexistence with immunodeficiency states is not a hallmark, but may occur. Remember: paracoccidioidomycosis is a disease of protean manifestations that may exhibit peculiar clinical aspects hindering proper diagnosis 338 Angela Restrepo, Angela M. Tobón, and Carlos A. Agudelo Fig. 18.6. Tissue biopsy with abundant P. brasiliensis yeasts enclosed within a granuloma. GMS. [Figure in color on CD-ROM]. sera. Most importantly, immunosupressed patients who do not raise antibodies can be diagnosed by antigen detection, and in addition, decreasing antigen titers correlate with their clinical improvement. Skin tests play no role in diagnosis (33,34). 7. TREATMENT Antimicrobial agents from three different classes are currently used to treat PCM, with varying success. They include the sulfonamides, the polyene amphotericin B, and certain azoles (Table 18.2). Use of the newer agents, including voriconazole, posaconazole, and caspofungin, has been reported in isolated cases, but all appear promising. It should be stated that additional measures such as adequate nutrition and suspension of alcohol intake and smoking are required to accelerate recovery and diminish fibrous sequelae. In advanced cases (approximately 10%) no response to treatment may occur (1–3). Sulfonamides are of low cost and appear effective in 70% of patients. They require prolonged periods of treatment (3 to 5 years) to avoid relapses and the development of resistance. Rapidly absorbed sulfonamides such as sulfadiazine are given at a dose of 4 g per day while those characterized by prolonged absorption are administered at half the dose (2 g per day). Trimethoprim–sulfamethoxazole also appears to be effective at a 160/800 mg per tablet, two tablets twice daily. Adverse effects more commonly seen with the sulfonamides include rash, crystalluria photosensitivity, emesis, and diarrhea (1,18). Amphotericin B is effective in about 70% of cases, but its use is now limited to severely ill patients, to those with CNS or cardiac disease, and those unable to tolerate azole agents. A total cumulative dose of 1 to 2 g, based on clinical response, 18. Paracoccidioidomycosis 339 Table 18.2 Antifungal use in paracoccidioidomycosis Antifungals Dose (daily) Route Duration Response Relapse (mean) rate (%) rate (%) Sulfonamides 4 grams PO 3–5 years 70 20 Amphotericin B 1 gram IV Based on 75 20 (cumulative clinical dose)a response Ketoconazole 200–400 mg PO 6–18 months 70 10 Itraconazole Capsules 200 mg PO 6 months 95 5 Suspension 100–200 mg PO 6 months 95 5 Parenteral 200 mg IVb Based on 95 5 clinical response PO, oral; IV, intravenous. aTo be followed by an oral medication. bTo be followed by an oral azole. is typically given, followed by maintenance therapy with oral medications. Infusional toxicity, electrolyte abnormalities, and renal dysfunction are common adverse effects. Lipid-based formulations of amphotericin B have been used in a few cases, limited likely by their high costs (1–3). With the exception of fluconazole, most of the systemically absorbed azole antifungals have proven effective in the therapy of PCM. Ketoconazole can be used successfully at a dose of 200 to 400 mg/day in adults or 5 mg/kg per day in children for 6 to 18 months; relapses occur in approximately 10% of the cases. Complications include hepatitis, gonadal dysfunction, and gastrointestinal toxicity. Interactions with several medications that are metabolized through the P450 cytochrome also limit the use of this drug. Since the introduction of itraconazole, now considered as the best option for the treatment of PCM, use of ketoconazole has decreased. Itraconazole is typically administered at a dose of 100 to 200 mg/day for approximately 6 months of therapy, based on clinical response and mycology laboratory data. Itraconazole has been shown to be effective in 95% of the patients with less adverse effects; relapses occur in 5% of the cases. Despite this high response rate, itraconazole has not reduced the fibrous pulmonary sequelae (35). Care should be taken with the administration of antacids and H2 blockers as they hinder proper absorption; ingestion with a cola drink (acid) appears to improve absorption (1–3). Itraconazole is also available as an oral solution with improved absorption compared to the capsular formulation. As for the solution, a loading dose similar to that of capsular itraconazole (200 mg, three times per day for 3–5 days) is administered and followed by a lower 200 mg/day dose. An intravenous formulation has made it possible to treat severely ill patients at 200 mg bid for four doses, followed by 200 mg daily until favorable changes occur in the patient’s clinical aspects (1,2,35,36). 340 Angela Restrepo, Angela M. Tobón, and Carlos A. Agudelo 8. PREVENTION Preventive measures are difficult to establish because the source of infection is unknown; the mycosis is not transmissible from person to person. Nonetheless, precaution against aerosols is recommended when falling trees or hunting armadillos in the forest (1). REFERENCES 1. Lacaz CS, Porto E, Martins JEC, et al. Paracoccidioidomicose. In: Lacaz CS, Porto E, Martins JEC, et al., eds. Tratado de micologia médica lacaz 2002, 9th ed. Sao Paulo: Servier, 2002:639–729. 2. Restrepo A, Tobón AM. Paracoccidioides brasiliensis. In: Mandell GL, Bennett JE, Dolin R, eds. Mandell, Douglas, and Bennett’s principles and practice of infectious diseases. 6th ed. Philadelphia: Elsevier Churchill Livingstone, 2005:3062–3068. 3. Restrepo A. Paracoccidioidomycosis. In: Dismukes WE, Pappas PG, Sobel JD, eds. Clinical mycology. New York: Oxford University Press, 2003:328–345. 4. San-Blas G, Niño-Vega G, Iturriaga T. Paracoccidioides brasiliensis and paracoccid- ioidomycosis: molecular approaches to morphogenesis, diagnosis, epidemiology, taxonomy and genetics. Med Mycol 2002;40:225–242. 5. Blotta MH, Mamoni RL, Oliveira SJ, et al. Endemic regions of paracoccidioidomycosis in Brazil: a clinical and epidemiologic study of 584 cases in the southeast region. Am J Trop Med Hyg 1999;61:390–394. 6. Paniagoa AM, Aguiar JI, Aguiar ES, et al. Paracoccidioidomicose: a clinical and epidemi- ological study of 422 cases observed in Mato Grosso do Soul. Rev Soc Bras Med Trop 2003;36:455–459. 7. Kalmar EM, Alencar FE, Alves FP, et al. Paracoccidioidomycosis: an epidemiologic survey in a pediatric population from the Brazilian Amazon using skin tests. Am J Trop Med Hyg 2004,71:82–86. 8. Rios-Gonçalves AJ, Londero AT, Terra GM, Rozenbaum E, Abreu TF, Nogueira SA. Paracoccidioidomycosis in children in the state of Rio de Janeiro (Brazil). Geographic distribution and the study of a “reservarea”. Rev Inst Med Trop Sao Paulo 1998;40:11–13. 9. Calle D, Rosero DS, Orozco LC, Camargo D, Castañeda E, Restrepo A. Paracoccidioidomy- cosis in Colombia: an ecological study. Epidemiol Infect 2001;126:309–315. 10. Zavascki AP, Bienardt JC, Severo LC. Paracoccidioidomycosis in organ transplant recipient: case report. Rev Inst Med Trop Sao Paulo 2004;46:279–281. 11. Paniagoa AMM, Carli ACF, Aguiar JIA, et al. Paracoccidioidomycosis in patients with human immunodeficiency virus: review of 12 cases observed in an endemic region in Brazil. J Infect 2005;51:248–252. 12. Horre R, Schumacher G, Alpers K, et al. A case of imported paracoccidioidomycosis in a German legionnaire. Med Mycol 2002;40:213–216. 13. Mayr A, Kirchmair M, Rainer J, et al. Chronic paracoccidioidomycosis in a female patient in Austria. Eur J Clin Microbiol Infect Dis 2004;23:916–919. 14. Franco M, Bagagli E, Scapolio S, da Silva Lacaz C. A critical analysis of isolation of Paracoccidioides brasiliensis from soil. Med Mycol 2000,38:185–191. 15. McEwen JG, Bedoya V, Patiño MM, Salazar ME, Restrepo A. Experimental murine paracoccidioidomycosis induced by the inhalation of conidia. J Med Vet Mycol 1987;25:165–175. 16. Cock AM, Cano LE, Vélez D, Aristizabal BH, Trujillo J, Restrepo A. Fibrotic sequelae in pulmonary paracoccidioidomycosis: histopathological aspects in BALB/c mice infected 18. Paracoccidioidomycosis 341 with viable and non-viable Paracoccidioides brasiliensis propagule. Rev Inst Med Trop S Paulo 2000;42:59–66. 17. Borges-Walmsley MI, Chen D, Shu X, Walmsley AR. The pathobiology of Paracoccid- ioides brasiliensis. Trends Microbiol 2002:10:80–87. 18. Pereira RM, Bucaretchi F, Barison Ede M, Hessel G, Tresoldi AT. Paracoccidioidomycosis in children: clinical presentation, follow-up and outcome. Rev Inst Med Trop S Paulo 2004;46:127–131. 19. Benard G, Kavakama J, Mendes-Giannini MJ, Kono A, Duarte AJ, Shikanai-Yasuda MA. Contribution to the natural history of paracoccidioidomycosis: identification of the primary pulmonary infection in the severe acute form of the disease–a case report. Clin Infect Dis 2005;40:e1–e4. 20. Benard G, Duarte AJ. Paracoccidioidomycosis: a model for evaluation of the effects of human immunodeficiency virus infection on the natural history of endemic tropical diseases. Clin Infect Dis 2000;31:1032–1039. 21. Marques Mello L, Silva-Vergara ML, Rodrigues V. Patients with active infection with Paracoccidioides brasiliensis present a Th2 immune response characterized by high Interleukin-4 and Interleukin-5 production. Hum Immunol 2002;63:149–154. 22. Mamoni RL, Blotta MH. Kinetics of cytokines and chemokines gene expression distin- guishes Paracoccidioides brasiliensis infection from disease. Cytokine 2005;32:20–29. 23. Souza AR, Gesztesi JL, del Negro GM, et al. Anti-idiotypic antibodies in patients with different clinical forms of paracoccidioidomycosis. Clin Diagn Lab Immunol 2000;7: 175–181. 24. Coutinho ZF, da Silva D, Lazera M, et al. Paracoccidioidomycosis mortality in Brazil (1980–1995). Cad Saude Publica 2002;18:1441–1454. 25. dos Santos JW, Debiasi RB, Miletho JN, Bertolazi AN, Fagundes AL, Michel GT. Asymp- tomatic presentation of chronic pulmonary paracoccidioidomycosis: case report and review. Mycopathologia. 2004;157:53–57. 26. Fumari K, Kavakama J, Shikanai-Yasuda MA, et al. Chronic pulmonary paracoccidioidomy- cosis (South American blastomycosis): high resolution CT findings in 41 patients. Am J Roentgenol 1999;173:59–64. 27. Leal AM, Bellucci AD, Muglia VF, Lucchesi FR. Unique adrenal gland imaging features in Addison’s disease caused by paracoccidioidomycosis. Am J Roentgenol 2003;181: 1433–1434. 28. Almeida SM, Queiroz-Telles F, Teive HA, Ribeiro CE, Werneck LC. Central nervous system paracoccidioidomycosis: clinical features and laboratorial findings. J Infect 2004;48:193–198. 29. Godoy H, Reichart PA. Oral manifestations of paracoccidioidomycosis. Report of 21 cases from Argentina. Mycoses 2003;46:412–417. 30. San Blas G, Niño-Vega G. Paracoccidioides brasiliensis: virulence and host response. In: Cihlar RL, Caldernone RA, eds. Fungal pathogenesis: principles and clinical applications. New York, NY: Marcel Dekker, Inc.; 2000:206–226. 31. Do Valle ACF, Costa RLB, Fialho Monteiro PC, Von Helder J, Muniz MM, Zancopé- Oliveira RM. Interpretation and clinical correlation of serological tests in paracoccid- ioidomycosis. Med Mycol 2001;39:373–377. 32. Del Negro GMB, Pereira CN, Andrade HF, et al. Evaluation of tests for antibody response in the follow-up of patients with acute and chronic forms of paracoccidioidomycosis. J Med Microbiol 2000;49:37–46. 33. Marques da Silva SH, Colombo AL, Blotta MH, Lopes JD, Queiroz-Telles F, Pires de Camargo Z. Detection of circulating gp43 antigen in serum, cerebrospinal fluid, and bronchoalveolar lavage fluid of patients with paracoccidioidomycosis. J Clin Microbiol 2003;41:3675–3680. 342 Angela Restrepo, Angela M. Tobón, and Carlos A. Agudelo 34. Gómez BL, Figueroa JI, Hamilton AJ, et al. Antigenemia in patients with paracoccid- ioidomycosis: detection of the 87-kilodalton determinant during and after antifungal therapy. J Clin Microbiol 1998;36:3309–3316. 35. Tobon AM, Agudelo CA, Osorio ML, et al. Residual pulmonary abnormalities in adult patients with chronic paracoccidioidomycosis: prolonged follow-up after itraconazole therapy. Clin Infect Dis 2003:37:898–904. 36. Restrepo A, Benard G. Paracoccidioidomycosis. In: Feigin R, Cherry J, Demmler G, Kaplan S, eds. Textbook of pediatric infectious diseases. 5th ed. Philadelphia: WB Saunders, 2004:2592–2601. SUGGESTED READINGS Bethlem EP, Capone D, Maranhao B, Carvalho CR, Wanke B. Paracoccidoidiomycosis. Curr Opin Pulm Med 1999;5:319–325. Bicalho RN, Santo MF, de Aguiar MCF, Santos VR. Oral paracoccidioidomycosis: a retro- spective study of 62 Brazilian patients. Oral Dis 2001;7:56–60. de Camargo ZP, de Franco MF. Current knowledge on pathogenesis and immunodiagnosis of paracoccidioidomycosis. Rev Iberoam Micol 2000;17:41–48. Franco M, Lacaz CS, Restrepo-Moreno A, del Negro G, eds. Paracoccidioidomycosis. Boca Raton, FL: CRC Press, 1994:1–410. Hahn RC, Hamdan JS. Effects of amphotericin B and three azole derivatives on the lipids of yeast cells of Paracoccidioides brasiliensis. Antimicrob Agents Chemother 2000;44: 1997–2000. Kashino SS, Fazioli RA, Cafalli-Favati C, et al. Resistance to Paracoccidioides brasiliensis infection is linked to a preferential Th1 immune response, whereas susceptibility is associated with absence of IFN-gamma production. J Interferon Cytokine Res 2000;20:89–97. Manns BJ, Baylis BW, Urbanski SJ, Gibb AS, Rabin HR. Paracoccidioidomycosis: case report and review. Clin Infect Dis 1996;23:1026–1032. Tobon AM, Orozco B, Estrada S, et al. Paracoccidioidomycosis and AIDS: report of the first two Colombian cases. |
Rev Inst Med Trop Sao Paulo 1998;40:377–381. Yamaga LY, Benard G, Hironaka FH, et al. The role of gallium-67 scan in defining the extent of disease in an endemic deep mycosis, paracoccidioidomycosis: a predominant multifocal disease. Eur J Nucl Med Mol Imaging 2003;30:888–894. 19 Sporotrichosis Carol A. Kauffman, MD 1. INTRODUCTION Sporotrichosis is a subacute to chronic mycotic infection of skin and subcutaneous tissues. Most cases of sporotrichosis arise from direct inoculation of the organism from soil, vegetation, or wood into the subcutaneous tissues. Subsequent spread along the lymphatics draining the primary lesion is common, but hematogenous spread is rare. The organism is occasionally inhaled from the soil, causing pneumonia. Immunosup- pressed hosts can develop disseminated infection, and alcoholism and diabetes mellitus appear to be risk factors for locally invasive osteoarticular sporotrichosis. The etiologic agent is Sporothrix schenckii, named after Dr. Schenck, who described the first case in Baltimore in 1898. The disease has a worldwide distribution, but most cases currently are reported from the Americas and Japan. Early in the twentieth century, the most important work on the mycological and clinical aspects of sporotrichosis was carried out in France by de Beurmann and Gougerot. Their monograph is still timely in its description of the disease (1). In 1903, they also developed the first effective antifungal therapy, potassium iodide, which although now relegated to a secondary role, is still useful for the treatment of lymphocutaneous sporotrichosis. 2. ETIOLOGIC AGENT S. schenckii is a dimorphic fungus that exists as a mould in the environment and as a yeast in tissues. The dimorphism is temperature dependent. In the environment and in the laboratory, at 25º to 27ºC, S. schenckii is a mould with thin, septate, branching hyphae that have conidia that can be either dark or hyaline and that tend to arrange themselves along the hyphae in “bouquet-like” arrangements (Fig. 19.1). In the laboratory, on Sabouraud’s dextrose agar, growth of a white to cream-colored mould occurs within 1 to 2 weeks. The colony becomes brown or black and assumes a wrinkled appearance over the ensuing weeks (Fig. 19.2). In tissues and in vitro at 37ºC, S. schenckii assumes a yeastlike form. The yeasts are 4 to 6 μm in diameter and may show budding; they are classically described as From: Infectious Disease: Diagnosis and Treatment of Human Mycoses Edited by: D. R. Hospenthal and M. G. Rinaldi © Humana Press Inc., Totowa, NJ 343 344 Carol A. Kauffman Fig. 19.1. Microscopic view of the mould form of Sporothrix schenckii grown at 25ºC on Sabouraud dextrose agar. Note the thin septate hyphae with conidiophores that bear oval conidia that appear “bouquet-like.” (Courtesy of Dr. D. R. Hospenthal.) [Figure in color on CD-ROM]. Fig. 19.2. Colony of Sporothrix schenckii grown at 25ºC on malt extract agar. Initially cream-colored, the colony darkens over time. [Figure in color on CD-ROM]. being cigar-shaped although round and oval forms are also seen (Fig. 19.3). In the laboratory, growth of the yeast phase is accomplished by incubation at 35º to 37ºC using enriched media, such as brain heart infusion (BHI) agar. The colony morphology of S. schenckii in the yeast phase is usually off-white and wrinkled. Some strains of S. schenckii do not grow well at 37ºC but do grow at 35ºC. These strains are generally found in fixed cutaneous lesions that do not manifest lymphangitic spread (2). 19. Sporotrichosis 345 Fig. 19.3. Smear of an ulcerated lesion caused by Sporothrix schenckii showing a large number of oval and cigar-shaped yeasts, 4 to 6 μm in diameter. Courtesy of Dr. K. Reed. [Figure in color on CD-ROM]. 3. EPIDEMIOLOGY S. schenckii is found throughout the world. Most cases are reported from the Americas and Japan. In the environment, S. schenckii is found in sphagnum moss, decaying wood, vegetation, hay, and soil (3). For infection to occur, one must be exposed to an environmental source, and the organism must be inoculated through the skin. This can occur with motor vehicle accidents, hay baling, landscaping, and in developing countries, just the activities of daily living (4–7). The typical person who develops sporotrichosis is a healthy man whose occupation or hobby takes him into the out-of-doors. Classically, landscapers and gardeners develop sporotrichosis because they are exposed to contaminated materials and their activities frequently lead to nicks and cuts on their extremities, allowing the organism easy access. Zoonotic transmission also occurs from infected animals or from soil transferred from the nails of burrowing animals, such as armadillos (8,9). Cats develop ulcerated skin lesions due to sporotrichosis and many die of the infection. These ulcers are teeming with organisms and are highly infectious. The persons most often infected are veterinarians, children, and their household caretakers, who are usually women. Sporotrichosis also has occurred in laboratory workers who, in the course of handling infected animals or culture material, have inoculated themselves or splashed material into their eyes (10). Outbreaks of sporotrichosis are not uncommon. The largest outbreak involved more than 3000 South African gold miners who were inoculated with S. schenckii from contaminated timbers in the mines (3). Other outbreaks have been traced back to contaminated sphagnum moss packed around trees and bushes, contaminated seedlings used in topiary creations, and hay used for Halloween parties (4,11–13). Outbreaks 346 Carol A. Kauffman have also been traced back to transmission from cats. A large outbreak of cat-associated sporotrichosis affecting mainly housewives in Rio de Janeiro has been ongoing since 1998 (14). 4. PATHOGENESIS AND IMMUNOLOGY Infection with S. schenckii is almost always initiated when the mould that is present in the environment is inoculated into the skin, usually through minor trauma. Inhalation of the conidia of S. schenckii is the presumed method of transmission in the uncommon syndrome of pulmonary sporotrichosis. Virulence factors of S. schenckii, other than the ability to grow at 37ºC, have not been clarified, but likely include extracellular proteinases and melanin (15). The host response is comprised primarily of neutrophils, monocytes, and macrophages, cells able to ingest and kill the yeast phase of S. schenckii (16). Antibody appears unimportant in immunity, but cell-mediated immunity is crucial in containing infection with S. schenckii (17,18). This appears further supported by the clinical observation that S. schenckii causes disseminated infection in acquired immunodeficiency syndrome (AIDS) patients, a complication rarely noted in normal hosts (19). It now appears that tumor necrosis factor (TNF) also plays a role in immunity, given the recent clinical observation that treatment with an antagonist for this cytokine has led to disseminated sporotrichosis (20). 5. CLINICAL MANIFESTATIONS The usual manifestation of sporotrichosis is localized lymphocutaneous infection. Most patients who present with typical lymphocutaneous sporotrichosis are healthy hosts. Extensive disseminated cutaneous lesions and spread to other structures, including joints, meninges, lungs, and other organs almost always occur in those who have certain underlying illnesses. Alcoholism and diabetes mellitus are two consistent risk factors for more severe sporotrichosis (3). Chronic obstructive pulmonary disease is almost always present in patients who have pulmonary sporotrichosis, and dissemi- nated sporotrichosis is rare unless cell-mediated immunity is suppressed (Table 19.1). 5.1. Lymphocutaneous Sporotrichosis The first manifestation of infection generally occurs several days to weeks after cutaneous inoculation of the fungus when a papule appears at the site of inoculation. This primary lesion becomes nodular, and most will eventually ulcerate. Drainage from the lesion is minimal, is not grossly purulent, and has no odor. Pain is generally mild, and most patients have no systemic symptoms. Over the next few weeks, new nodules, that often ulcerate, appear proximal to the initial lesion along the lymphatic distribution (Fig. 19.4). The differential diagnosis for this form of sporotrichosis includes infection with M. marinum or another atypical mycobacterium, Leishmania species, and Nocardia brasiliensis (21). Rarely, other bacterial, fungal, and even viral infections cause a similar lymphocutaneous syndrome (22). Fixed cutaneous sporotrichosis is uncommon in North America, but common in South America (Fig. 19.5). Patients with this form of sporotrichosis manifest only a single lesion, often on the face, which can be verrucous or ulcerative (6). The lesion 19. Sporotrichosis 347 Table 19.1 Clinical manifestations of sporotrichosis Clinical syndrome Known risk factors Initiation of infection Lymphocutaneous None Local inoculation Fixed cutaneous None Local inoculation Osteoarticular Alcoholism, diabetes Local inoculation or hematogenous spread Pulmonary COPD, alcoholism Inhalation Meningitis AIDS Hematogenous spread Other focal disease (eye, None known Hematogenous breast, larynx, pericardium, spread or local epididymis, rectum, spleen, inoculation liver) Disseminated AIDS Hematogenous spread COPD, chronic obstructive pulmonary disease. Fig. 19.4. Typical skin lesions in lymphatic distribution seen in a patient who was a horticulturist and had inoculation of Sporothrix schenckii in the subcutaneous tissue of the wrist. (Reproduced with permission from C. Watanakunakorn, Clinical Infectious Diseases, University of Chicago Press, 1996.) [Figure in color on CD-ROM]. may regress and flare periodically, and can be present for years until it is treated. Pain and drainage are not prominent symptoms. 5.2. Pulmonary Sporotrichosis Pulmonary sporotrichosis is usually a subacute to chronic illness (23). The symptoms mimic those of reactivation tuberculosis. Patients have fever, night sweats, weight loss, and fatigue; dyspnea, cough, purulent sputum, and hemoptysis also occur frequently. Chest radiography shows unilateral or bilateral fibronodular or cavitary disease; the 348 Carol A. Kauffman Fig. 19.5. Fixed cutaneous skin lesion of sporotrichosis. In this form of the disease, lymphatic spread does not occur, and the lesion may remain for months to years until treated. (Courtesy of Dr. P. Pappas. Reproduced with permission from C.A. Kauffman, Clinical Infec- tious Diseases, University of Chicago Press, 1999.) [Figure in color on CD-ROM]. Fig. 19.6. Chest radiograph of a patient with pulmonary sporotrichosis. The patient was an alcoholic who also had diabetes mellitus and chronic obstructive pulmonary disease. 19. Sporotrichosis 349 upper lobes are preferentially involved (Fig. 19.6). Sporotrichosis must be differentiated from tuberculosis, chronic cavitary histoplasmosis or blastomycosis, and sarcoidosis. Some, but not all, patients with pulmonary sporotrichosis have disease elsewhere, especially in the skin and osteoarticular structures. 5.3. Osteoarticular Sporotrichosis Osteoarticular sporotrichosis is an uncommon manifestation of infection with S. schenckii that can occur after local inoculation, but more often arises from hematogenous spread. It is found most often in middle-aged men and appears to occur more frequently in alcoholics. Overlying cutaneous lesions may or may not be present, and one or more joints may be involved. Most commonly, the knees, elbows, wrists, and ankles are infected (24). Bone involvement usually occurs contiguous to an infected joint (Fig. 19.7). Bursitis and tenosynovitis, the latter presenting as nerve entrapment, also have been described (25). 5.4. Meningitis and Disseminated Infection Meningitis, a rare manifestation of sporotrichosis that occurs almost always in those with cellular immune defects, is usually chronic and must be differentiated from tuberculosis or cryptococcosis (26). Fever and headache are prominent symptoms, and the cerebrospinal fluid (CSF) findings are those of a lymphocytic meningitis with mild hypoglycorrhachia. Meningitis may be an isolated finding or a manifestation of widespread dissemination. Disseminated sporotrichosis is very uncommon, with most cases having been reported in patients with AIDS (27). S. schenckii has been reported Fig. 19.7. Elbow radiograph of a patient who had osteoarticular sporotrichosis manifested by infection of both elbows and one knee. There is destruction of the joint and adjacent osteomyelitis of the radius, ulna, and humerus. 350 Carol A. Kauffman very rarely to cause infection of eye, larynx, breast, pericardium, spleen, liver, bone marrow, lymph nodes, rectum, and epididymis (28). 6. DIAGNOSIS Culture of S. schenckii is the gold standard for establishing the diagnosis of sporotri- chosis. Biopsy or aspiration material from a cutaneous lesion should be sent to the laboratory for both culture and histopathology. Sputum, synovial fluid, or CSF should be obtained, when appropriate, for smear and culture. Material obtained for culture should be inoculated onto Sabouraud’s agar or blood agar and incubated at room temperature to allow growth of the mould phase of S. schenckii. Growth usually occurs within a week, but can take several weeks. The characteristic arrangement of conidia on the hyphae makes the diagnosis likely, but conversion to the yeast phase at 35º to 37ºC, which may take several weeks, allows definitive identification of the organism as S. schenckii. The histopathology of sporotrichosis reveals a mixed granulomatous and pyogenic inflammatory process. The organism is an oval to cigar-shaped yeast, 3 to 5 μm in diameter, and can exhibit multiple buds. However, it is difficult |
to visualize the organisms within tissues, even with the use of methenamine silver or periodic acid Schiff stains (see Fig. 3.10, Chapter 3). In some cases, a tissue reaction that may represent antigen–antibody complexes, called an asteroid body, can be seen. In an asteroid body, the basophilic yeast is surrounded by eosinophilic material radiating outward like spokes on a wheel. This is also known as the Splendore–Hoeppli phenomenon, which is not specific for sporotrichosis, but can be seen in various parasitic, fungal, and bacterial infections, and may be due to antigen–antibody complexes. Serology is not useful in the diagnosis of sporotrichosis. In the special case of sporotrichal meningitis, a latex agglutination assay and an enzyme immunoassay on CSF have been reported to be both sensitive and specific, although neither test is readily available (29). 7. TREATMENT In general, most patients who have sporotrichosis are treated with oral antifungal agents. Those patients who have disseminated infection, meningitis, or severe pulmonary involvement should be treated initially with intravenous amphotericin B. Guidelines for the management of the various forms of sporotrichosis have been updated by the Infectious Diseases Society of America (30). The suggestions that follow are modified from these Guidelines (Table 19.2). 7.1. Lymphocutaneous Sporotrichosis Itraconazole is the drug of choice for the treatment of this form of sporotrichosis (30–32). The dosage should be 200 mg/day, which is best given as the oral solution to achieve higher serum concentrations. If itraconazole capsules are used the patient cannot be taking any acid-inhibiting drugs, such as antacids, proton pump inhibitors, or H2 blockers, and should take the capsules with food to ensure adequate absorption. Treatment should continue until the lesions have resolved; this usually takes 3 to 6 months. Saturated solution of potassium iodide (SSKI) has been used successfully to treat lymphocutaneous sporotrichosis for decades. It still is not clear how SSKI inhibits 19. Sporotrichosis 351 Table 19.2 Treatment of sporotrichosis Clinical syndrome Primary therapy Alternate therapy Duration Lymphocutaneous Itraconazole 100-200 mg SSKI, titrated dose 3–6 months and cutaneous daily Fluconazole 400 mg/day Terbinafine 1000 mg/day Hyperthermia Pulmonary Itraconazole 400 mg/day Amphotericin B, 1–2 years 1–2 g total Osteoarticular Itraconazole 400 mg/day Amphotericin B, 1–2 yr 1–2 g total Meningitis Amphotericin B 1–2 g Itraconazole 400 See belowa total or equivalent mg/day dosage of lipid formulation Disseminated Amphotericin B 1–2 g Itraconazole 400 See belowb total or equivalent mg/day dosage of lipid formulation aTherapy for meningeal sporotrichosis has not been studied. Initial therapy should be with amphotericin B or a lipid formulation of amphotericin B; therapy can be changed to itraconazole after 4–6 weeks if the patient is doing well. The duration of treatment is unknown, and some patients may require life-long suppressive therapy. bIn patients with HIV infection and disseminated sporotrichosis, therapy can be changed to itraconazole after the patient’s condition has stabilized; life-long suppressive therapy with itraconazole is indicated in these patients. S. schenckii (33). The initial dose is 5 to 10 drops three times daily, increasing weekly to a maximum of 40 to 50 drops three times daily, as tolerated. Side effects are very common and include nausea, rash, metallic taste, fever, and salivary gland swelling. Several other options exist if the patient is unable to tolerate itraconazole or SSKI. Fluconazole at a dosage of 400 mg daily can be used (34). Voriconazole appears to be less active than itraconazole and should not be used to treat sporotrichosis (35). High doses of terbinafine (500 mg twice daily) appear to be effective for lymphocutaneous sporotrichosis (36). However, experience is limited, and this should be tried only in those who fail standard therapy. Local hyperthermia can be used to treat cutaneous sporotrichosis (37). A variety of different warming devices are available, but each must be used faithfully for months to effect improvement in cutaneous lesions (38). 7.2. Pulmonary Sporotrichosis Pulmonary sporotrichosis can be quite recalcitrant to therapy. If the patient is seriously ill, amphotericin B, preferably as a lipid formulation, should be used initially (23,30). The daily dosage of amphotericin B is 0.7 mg/kg daily, and for the 352 Carol A. Kauffman lipid formulations it is 3 to 5 mg/kg daily. When the patient is stable, therapy can be changed to oral itraconazole, 200 mg twice daily. The duration of therapy should be at least 1 year and perhaps longer for some patients. Surgical resection is an option for patients who have a single focal lesion that has not responded to medical management. 7.3. Osteoarticular Sporotrichosis This form of sporotrichosis, which is almost always chronic and not life-threatening, can be treated with an oral antifungal agent (24). Itraconazole is the agent of choice, and the dosage is 200 mg twice daily (30). Therapy should continue for 1 to 2 years. Although inferior to itraconazole in the treatment of this infection, if the patient cannot tolerate itraconazole, fluconazole at a dosage of 800 mg daily can be tried (34). Amphotericin B (0.7 mg/kg per day) or a lipid formulation of amphotericin B (3 to 5 mg/kg per day) is the only remaining option. Intraarticular, amphotericin B has been reported to be effective, but is not recommended (39). Even if cure occurs, joint function rarely is recovered. 7.4. Meningitis and Disseminated Infection Amphotericin B is the drug of choice for patients with these life-threatening forms of sporotrichosis (30). For meningitis, it is recommended that a lipid formulation of amphotericin B be given at a dosage of 5 mg/kg daily for 4–6 weeks. Itraconazole can be used after the patient has responded to amphotericin B if long-term suppressive therapy is needed. REFERENCES 1. de Beurmann L, Gougerot H. Les Sporotrichosis. Paris. Felix Alcan, 1912. 2. Kwon-Chung KJ. Comparison of isolates of Sporothrix schenckii obtained from fixed cutaneous lesions with isolates from other types of lesions. J Infect Dis 1979;139:424–431. 3. Kauffman CA. Sporotrichosis. Clin Infect Dis 1999;29:231–237. 4. Dixon DM, Salkin IF, Duncan RA, et al. Isolation and characterization of Sporothrix schenckii from clinical and environmental sources associated with the largest U.S. epidemic of sporotrichosis. J Clin Microbiol 1991;29:1106–1113. 5. Zhang X, Andrews JH. 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Systemic sporotrichosis treated with itraconazole. Clin Infect Dis 1993;17:210–217. 25. Atdjian M, Granda JL, Ingberg HO, Kaplan BL. Systemic sporotrichosis polytenosynovitis with median and ulnar nerve entrapment. JAMA 1980;243:1841–1842. 26. Donabedian H, O’Donnell E, Olszewski C, MacArthur RD, Budd N. Disseminated cutaneous and meningeal sporotrichosis in an AIDS patient. Diagn Microbiol Infect Dis 1994;18:111–115. 27. Al-Tawfiq JA, Wools KK. Disseminated sporotrichosis and Sporothrix schenckii fungemia as the initial presentation of human immunodeficiency virus infection. Clin Infect Dis 1998;26:1403–1406. 28. Wilson DE, Mann JJ, Bennett JE, Utz JP. Clinical features of extracutaneous sporotrichosis. Medicine (Baltimore) 1967;46:265–279. 29. Scott EN, Kaufman L, Brown AC, Muchmore HG. Serologic studies in the diagnosis and management of meningitis due to Sporothrix schenckii. N Engl J Med 1987;317:935–940. 30. Kauffman CA, Bustamante B, Chapman SW, Pappas PG. 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Comparative evaluation of the efficacy and safety of two doses of terbinafine (500 and 1000 mg day−1) in the treatment of cutaneous or lymphocutaneous sporotrichosis. Mycoses 2003;47:62–68. 37. Hiruma M, Kagawa S. The effects of heat on Sporothrix schenckii in vitro and in vivo. Mycopathologia 1998;84:21–30. 38. Hiruma M, Kawada A, Noguchi H, Ishibashi A, Conti Diaz IA. Hyperthermic treatment of sporotrichosis: experimental use of infrared and far infrared rays. Mycoses 1992;35:293–299. 39. Downs NJ, Hinthorn DR, Mhatre VR, Liu C. Intra-articular amphotericin B treatment of Sporothrix schenckii arthritis. Arch Intern Med 1989;149:954–955 SUGGESTED READINGS Kauffman CA. Old and new therapies for sporotrichosis. Clin Infect Dis 1995;21:981–985. Kauffman CA, Bustamante B, Chapman SW, Pappas PG. Clinical practice guidelines for the management of sporotichosis: 2007 update by IDSA. Clin Infect Dis (In press). Kwon-Chung KJ, Bennett JE. Sporotrichosis. In: Medical Mycology. Philadelphia: Lea & Febiger, 1992:707–729. 20 Dermatophytosis (Tinea) and Other Superficial Fungal Infections Aditya K. Gupta, MD, PhD and Elizabeth A. Cooper, HBSc 1. INTRODUCTION Superficial dermatophyte infection has been reported under a variety of different terminologies since the early days of recorded human civilization (1). However, it has only been during the past 200 years with the development of modern science that the contagious nature of the disease could be related to the presence of fungal organisms (1). The current taxonomy of dermatophytes used today, dividing organisms into Trichophyton, Microsporum, and Epidermophyton species, was not developed until 1934 (1). Topical preparations have been the historic method of treatment for dermato- phyte infections. The first effective oral medication for dermatophytes, griseofulvin, was not developed until 1958 (1). Thus, although the term “tinea” used for dermato- phyte infection has ancient roots (1), the current standards of medical treatment for dermatophyte infection are a recent development, and the field continues to develop as more fungal knowledge and more effective therapies become available. Dermatophytes require keratin for growth and therefore infect hair, nails, and super- ficial skin, with clinical manifestations named for the area affected: tinea capitis (scalp); tinea corporis (body); tinea cruris (groin); tinea pedis (feet); tinea manuum (hands); tinea |
barbae (affecting the beard area, in males); tinea faciei (face); and tinea unguium (nails) (2). Tinea infections have alternately been called “ringworm,” because of the lesions that present as a circular or oval clearing surrounded by a red, scaly, elevated border (“ring”). Tinea unguium is the subset of onychomycosis infections caused by dermatophytes, as opposed to nail infections caused by Candida or nondermatophyte moulds. Besides the dermatophytoses, superficial infections may also result from infection with other fungi, including the Malassezia species of yeast. Malassezia feed on lipids found in areas where sebaceous gland activity is highest and in individuals with high levels of sebaceous secretions. Although first recognized as a pathogen in 1846, laboratory culture was not successful until it was recognized that the organisms had a lipid requirement, in 1927 (3). Initially only two species were described, under the genus From: Infectious Disease: Diagnosis and Treatment of Human Mycoses Edited by: D. R. Hospenthal and M. G. Rinaldi © Humana Press Inc., Totowa, NJ 355 356 Aditya K. Gupta and Elizabeth A. Cooper name Pityrosporum, and only three species were recognized as of 1970 (3). Difficulty in identification was also complicated by the existence of both yeast and mycelial forms; conversion between forms was not induced in the laboratory until 1977. Genetic research in the 1990s confirmed at least seven species of Malassezia existed, and more have been discovered since. Because of the issues of species identification, definitive understanding of Malassezia infection has been difficult. Malassezia is known to cause pityriasis (tinea) versicolor and is associated with seborrheic dermatitis, and thus these superficial conditions will be included here along with the dermatophyte infections. Other rare superficial fungal infections, including those secondary to Candida (Chapter 7), white piedra (caused by Trichosporon, Chapter 8), black piedra, and tinea nigra (Chapter 11), are discussed elsewhere in this text, as are the nonsuperficial infections caused by Malassezia (Chapter 8). 2. ETIOLOGIC AGENTS Three fungal genera cause tinea infections: Microsporum, Trichophyton, and more rarely, Epidermophyton (2). Species may be grouped by their source of human infection, other humans (anthropophilic), animals (zoophilic), or less commonly, soil (geophilic). Infections may be transmitted between humans or, much more rarely, from animals or soil to humans. The major causative species differ geographically and may change in prevalence over time owing to population movements from immigration or travel. Anthropophilic dermatophytes are the most frequent causes of onychomycosis and other superficial dermatophytoses, and the most frequently seen agents of infection are T. rubrum and T. mentagrophytes. T. tonsurans is currently the most frequent cause of tinea capitis in North America. M. canis is a zoophilic organism frequently picked up by humans from contact with animals such as dogs and cats. The Malassezia yeast species are associated with the superficial fungal infec- tions pityriasis (tinea) versicolor (PV) and seborrheic dermatitis (SD). The currently recognized species are M. furfur, M. pachydermatis, M. sympodialis, M. globosa, M. slooffiae, M. restricta, and M. obtusa (4). M. pachydermatis is typically associated with animal infection rather than human infection (3). Up to five new species have been described in the recent literature: M. dermatis, M. equi, M. japonica, M. yamatoensis, and M. nana (4). The most common Malassezia species contributing to PV lesions are M. globosa (50% to 60%), M. sympodialis (3% to 59%), M. furfur, and M. slooffiae (1% to 10%) (5). 3. EPIDEMIOLOGY Dermatophytosis is a frequent diagnosis, and it has been estimated that the risk of acquiring such infection is 10% to 20% in an individual’s lifetime (6). While some types of infections are well studied (tinea capitis, onychomycosis), other types are less well defined. Overall rates of dermatophyte infection in subjects seeking outpatient treatment in the United States have been measured using the National Ambulatory Medical Care Survey (NAMCS) from 1990 to 1994 (7). This survey modeled an estimated 21.6 million physician office visits for fungal infections during this period, breaking down into types of infection as follows: tinea corporis—27.2%, 20. Dermatophytosis and Other Superficial Fungal Infections 357 tinea cruris—16.9%, tinea pedis—16.7%, tinea unguium—15.6%, tinea of hair and beard—6.9%, and tinea manuum—1.0%. 3.1. Tinea Pedis/Manuum Tinea pedis is estimated to affect 10% of the world population (8). Infections are more frequent in tropical climates and may also be associated with use of occlusive footwear (8). Males are more often affected than females for both tinea pedis and tinea manuum, with most infections affecting the web space between the fourth and fifth toes (8). Children do not often develop tinea pedis. Patients with atopic dermatitis or immunosuppressive disorders may be predisposed to developing tinea pedis. Predis- posing factors for tinea manuum include manual work that results in repeated trauma to the hands, hyperhidrosis, and the frequent use of alkaline soaps. 3.2. Tinea Corporis/Cruris Tinea corporis and tinea cruris are found commonly, with worldwide distribution (9). Little data on prevalence in North America has been published, but tinea corporis was found to be the most common dermatophytosis for which patients sought treatment during the National Ambulatory Medical Care Survey from 1990 to 1994 (27.2% of all dermatophytoses, with an estimated 2.3 million physician visits made) (7). A subset of tinea corporis affecting only the nonbearded regions of the face, tinea faciei, makes up 3% to 4% of tinea corporis cases, and is more frequently seen in warm, humid climates (10). 3.3. Tinea Capitis In North America, the genus Trichophyton, particularly T. tonsurans, is the predom- inant cause of tinea capitis infection (1). In Western Europe, M. canis and T. violaceum are the most common pathogens of tinea capitis; T. tonsurans is dominant in the Caribbean and South America; M. canis, T. mentagrophytes, and T. violaceum dominate in the Middle East (1). Tinea capitis is most prevalent in children older than 6 months of age and before puberty (1). African Americans develop tinea capitis at much higher frequencies than the general US population (1). Trichophyton species affect males and females equally, although M. audouinii and M. canis affect males more than females (1). Infection spread may increase in conditions of overcrowding, poverty, and poor hygiene. 3.4. Onychomycosis Onychomycosis has an estimated prevalence of 6.5% to 12.8% in North America, accounting for up to 50% of all nail disease (11,12). It is more common in males than females, and in people older than 60 years of age. Other risk factors include trauma to the nail, diabetes, peripheral arterial disease, and immunocompromised status (12). Tinea pedis may be present in patients with toenail onychomycosis (12). 3.5. Pityriasis Versicolor Pityriasis versicolor (PV) has worldwide distribution. Prevalence in tropical climates has been reported at 30% to 40%, compared to 1% to 4% in temperate climates (3,13). 358 Aditya K. Gupta and Elizabeth A. Cooper PV does not typically affect prepubescent children, but is more frequent in adults when sebaceous gland activity is most active (5,13). Equal prevalence between the sexes has been noted (5). 3.6. Seborrheic Dermatitis Seborrheic dermatitis (SD) is a chronic, recurrent disorder affecting between 1% and 5% of immunocompetent adults (3,13,14). Typically referred to as dandruff, the mild form affects a large proportion of the North American population, but reported numbers are likely underestimated as people to do not tend to seek medical advice for dandruff. Males are more frequently affected than females, and the disease is more severe in winter, improving with summer sun exposure (15). SD chiefly affects adolescents, young adults, and adults older than age 50 (16). Incidence may increase in immuno- compromised populations such as in persons infected with human immunodeficiency virus (HIV), in whom estimates of incidence of SD are as high as 83% (3). 4. PATHOGENESIS AND IMMUNOLOGY The dermatophytes colonize keratinized tissue of the stratum corneum; invasion by anthropophilic species usually result in less inflammation than that of zoophilic or geophilic species (17). The epidermis functions as a barrier to microorganisms, and commensal flora may also help reduce infection by pathogens (3). Entry into the stratum corneum may result from trauma to the skin or some other breach of the skin barrier. Excessive sweating and occlusive clothing/footwear aid in providing a warm, moist environment conducive to tinea infection. Infection may be transferred from one area of the body to another. Infection may also be transmitted between individuals by direct or indirect contact with scales containing fungal arthroconidia from infected individuals, such as seen in individuals participating in contact sports including wrestling and rugby (9). Fungicidal proteins are present in the epidermis, and some skin lipids in the scalp and hair are fungicidal to certain, but not all, dermatophyte species (3). Dermatophyte glycopeptides prompt development of delayed hypersensitivity, although patients with inflammatory infection are more likely to demonstrate such a reaction than patients with noninflammatory chronic infection (18). Dermatophytes and other microorganisms can activate the alternate pathway of the complement cascade of immune response, causing production of molecules that prompt the chemotactic activity of neutrophils into the skin (3). Immunoglobulins are secreted onto the skin surface via sweat, and commensal organisms including Malassezia species have been found to be coated with such immunoglobulins (3). Malassezia organisms are a normal part of human commensal skin flora, found particularly in sebaceous skin such as the chest, back, and head, and in the yeast form rather than the mycelial form (3). Malassezia species vary in the antigens presented, and can alter their expressed antigens throughout the growth cycle (3). The ability of Malassezia to elicit activities of the human immune system is not clear (3). People and animals, though uninfected, may still be asymptomatic pathogen carriers. Fomites also play a significant role in transmission. Autoinoculation may also occur, for example, tinea pedis spreading to tinea cruris, tinea capitis to tinea corporis, or 20. Dermatophytosis and Other Superficial Fungal Infections 359 onychomycosis to tinea pedis (19). High levels of perspiration may be predisposing to infection, as fungal arthroconidia persist to a greater extent on the scalp with higher levels of oils (9). 5. CLINICAL MANIFESTATIONS Clinical presentations and differential diagnoses for the various superficial infections are summarized in Table 20.1. Table 20.1 Clinical presentations and differential diagnoses of common dermatophyte and superficial fungal infections Condition Presentation Differential diagnosis Tinea pedis Interdigital: Scaling, fissuring, Candidiasis, erythrasma, bacterial maceration, erosions, hyperhidrosis, infection, psoriasis, contact pruritus, odor dermatitis, dyshidrotic eczema, Moccasin: Fine silvery scales with Reiter’s syndrome underlying pink or red skin on soles, heels, sides of feet Vesicobullous: Inflammatory vesicular or bullous lesions, particularly at in-step Tinea manuum Dry, scaly, hyperkeratotic skin Contact dermatitis, atopic particularly of the palmar area, dermatitis, pompholyx, minimal erythema psoriasis, lamellar dyshidrosis Tinea corporis Annular erythematous plaques with Impetigo, nummular dermatitis, raised leading edges and scaling, secondary or tertiary syphilis, over glabrous skin of trunk; may be psoriasis, lichen planus, central clearing seborrheic dermatitis, pityriasis rosea, pityriasis rubra pilaris, candidal intertrigo, atopic dermatitis, cutaneous lupus, pityriasis versicolor Tinea cruris Annular erythematous plaques with Psoriasis, seborrheic dermatitis, raised leading edges and scaling, candidiasis, erythrasma, lichen over pubic area, perineal and simplex chronicus, Darier’s perianal skin, typically not affecting disease, pemphigus vegetans the scrotum or labia majora Tinea capitis Noninflammatory: Erythematous Seborrheicdermatitis, psoriasis, papules around hair shaft spreading atopic dermatitis, tinea out with fine scaling in noticeable amiantacia, alopecia patches and partial or complete areata, trichotillomania, alopecia lupus erythematosus, lichen planopilaris, traction folliculitis, bacterial pyoderma (Continued) 360 Aditya K. Gupta and Elizabeth A. Cooper Table 20.1 (Continued) Condition Presentation Differential diagnosis Black dot: Noticeable black dots where hair breakage at scalp level occurs, scaling with little inflammation (particularly with T. tonsurans or T. violaceum) Inflammatory: Kerion with pustules, loose hair, discharge of pus Favic: Large yellow crusts on the scalp Onychomycosis Distal lateral subungual (DLSO): Psoriasis, chronic onycholysis, Infection at the distal end of nail chronic paronychia, trauma, plate; discoloration and thickening hemorrhage, onychogryphosis, of nail plate, onycholysis, subungual lichen planus, alopecia debris areata, subungual malignant Superficial white (SWO): White spots melanoma, subungual or patches on the surface of the nail squamous cell carcinoma plate Proximal subungual (PSO): Infection of the proximal nail fold, and extending distally, typically whitish in color Endonyx: Milky white discoloration of the nail plate without hyperkeratosis, onycholysis; may show lamellar splitting of the nail plate (typically caused by T. soudanense or T. violaceum) Ptyriasis Well-defined, hyperpigmented or Vitiligo, chloasma, tinea versicolor hypopigmented lesions of areas with corporis, pityriasis rotunda, high concentrations of sebaceous erythrasma glands such |
as scalp, chest, back, upper arms and face; showing fine scaling in most cases (caused by Malassezia species) Seborrheic Red, flaky, greasy-looking patches of Psoriasis, atopic dermatitis, dermatitis skin on scalp, nasolabial folds, tinea capitis, rosacea, lupus eyebrows and ears: “dandruff’ of the erythematosus scalp; may also affect groin, axillae, anterior chest; pruritus, irritation may be associated (associated with Malassezia infection) 20. Dermatophytosis and Other Superficial Fungal Infections 361 5.1. Tinea Pedis/Manuum Also known as “athlete’s foot,” there are three common presentations recognized in tinea pedis: interdigital, moccasin, and vesicobullous (8). Interdigital is the most common presentation, and typically infects the toe webs, particularly between the fourth and fifth toes (Fig 20.1) (8). Interdigital infection may show fissuring, scaling, maceration, and erosions. Hyperhidrosis, pruritus, and foul odor may also be present. Dermatophytosis simplex is an uncomplicated form of interdigital tinea pedis, contrasted with dermatophytosis complex which is associated with concomitant bacterial infection showing inflammation, maceration and odor, and may be facilitated by breakdown of the skin in preliminary infection stages (8). Moccasin tinea pedis manifests as fine silvery scales with underlying pink to red skin, on the soles, heels and sides of feet (Fig. 20.2) (8). More severe cases may show cracked, inflamed skin, erythema, and odor. This infection is most commonly produced by T. rubrum. Fig. 20.1. Interdigital tinea pedis. [Figure in color on CD-ROM]. 362 Aditya K. Gupta and Elizabeth A. Cooper Fig. 20.2. Moccasin tinea pedis, with close-up of fine scaling. [Figure in color on CD-ROM]. Vesicobullous tinea pedis is the least common type, and shows as acute and highly inflammatory vesicular or bullous lesions, typically at the in-step, but inflammation may spread over the whole sole (8). This type of infection is associated with T. mentagrophytes infection. Tinea manuum is a rare form that primarily affects the palmar areas of the hands, and presents as chronic, dry, scaly, hyperkeratotic skin with minimal erythema (8). Infections are most frequently caused by T. rubrum. Tinea manuum may accompany tinea pedis or onychomycosis, and a two feet–one hand syndrome has been noted to occur (20). This syndrome consists of development of tinea manuum from excoriation of infected soles (tinea pedis) and/or toenails (onychomycosis). Typically one hand is dominant in the excoriation, leading to infection in that hand which may also lead to fingernail onychomycosis. 20. Dermatophytosis and Other Superficial Fungal Infections 363 5.2. Tinea Corporis/Cruris Tinea corporis is a superficial dermatophyte infection of the glabrous skin, excluding the scalp, beard, face, hands, feet, and groin (Fig. 20.3) (9). It is more common in men than in women, and is also common in children (9). Tinea faciei is a subset of tinea corporis affecting only the facial area, excluding the beard region (10). Also known as “jock itch,” tinea cruris is a dermatophyte infection of the genitalia, pubic area, perineal skin, and perianal skin (Fig. 20.4). The scrotum and labia majora are typically not affected. Infection is more common in men than in women (9). Tinea cruris and tinea corporis present as annular erythematous plaques with raised leading edges and scaling; central clearing of the lesion may be noticed, but nodules may remain present throughout the lesion (9). Infection is typically associated with pruritus. Infection may also present as an erythematous papule or series of vesicles. Significant inflammation may result from infection with zoophilic organisms such as T. verrucosum, producing large pustular lesions or a kerion or associated with formation of frank bullae causing tinea corporis bullosa (9). Infection may also spread down the hair shaft into the dermis producing inflammatory papules and pustules (9). Tinea faciei has a broad range of presentations. Infection may begin as flat, scaly macules that develop into a raised border advancing outward in all direction, with or without development of papules, vesicles, and crusts (Fig. 20.5) (10). Lesions may not be annular. The central area may become either hypopigmented, or hyperpigmented. Lesions may occur singly or in multiple patches, and may extend to other parts of the body (10). Tinea imbricata or Tokelau is a chronic infection of glabrous skin caused by the anthropophilic dermatophyte T. concentricum , presenting as distinctive scaly, concentric, overlapping plaques that may cover large areas of the body (21). Lesions typically begin on the face, and spread to large areas of the body. The infection is endemic to Polynesia, Central and South America, particularly in rural areas. Fig. 20.3. Tinea corporis. [Figure in color on CD-ROM]. 364 Aditya K. Gupta and Elizabeth A. Cooper Fig. 20.4. Tinea cruris. [Figure in color on CD-ROM]. 5.3. Tinea Capitis Infection of the scalp involves hyphal proliferation in the stratum corneum that extends into the hair follicle orifice and hair shaft (1). Exposure of the scalp to the inoculum from an infected individual, animal, or other source of organism may result in infection where trauma or some other factor (tight braiding exposing the scalp, application of hair oil that facilitates adherence of arthroconidia) aids arthroconidia implantation (1). Inflammatory tinea capitis is associated with zoophilic or geophilic species such as M. canis or M. gypseum, but may also occur with T. verrucosum, T. schoenleinii, T. tonsurans, and M. audouinii (1). A kerion may be produced: an oozing Fig. 20.5. Tinea faciei. [Figure in color on CD-ROM]. 20. Dermatophytosis and Other Superficial Fungal Infections 365 mass with pustules, loose hair and discharge of pus. Signs of systemic illness may be present, including fever and lymphadenopathy. Noninflammatory or epidemic tinea capitis may begin as a small erythematous papule around the hair shaft which spreads outwards, developing fine scaling in noticeable patches (Fig. 20.6) (1). Partial or complete alopecia may result where brittle hair breaks off a few millimeters from the scalp. Affected hair may appear grey due to coating with arthroconidia. Non-inflammatory infection is associated with M. audouinii and M. ferrugineum; however T. tonsurans and M. canis may infrequently cause noninflammatory infection. Black dot tinea capitis is most frequently associated with T. tonsurans or T. violaceum infection, and results from hair breakage at the level of the scalp, showing diseased hair in the follicle as a “black dot” (1). Scaling is typically present with little inflammation, although inflammatory kerions are possible (1). Favic infection is rare in North America and most often caused by T. schoenleinii, leading to large yellow crusts (1,22). 5.4. Onychomycosis The most common presentation of onychomycosis is distal lateral subungual onychomycosis (DLSO), which presents as a nail with discoloration and varying degrees of hyperkeratosis, onycholysis (separation of nail from nail bed), subungual Fig. 20.6. Noninflammatory tinea capitis. [Figure in color on CD-ROM]. 366 Aditya K. Gupta and Elizabeth A. Cooper Fig. 20.7. Top left and right: onychomycosis; bottom left:onycholysis; bottom right: psoriasis. [Figure in color on CD-ROM]. debris, and thickening (23,24) (Fig. 20.7). DLSO begins at the distal edge of the nail (hyponychium) and travels proximally through the stratum corneum of the nail bed and involving the nail plate (Fig. 20.8). The most severe grades of DLSO may progress to total dystrophic onychomycosis (TDO), where the nail plate becomes friable and crumbles away to varying degree, leaving exposed thickened nail bed and subungual debris. Within the spectrum of DLSO presentations, infections may spread relatively evenly across the nail plate. Alternatively, infection may penetrate only the lateral edge or edges of the nail plate (lateral infection), or may penetrate longitudinally in a “spike” formation (25). Infection may also develop as a dermatophytoma, where debris and fungus clump densely to form a thick, hyperkeratotic mass (25). These presentations may not respond as well to therapy as a more diffuse DLSO presentation. Infrequently, infection may present as superficial white onychomycosis (SWO), proximal subungual onychomycosis (PSO), or endonyx onychomycosis (23,24). SWO involves infection of the superficial nail plate, showing patches of white discoloration on the nail surface. Multiple nails may be affected, and varying degrees of nail plate area may be covered (Fig. 20.9). The rare presentation PSO results from invasion of the proximal nail fold and extending distally along the underside of the nail plate as a white patch of infection (Fig. 20.10). PSO is more common in HIV-infected persons than in the healthy population, and may serve as a marker for degree of immunodeficiency (26). Endonyx infection presents as a diffuse milky white discoloration of the nail in 20. Dermatophytosis and Other Superficial Fungal Infections 367 Fig. 20.8. Routes of infection causing the typical presentations of onychomycosis. [Figure in color on CD-ROM]. the absence of hyperkeratosis and onycholysis, with nail plate surface and thickness remaining normal (23). Alternately, the nail may show lamellar splitting of the nail plate, with invasion of superficial and deeper layers of the nail plate, without excessive thickening or discoloration of the nail unit (24). Endonyx infections are usually caused by T. soudanense or T. violaceum (23,24). 5.5. Pityriasis Versicolor Pityriasis versicolor presents as well-defined lesions, with a fine scale from desqua- mation, that are either hyperpigmented (pink, tan, dark brown, or black) or hypopig- mented (white, or lighter than normal skin). Hypopigmentation may not always exhibit Fig. 20.9. Typical presentation of superficial white onychomycosis (SWO) on the third toenail with DLSO presented in the great toenail. [Figure in color on CD-ROM]. 368 Aditya K. Gupta and Elizabeth A. Cooper Fig. 20.10. Proximal subungual onychomycosis (PSO) developed during occlusion by the neighboring digit. [Figure in color on CD-ROM]. scaling (Fig. 20.11) (27). It is a cosmetic disorder that is largely asymptomatic, with the exception of possible mild pruritus (5). There is a large variation in lesion size from macules to entire trunk coverage (28). Lesions are predominant in areas with a high number of sebaceous glands such as the scalp, chest, and back, as well as upper arms and face (5,27). Facial lesions are more common in children than adults (3). Hypopig- mentation may occur independently or following the hyperpigmented stage (27). 5.6. Seborrheic Dermatitis Seborrheic dermatitis presents as red, flaking, greasy-looking patches of skin on the scalp and hair-bearing areas of the face such as the nasolabial folds, eyebrows, and ears (Fig. 20.12) (15). SD is a more severe form of dandruff involving body sites of abundant sebaceous gland activity. It may occur on the groin, axillae, anterior chest, or inside/behind the ears (15,29). Dandruff can appear as loosely adherent white or gray flakes, while severe SD may be thick, oily, yellow-brown crusts. Pruritus, irritation, and 20. Dermatophytosis and Other Superficial Fungal Infections 369 Fig. 20.11. Tinea versicolor showing hyperpigmentated lesions (upper photos) and hypopigmented lesions (lower photo). [Figure in color on CD-ROM]. Fig. 20.12. Seborrheic dermatitis—severe presentation. [Figure in color on CD-ROM]. a tight, dry feeling may be associated with the afflicted area (30). Some cases present with little erythema, while others present as a sore scalp with occasional pustules (14). 6. DIAGNOSIS Definitive diagnosis of tineas require confirmation of dermatophyte organisms by microscopic examination and laboratory fungal culture methods. For skin infections, scrapings or swabs can be taken from the leading edge of a lesion (1,29). Nail clippings and subungual debris can similarly be investigated. Potassium hydroxide (KOH) is 370 Aditya K. Gupta and Elizabeth A. Cooper added to the samples to dissociate hyphae from keratinocytes, and the samples are examined by microscopy (1,31). Microscopy can indicate the presence of dermato- phytes via the presence of hyphae, however dermatophyte species cannot be distin- guished by microscopy, thus cultures are required to confirm the causative species. Malassezia species may show a distinctive “spaghetti and meatball” form (mixture of yeasts and short hyphae) on microscopic examination (3). Microscopic examination of hairs may help differentiate types of tinea capitis infection: ectothrix infection can be distinguished from endothrix infection where arthroconidia appear as chains of the surface of the hair shaft or as a mosaic sheath around the hair (1). Inspection under the Wood’s light (filtered ultraviolet light with a peak of 365 nm) may aid in diagnosis (1). Ectothrix infections with M. audouinii, M. canis, and M. ferrugineum show bright green fluorescence under the Wood’s light. T. schoenleinii shows dull green fluorescence. T. tonsurans, however, does not fluoresce, and the utility of the Wood’s lamp for diagnosis is currently limited in countries where this is the major infecting agent. 7. TREATMENT Treatment for superficial fungal infections varies widely, although the antifungal medications used typically belong to either the azole or allylamines drug classes (Table 20.2). Topical use of |
these drugs can be effective in infections of limited area. Some of the newer topical antifungals exhibit anti-inflammatory and antibac- terial activities as well as antifungal activity, and may therefore be a suitable choice for infections showing inflammation or concomitant bacterial infection. Five main systemic agents are available: terbinafine, itraconazole, fluconazole, griseofulvin, and ketoconazole. Oral formulations may be required for infections of more severe or more widespread presentation. Oral therapy may alternately be preferred for immunocom- promised patients, in whom prompt, thorough resolution of infection is mandatory. Oral therapy may also be a more convenient choice for the patient than daily topical therapy applications. Safety of therapy is more of a concern for oral treatment than topical treatment, as serum absorption tends to be minimal with topical drug use for dermatophy- tosis. With topical agents, most adverse events are skin reactions at the application site that are mild and transient. Owing to the large number of topical medications, adverse events will not be discussed here. Adverse events with oral medications are discussed where relevant. The oral antifungal medications are commonly associated with the potential for severe hepatic toxicity, rare serious skin events such as Stevens– Johnson syndrome, and possible drug–drug interactions due to metabolism through the cytochrome P-450 system. Current country-specific prescribing information for any dermatophytosis medication should be consulted before providing any medication. Relapse of therapy has been noted with most types of dermatophyte infection. Patients must be encouraged to complete a full treatment cycle, as infection can be present without visible symptoms. Treatment must include microscopic exami- nation and culture to confirm elimination of the pathogen. Infection transmission from symptom-free carriers such as family members and pets may need to be controlled with adjunct therapies and techniques; fomites such as hats and combs must also be treated. Table 20.2 Treatment options available for dermatophytoses and other superficial fungal infectionsa Terbinafine Itraconazole Fluconazole Ketoconazole Griseofulvin Topicals Tinea pedis/ Creamb: Apply Oral: 200 mg Oral: 150 mg 2% Creamb: Microsizeb: Cipclopiro 0.77% Clotrimazoleb manuuma twice daily × bid × 1 once weekly Apply once 1 g/day cream or gelb: Miconazoleb 1–4 weeks week × 2–6 weeks daily × 6 Ultramicrosize: Twice daily × 4 Butenafineb 1% Solutionb: weeks 660 or 750 weeks Econazoleb Apply twice Oralb: 200–400 mg/day × 4–8 Antifungal daily × 1 mg/day × >4 weeks powder for week weeks prevention Oral: 250 mg/day × 2 weeks Tinea Creamb: Apply Oral: Oral:150–300 2% Creamb: Microsizeb: 500 Cipclopiro 0.77% Clotrimazole corporis/cruris twice daily × 200 mg/day mg once Apply once mg/day cream or gelb: Miconazole 1–4 weeks × 1 week weekly × 2–4 daily × 2 Ultramicrosize: Twice daily × 4 Butenafine 1% Solutionb: weeks weeks 330–375 weeks Econazole Apply twice Oralb: 200–400 mg/day × 2–4 daily × mg/day × 4 weeks 1 week weeks Oral: 250 mg/day × 2–4 weeks Tinea capitis See Table 20.3 See Table 20.3 See Table 20.3 Only effective See Table 20.3 Selenium sulfide Corticosteroid for pediatric for pediatric for pediatric against for pediatric shampoo 1% as adjunct dosing dosing dosing Trichophyton. dosing adjunct therapy therapy for 2% shampoo severe used as inflammatory adjunct varieties therapy (Continued) Table 20.2 (Continued) Terbinafine Itraconazole Fluconazole Ketoconazole Griseofulvin Topicals Onychomycosis Oralb: 250 Oralb: Continuous Oral: 150mg Oral: 200–400 Microsizeb 1 † Cipclopiro 8% Amorolfine mg/day therapyb: 200 once weekly mg/day × 6 g/day lacquer: once 5% lacquer Toenail: 12 mg/day × 12 Toenail: 9–15 months-Not Ultrami- daily × 48 – Not weeks weeks Pulse months recommended due crosize weeks approved in Fingernail: therapy: 200 mg Fingernail: to hepatotoxicity 660 or 750 North 6 weeks bid for 1 week, 4–9 months risk mg/day× America followed by 3 4–12 itraconazole–free months weeks Toenails: 3 pulses Fingernailsb only: 2 pulses Pityriasis 1% Solutionb: Oral: 200 mg/day × 2% Shampoo: 5 †2% Creamb: Apply Not effective Cipclopirob Clotrimazoleb versicolor Apply twice 5–7 days days once daily × 0.77% cream Miconazoleb daily × 1 Oral: 300 mg 2 weeks Selenium Butenafineb week once weekly Oral: 200 mg/day × sulfideb Econazoleb Oral: not × 2 weeks 2 weeks, 10 days effective or 5 days; 400 mg per week × 2 weeks; 400 mg per day × 3 days; 3 doses of 400 mg given every 12 hours Seborrheic 1% Solution: Oral: 200 2% Shampoo: 2% Creamb: Apply Not Cipclopiroxb: 0.77% Zinc dermatitis Once daily × 4 mg/day × 1 Twice a week twice daily × effective cream, shampoo, pyrithioneb weeks week × 4 weeks 4 weeks or gel Metronidazole Oral: 250 mg/day Shampoob: Twice Selenium sulfideb Bifonazole × 4 weeks a week × 4 weeks Coal tarb Miconazole Oralb: 200–400 Hydrocortisoneb mg/day × 4 weeks bid, twice daily. aThere are no approved treatments specifically for tinea manuum; treatments shown are for tinea pedis which are effective in the treatment of tinea manuum. bFDA-approved indications. 374 Aditya K. Gupta and Elizabeth A. Cooper 7.1. Tinea Pedis/Manuum Griseofulvin and topical terbinafine, butenafine, miconazole, econazole, ketoconazole, clotrimazole, and ciclopirox are US Food and Drug Administration (FDA)-approved treatments (Table 20.2) (6,17). Studies have shown that oral terbinafine and itraconazole may be the most effective treatments, and a higher cure rate has been shown with topical allylamines than with topical azoles (17,32). Topical formulations may be used for milder, limited presentations. For widespread or more severe presentations, oral formulations may be required. There is a higher relapse rate when using topical agents (32). Broad-spectrum topical agents may be useful, and agents with antibacterial activity may be preferred for presentations where it is suspected bacterial infection is superim- posed on fungal infection (e.g., miconazole nitrate 1%, ciclopirox olamine 1%, naftifine hydrochloride 1%, sulconazole nitrate 1%). Formulations allowing once daily appli- cation may be preferred to twice daily usage, to aid patient compliance (e.g., naftifine 1% cream, bifonazole 1%, ketoconazole cream 2%). Chronic infection may warrant the use of oral antifungals, particularly if previous topical regimens have failed. Oral itraconazole, terbinafine, and fluconazole have been used successfully in the treatment of tinea pedis, although none of these agents is currently approved by the FDA for use in tinea pedis. These oral agents are preferred over ketoconazole, owing to the potential for hepatic side effects with ketoconazole use. Oral griseofulvin has lower efficacy than the newer antifungals, poor keratin adherence and has activity limited to dermatophytes, which may be a limitation where superimposed bacterial infection is present (8). There are no approved treatments specifically for tinea manuum; treatments for tinea pedis are effectively used to treat tinea manuum. Tinea pedis may frequently recur. Proper foot hygiene may help prevent reinfection. Patients should avoid walking barefoot in communal areas such as bathrooms, showers, or swimming areas, and ensure that feet are dried thoroughly after bathing, showering, or swimming (33). In addition, patients should avoid occlusive footwear or alternate shoes every 2 to 3 days, and change socks often (33). 7.2. Tinea Corporis/Cruris Griseofulvin and topical terbinafine, butenafine, econazole, miconazole, ketoconazole, clotrimazole, and ciclopirox are FDA-approved treatments (Table 20.2) (19). Topical formulations may be used for infections of smaller areas. (e.g., sulconazole, oxiconazole, miconazole, clotrimazole, econazole, ketoconazole) (9). Oral therapy may be required where larger areas are involved, or where infection is chronic/recurrent. Topical corticosteroid use is not recommended, as it may lead to suppression of physical signs of infection, with lack of symptoms being wrongly associated with clearance of infection, leading to treatment relapse (9). Oral itraconazole, terbinafine, and fluconazole have been used successfully in the treatment of tinea corporis/cruris, although none of these agents is currently approved by the FDA for use in these indications. These oral agents are preferred over ketoconazole, owing to the potential for hepatic side effects with ketoconazole use, and griseofulvin is not recommended as it does not adequately bind the keratin in the stratum corneum, reducing efficacy (9). 20. Dermatophytosis and Other Superficial Fungal Infections 375 Tinea faciei are typically cleared with topical treatment. Topical ciclopirox and terbinafine may provide good anti-inflammatory effects as well as antifungal activity (10). Miconazole or similar azoles may also be effective. Azoles should be used for 3 to 4 weeks, or at least 1 week after resolution of lesions. Resistant lesions, cases of extensive disease, or more severe cases of infection may require oral therapy (10). Tinea imbricata is best treated with oral terbinafine or griseofulvin, although a high rate of recurrence has been noted (21). Itraconazole and fluconazole have not been effective. Adjunctive therapy with keratolytic creams such as Whitfield’s ointment (benzoic and salicylic acids) may increase treatment efficacy (21). 7.3. Tinea Capitis Oral therapy is required to adequately treat tinea capitis. Topical antifungals such as antifungal shampoos (selenium sulfide, povidone iodine, zinc pyrithione) may be used as adjunct therapy with or without oral antifungals to prevent reinfection or to treat asymptomatic carriers (1,33). As most infections occur in children, dosing regimens are modified from the typical adult regimens provided in other indications, and are usually given on a weight-based schedule (Table 20.3). Further, infections with Microsporum may require higher dosing than infections with Trichophyton, or longer regimens of therapy (1,34). Griseofulvin is the only FDA-approved oral treatment; however, terbinafine, itraconazole, and fluconazole have frequently been used in the successful resolution of tinea capitis. Shorter treatment durations are required for itraconazole, terbinafine, and fluconazole than for griseofulvin (1). Liquid formulations are available for griseofulvin, itraconazole, and fluconazole, and may aid in pediatric dosing, although dosing regimens may vary from that suggested for tablet/capsule formulations. Infected children do not need to be kept out of school once treatment is initiated, particularly children in higher grades (33,35). Infection transmission from symptom- free carriers such as family members and pets may need to be controlled by using adjunct therapies and objects which may carry fomites such as hats, combs, pillows, blankets, and scissors may need to be disinfected with bleach (33). An “id” reaction has been observed with tinea capitis patients after initiation of drug therapy, and can be confused with allergic drug reaction (1). An “id” reaction may present as symmetrical, skin colored, or erythematous papules and plaques on the face, neck, and upper body, but can be generalized. The reaction may also be present before initiation of treatment. 7.4. Onychomycosis Onychomycosis is difficult to cure and has a high rate of recurrence (6,11). Typically, oral therapy is required to adequately treat onychomycosis infections (Table 20.2). Following successful treatment of infection, the infected nail area must be grown out, gradually becoming replaced by normal healthy nail material. This process may take from 9 to 18 months, depending on nail growth rate. Fingernails may show better treatment success rates than toenails, as they grow faster (36). Where the nail has been injured or shows other abnormal growth patterns, nail outgrowth may be slow, and the nail may never regain a normal appearance. Further, relapse of infection is Table 20.3 Pediatric tinea capitis dosing regimens Regimen Durationa Weight (kg) 10–20 21–30 31–40 41–50 50+ Terbinafine 5 mg/kg per dayb 2-4 weeks 62.5 mg/day 125 mg/day 125 mg/day 250 mg/day 250 mg/day (continuous) Itraconazole 5 mg/kg per day 2-4 weeks 100 mg every 100 mg/day 100 mg once 200 mg/day 200 mg/day (continuous) other day daily alternating with twice daily Itraconazole Capsules: 1–3 pulses 100 mg every 100 mg/day 100 mg once 200 mg/day 200 mg bidd (pulse)c 5 mg/kg per day other day daily alternating with twice daily Oral suspension: 1–3 pulses 3 mg/kg per day Fluconazole Oral suspension: 20 days (continuous) 6 mg/kg per day Fluconazole Oral suspension: 8–12 weeks (pulse)e 6 mg/kg per day Griseofulvin Microsize: 6–12 weeks (continuous) 20–25 mg/kg per day Ultramicrosize: 6–12 weeks 10–15 mg/kg per day Oral suspension: 6–12 weeks 15–25 mg/kg perdayf bid, twice daily. aDurations of treatment are for Trichophyton tonsurans infection. Longer durations are often required for Microsporum canis infections. bDrugs are given by once daily dosing unless otherwise specified. cItraconazole pulses are given for 1 week, with 3 weeks “off” before starting the next pulse. dItraconazole adult dose 200 mg bid (approved for pulse use in fingernail onychomycosis). No standard has been established in clinical trials for tinea capitis for children >50 kg, and use varies from once-daily as with continuous regimen to twice-daily 200-mg dosing. eFluconazole pulses are 1 day on, 6 days off, before beginning next pulse. f Dosing based on Grifulvin V suspension 125 mg/5 ml. 20. Dermatophytosis |
and Other Superficial Fungal Infections 377 frequently noted. Patient expectations should be discussed, so the patient understands that successful treatment is unlikely to occur quickly, that long-term follow-up is necessary to catch any relapses in the early stages, and that the nail may not return to a normal appearance even though the infection may clear. Topical therapy may be effective in mild to moderate cases of infection; ciclopirox 8% lacquer is the only topical therapy currently approved for onychomycosis by the FDA. Amorolfine 5% nail lacquer has not been approved for use in North America (36–38). Routine nail débridement may be needed to provide effective delivery of drug to the infected area and to reduce the burden of fungal material needing treatment. The newer oral agents terbinafine and itraconazole are most frequently used for onychomycosis. Ketoconazole is not often used, owing to the potential for hepatic side effects. Griseofulvin is also not recommended, as the required regimens are significantly longer than those of the itraconazole or terbinafine and efficacy is low (39). Fluconazole has shown high efficacy, low relapse rates, and usefulness with yeast coinfection; however, there have been few studies on this treatment method (19,36). The approved oral therapy regimens for onychomycosis are as follows: terbinafine 250 mg/day for 12 weeks (toenails) or 6 weeks (fingernails only); itraconazole 200 mg/day for 12 weeks (toenails with or without fingernail involvement); and itraconazole 200 mg twice daily as pulse therapy (one pulse: 1 week of itraconazole followed by 3 weeks without itraconazole) using two pulses (fingernails only). Although only a continuous regimen of itraconazole is FDA-approved for toenail onychomycosis, the current standard of care of toenail onychomycosis for US dermatologists is a pulse itraconazole regimen (one pulse: 1 week of itraconazole followed by 3 weeks without itraconazole; three pulses given). Both terbinafine and itraconazole are readily taken up in the nail from the nail bed and matrix and may remain in the nail for a significant period after dosing is completed. Itraconazole action tends to be fungistatic, while terbinafine is fungicidal (39). Mycological cure rates (KOH negative and culture negative) for terbinafine use are estimated at 76% in a meta-analysis of clinical trial data from the medical literature (40). By comparison, itraconazole mycological cure rates are 59% (continuous therapy) and 63% (pulse therapy). Clinical response rates (infection cleared or showing marked improvement) were as follows: terbinafine—66%, itraconazole continuous therapy—70%, and itraconazole pulse therapy—70% (40). Itraconazole may be associated with more drug interactions than terbinafine owing to its metabolism through the CYP 3A4 pathway, limiting its use in some patients. Itraconazole is also prohibited in patients showing ventricular dysfunction such as current or past congestive heart failure (40). A current country-specific product monograph should be consulted for complete listing of known drug interactions, warnings, and monitoring requirements before prescribing. Rare cases of hepatic injury have been reported, and monitoring of hepatic enzymes is recommended for subjects with preexisting hepatic abnormality or a history of liver toxicity with use of other medications (39). Capsules must be taken with a meal or cola beverage to ensure adequate absorption (39). Terbinafine may interfere with metabolism of CYP 2D6 substrates, and some other drug interactions have been noted. A current country-specific product monograph 378 Aditya K. Gupta and Elizabeth A. Cooper should be consulted for complete listing of known drug interactions, warnings, and monitoring requirements before prescribing. Rare cases of hepatic injury have been reported with terbinafine. Terbinafine is not recommended for patients with existing liver disease, and all patients should be screened for hepatic enzyme abnormal- ities (alanine transaminase [ALT] and aspartate transaminase [AST]) before initiating terbinafine (39). Terbinafine may be taken in fasted or fed state without affecting absorption. Routine nail débridement may be complementary for subjects using oral therapy as well as topical therapy, particularly where the nail is thickened, or where the infection presents as a dermatophytoma, spike, or lateral infection. Caution must be taken not to damage the underlying skin during débridement, particularly in subjects who are vulnerable to severe lower limb complications, such as diabetics or individuals with reduced lower limb profusion. As with tinea pedis, proper foot and nail hygiene may help prevent reinfection. Patients should avoid walking barefoot in communal areas such as bathrooms, showers, or swimming areas, and ensure that feet are dried thoroughly after bathing, showering, or swimming (33). Nails should be kept short and clean. Shoes should fit properly and socks should be made from absorbent material such as cotton. 7.5. Pityriasis Versicolor A variety of topical agents may be used to treat PV (Table 20.2). Topical azoles (ketoconazole, fluconazole, bifonazole, clotrimazole, miconazole) have been effective in treating Malassezia, both in cream formulation or shampoos (5). Terbinafine solution, cream, gel, or spray has also been effective (5). Topical ciclopirox provides both antifungal and anti-inflammatory activity against Malassezia. Systemic therapies may be warranted in severe cases, or cases with widespread body involvement. Patients may also prefer a short-duration oral therapy to frequent appli- cation of a topical agent. Oral therapy with ketoconazole, itraconazole, and fluconazole has been effective for PV, and the regimens reported in the literature provide similar, high efficacy rates (5). Oral terbinafine and griseofulvin are not effective for PV (5). Relapse of PV is common owing to endogenous host factors: recurrence rates have been reported as high as 60% to 90% in 2 years posttreatment (41). Both ketoconazole (single 400-mg dose or 200 mg daily for 3 days once monthly) and itraconazole (single 400-mg dose once monthly for 6 months) have been used in prophylactic regimens for PV (5). Individual treatments for hyper- and hypopigmented variations of PV do not exist. Although fungal organisms may be eradicated after 2 weeks of therapy, it may take significantly longer before the skin’s normal pigmentation is restored, particularly with hypopigmented lesions (5). 7.6. Seborrheic Dermatitis There is no definitive cure for SD; it is a recurrent disease requiring prophylactic treatment (14). Topical corticosteroid lotions have typically been used as treatment but are being replaced by antifungal treatments in the form of shampoos, gels, and creams (Table 20.2). 20. Dermatophytosis and Other Superficial Fungal Infections 379 Topical ketoconazole (cream, shampoo, gel, emulsion) is the most prescribed azole for SD (42). Bifonazole, miconazole, and fluconazole may also be effective. Low-potency corticosteroids may be useful in providing anti-inflammatory treatment, although many newer antifungal agents such as ciclopirox may also provide anti- inflammatory activity comparable to corticosteroids (42). Ciclopirox (cream, gel or shampoo) provides effective antifungal treatment, and also has antibacterial and anti- inflammatory activities (43,44). Zinc pyrithione shampoos are safe and effective in controlling dandruff and SD of the scalp, and exhibit strong keratolytic and antifungal activity against Malassezia (15,45). Some patients benefit as well from non-antifungal, keratolytic agents (selenium sulfide, sulfur, salicylic acid) or antiproliferative (coal tar) shampoos (14,30). Tar shampoos often cause sensitivity of the skin to sunlight and are not as favorable cosmetically (30). Topical 1% terbinafine solution has been effectively used for scalp SD (42). Oral therapy should be reserved for severe inflammatory SD, widespread SD, or SD that has been refractory to topical treatment (42). Oral ketoconazole and oral itraconazole have been used effectively for SD. 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J Cutan Med Surg 2004;8:25–30. 38. Lecha M, Effendy I, Feuilhade de Chauvin M, Di Chiacchio N, Baran R. Treatment options- development of consensus guidelines. J Eur Acad Dermatol Venereol 2005;19(Suppl 1):25–33. 20. Dermatophytosis and Other Superficial Fungal Infections 381 39. Gupta AK, Ryder JE. The use of oral antifungal agents to treat onychomycosis. Dermatol Clin 2003;21:469–479. 40. Gupta AK, Ryder JE, Johnson A.M. Cumulative meta-analysis of systemic antifungal agents for the treatment of onychomycosis. Br J Dermatol 2004;150:537–544. 41. Faergemann J. Pityrosporum infections. J Am Acad Dermatol 1994;31:S18–S20. 42. Gupta AK, Bluhm R, Cooper EA, Summerbell RC, Batra R. Seborrheic dermatitis. Dermatol Clin 2003;21:401–412. 43. Aly R, Katz HI, Kempers SE, et al. Ciclopirox gel for seborrheic dermatitis of the scalp. Int J Dermatol 2003;42:19–22. 44. Lebwohl M, Plott T. Safety and efficacy of ciclopirox 1% shampoo for the treatment of seborrheic dermatitis of the scalp in the US population: results of a double-blind, vehicle- controlled trial. Int |
J Dermatol 2004;42:17–20. 45. Rogers JS, Moore AE, Meldrum H, Harding CR. Increased scalp skin lipids in response to antidandruff treatment containing zinc pyrithione. Arch Dermatol Res 2003;295:127–129. SUGGESTED READINGS Baran R, Gupta AK, Pierard GE. Pharmacotherapy of onychomycosis. Expert Opin Pharma- cother 2005;6:609–624. Crespo-Erchiga V, Florencio VD. Malassezia yeasts and pityriasis versicolor. Curr Opin Infect Dis 2006;19:139–147. Gupta AK, Chaudhry M, Elewski B. Tinea corporis, tinea cruris, tinea nigra, and piedra. Dermatol Clin 2003;21:395–400. Gupta AK, Chow M, Daniel CR, Aly R. Treatments of tinea pedis. Dermatol Clin 2003;21: 431–462. Gupta AK, Cooper EA, Ryder JE, Nicol KA, Chow M, Chaudhry MM. Optimal management of fungal infections of the skin, hair, and nails. Am J Clin Dermatol 2004;5:225–237. Gupta AK, Summerbell RC. Tinea capitis. Med Mycol 2000;38:255–287. Hay R. Literature review. Onychomycosis. J Eur Acad Dermatol Venereol 2005;19 (Suppl 1):1–7. Roberts BJ, Friedlander SF. Tinea capitis: a treatment update. Pediatr Ann 2005;34:191–200. 21 Subcutaneous Fungal Infections (Chromoblastomycosis, Mycetoma, and Lobomycosis) Michael B. Smith, MD and Michael R. McGinnis, PhD 1. INTRODUCTION Fungal infections involving subcutaneous tissue often develop following a penetrating wound through the skin. The etiologic agents are usually soil fungi or decomposers of plant material and infection typically occurs when woody plant material such as splinters and thorns penetrate the cutaneous barrier. With the exception of Sporothrix schenckii, the pathogens that cause infection in subcutaneous tissue are not dimorphic, that is, they do not grow in different morphological forms in vitro and in vivo. In tissue, they may form hyphae, yeast cells, muriform cells, or be organized into microcolonies called sclerotia (syn. grains, granules) (1). The combination of tissue morphology of the fungus, whether or not the fungus contains melanin in its cell wall (dematiaceous), and the clinical presentation, determines the specific type of subcu- taneous mycosis (Table 21.1). This information guides management approaches and assists with determining the prognosis for the patient. Fungal infections involving subcutaneous tissue are frequently grouped as (1) mycetoma, which can be caused by both dematiaceous and nondematiaceous fungi (2); (2) chromoblastomycosis, in which the etiologic agent develops dematia- ceous muriform cells with thickened melanized cell walls in subcutaneous microab- scesses (3); (3) subcutaneous phaeohyphomycosis, characterized by dematiaceous yeast cells, pseudohyphae, and irregular shaped hyphae to well-developed septate hyphae (may be present in any combination); or (4) hyalohyphomycosis, an infection that is similar to phaeohyphomycosis except that the fungi that cause this disease are not dematiaceous. Sporotrichosis (Chapter 19), subcutaneous hyalohyphomy- cosis (Chapter 10), subcutaneous phaeohyphomycosis (Chapter 11), and subcutaneous zygomycosis (including entomophthoramycosis; Chapter 12) are discussed elsewhere in this text. Rhinosporidiosis, caused by a protoctistan classified in a Mesomyce- tozoa clade, is not discussed. Mycetoma and chromoblastomycosis are considered together because these two infections have a number of clinical and pathological From: Infectious Disease: Diagnosis and Treatment of Human Mycoses Edited by: D. R. Hospenthal and M. G. Rinaldi © Humana Press Inc., Totowa, NJ 383 384 Michael B. Smith and Michael R. McGinnis Table 21.1 Overview of the major subcutaneous fungal infections Disease Etiology Key histological features Comments Chromoblastomycosis Cladophialophora, Dematiaceous muriform Fonsecaeaa cells in subcutaneous micro-abscesses Hyalohyphomycosis Aspergillus, Hyaline, septate hyphae, yeast See Chapter 10 Fusariuma cells, pseudohyphae, or any of the above Lobomycosis Lacazia Hyaline yeast cells in a chain with connectors between the cells Mycetoma Madurella, Sclerotia (grains, granules) Scedosporiuma composed of dematiaceous or hyaline fungal hyphae Phaeohyphomycosis Exophialaa Dematiaceous septate hyphae, See Chapter 11 yeast cells, pseudohyphae, or any of the above Rhinosporidiosis Rhinosporidium Hyaline sporangia with Etiologic agent sporangiospores of different recently sizes that are released into the proven not to environment through a pore; be a fungus see Figure 3-3, Chapter 3 Sporotrichosis Sporothrix Oval budding yeast cells See Chapter 19 Zygomycosis Basidiobolus, Hyaline, sparsely septate, See Chapter 12 (Entomophtho- Conidiobolus random branching, irregular ramycosis) diameter hyphae Zygomycosis (not Apophysomyces, Hyaline, sparsely septate, See Chapter 12 Entomophtho- Rhizopusa random branching, irregular ramycosis) diameter hyphae aExamples of major etiological agents; not inclusive. aspects in common with each other. Chromoblastomycosis has been referred by some as essentially a “mini mycetoma” because of its pathologic and clinical similarities to mycetoma. Both of these infections demonstrate the presence of tumefaction, draining sinuses, and aggregates of the etiologic agents within the subcutaneous tissue. Lobomy- cosis, which is caused by Lacazia loboi, is an infection that occurs in people living near water in Central and South America. In addition to human cases, this fungus causes cutaneous–subcutaneous infection in some animals (e.g., dolphins) (4). 2. ETIOLOGIC AGENTS 2.1. Chromoblastomycosis Chromoblastomycosis is caused by members of the genera Cladophialophora, Exophiala, Fonsecaea, and Phialophora, which belong to the ascomycete family Herpotrichiellaceae (Chaetothyriales) (Table 21.2). The genus Fonsecaea currently 21. Subcutaneous Fungal Infections 385 Table 21.2 Etiologic agents of human chromoblastomycosis, mycetoma, and lobomycosis Chromoblastomycosis Cladophialophora carrionii Exophiala dermatitidis Exophiala jeanselmei Exophiala spinifera Fonsecaea compacta Fonsecaea pedrosoi Phialophora verrucosa Rhinocladiella aquaspersa Mycetoma Acremonium kiliense Acremonium recifei Aspergillus flavus Aspergillus hollandicus Emericella nidulans Fusarium falciforme Corynespora cassicola Curvularia lunata Cylindrocarpon cyanescens Cylindrocarpon destructans Exophiala jeanselmei Fusarium falciforme Fusarium solani Fusarium verticillioides Leptosphaeria senegalensis Leptosphaeria tompkinsii Madurella grisea Madurella mycetomatis Neotestudina rosatii Phaeoacremonium inflatipes Phialophora verrucosa Polycytella hominis Pseudallescheria boydii Pseudochaetosphaeronema larense Pyrenochaeta mackinnonii Pyrenochaeta romeroi Rhinocladiella atrovirens Lobomycosis Lacazia loboi contains three species, two of which are primary agents of chromoblastomycosis, F. compacta and F. pedrosoi. Fonsecaea pedrosoi is the primary etiologic agent of chromoblastomycosis throughout the world. On the basis of molecular analysis, a new species called F. monophora has been proposed for isolates that were previously identified as F. pedrosoi which cause CNS infections (5). This latter infection is a phaeohyphomycosis because the fungus grows as dematiaceous hyphae in tissue. 386 Michael B. Smith and Michael R. McGinnis The genus Cladophialophora contains a number of species, including C. bantiana, the etiologic agent of CNS phaeohyphomycosis. Recently, Cladosporium carrionii, an important agent of chromoblastomycosis, was reclassified as Cladophialophora carrionii. This species is geographically more localized than F. pedrosoi. 2.2. Mycetoma Mycetoma is caused by a variety of genera, the frequency of which vary geograph- ically. The genus Pseudallescheria contains a number of species. Pseudallescheria boydii (anamorph Scedosporium apiospermum) is the major species of medical interest. It is unknown if the other species of Pseudallescheria can cause mycetoma. At this time, it is thought that P. boydii is the only etiologic agent of this genus that causes mycetoma. Pseudallescheria is classified in the order Microascales. The genus Madurella was originally established for sterile dematiaceous fungi causing mycetoma. Based on rDNA small subunit and internal transcribed spacer (ITS) sequence data, Madurella has been determined to be a heterogeneous group of fungi (6). Madurella mycetomatis, which worldwide is the most common cause of mycetoma, is in the Sordariales, whereas M. grisea is in the Pleosporales. The Ascomycetes orders Hypocreales, Eurotiales, and Dothideales contain several etiologic agents of mycetoma. 2.3. Lobomycosis Lacazia loboi (previously named Loboa loboi) is a noncultured fungus that resembles Paracoccidioides brasiliensis in vivo. It is the sole etiologic agent of lobomycosis, regardless of human or animal origin. Based on its morphology in tissue and its phylogenetic distinctness, the fungus is classified with the dimorphic Ascomycetes in the Onygenales. 3. EPIDEMIOLOGY Chromoblastomycosis, lobomycosis, and mycetoma have a number of features in common. Infected patients typically live in tropical and subtropical regions. The etiologic agents are introduced by localized trauma to the skin by woody plant material or other injuries such as insect bites. They are more commonly seen in individuals working in agriculture, mining, fishing, farming, and similar occupations where they are in constant contact with the environment. Infections occur more commonly in males, which is likely a reflection of occupational exposure. Mycetoma tends to occur in arid areas with short rainy seasons and a low relative humidity. Lobomycosis, unlike the other two infections, is not worldwide in occurrence. It is restricted to South and Central America, especially in communities located along rivers. Lobomycosis tends to develop on exposed and cooler areas of the body such as the extremities and the ears. This contrasts with chromoblastomycosis and mycetoma, which most frequently occur on the legs and feet. 4. PATHOGENESIS AND IMMUNOLOGY The three infections are characterized by chronic, granulomatous, slowly progressing, cutaneous–subcutaneous inflammatory processes resulting in disfiguring lesions. The duration of infection prior to diagnosis can be years. Many patients recall 21. Subcutaneous Fungal Infections 387 splinters or similar wounds at the infection site prior to the development of the infection. Chromoblastomycosis does not involve bones and it does not usually spread through the lymphatic system. Mycetoma, in contrast, often extends to bone where it causes extensive destruction. Lobomycosis remains localized to the dermis and subcutaneous tissue and does not cause disseminated infections. 5. CLINICAL MANIFESTATIONS 5.1. Chromoblastomycosis Chromoblastomycosis may exhibit a range of different types of lesions that vary depending on the duration and body site of the infection (7). After local injury, small verrucous papules develop. Localized spread or extension of the lesions can occur by autoinoculation. The slow growing lesions can develop into clusters of large hyperker- atotic verrucous plaques that produce ulceration, cystic areas, and eventually, scarring (Fig. 21.1). Scarring can become so extensive that the flexibility of limbs can be compromised. Muriform cells occur in subcutaneous areas of epitheliomatous hyper- plasia and microabscesses, and are expelled to the surface by transepithelial elimination. This results in “black dots” on the lesion surface which are accumulations of necrotic tissue and fungal cells. This process is similar to mycetoma, which is characterized by “sclerotia” being transported to the surface by draining sinuses associated with edema (tumefaction). For this reason, chromoblastomycosis has been referred to as a mini-mycetoma. Fig. 21.1. Chromoblastomycosis caused by Fonsecaea pedrosoi. (Courtesy of Dr. C. Halde.) [Figure in color on CD-ROM]. 388 Michael B. Smith and Michael R. McGinnis Fig. 21.2. Mycetoma caused by Madurella mycetomatis. (Courtesy of Dr. C. Halde.) [Figure in color on CD-ROM]. 5.2. Mycetoma Fungal mycetoma develops more slowly than mycetoma caused by bacterial agents (actinomycotic mycetoma) (8). Encapsulated lesions with clearly defined margins slowly develop after introduction of the fungus on a splinter or thorn. A subcutaneous painless swelling develops that is usually soft, firm, and lobate. Additional foci often Fig. 21.3. Lobomycosis caused by Lacazia loboi. Courtesy of Dr. P. Taborda. [Figure in color on CD-ROM]. 21. Subcutaneous Fungal Infections 389 develop, suppurate, and drain through multiple sinus tracts. Additional sinuses develop that are interconnected with each other, sterile deep abscesses, and the skin. Sclerotia of different colors (e.g., white grain, black grain) that are formed by specific fungi find their way to the surface. Even though mycetoma is painless owing to anesthetic effect or nerve damage, pain may occur when bone expands due to the fungal sclerotial mass, or when secondary infections develop. Mycetoma can produce functional disability, distortion, and deformity of limbs (Fig. 21.2). 5.3. Lobomycosis Lesions of lobomycosis develop slowing and become cutaneous nodules or plaques that may be smooth, verrucous, or ulcerative, and eventually can become surrounded by keloidal scar tissue (4). The lesions may be solitary or multiple; painless or slightly pruritic; with the fungus spreading contiguously or via lymphatic channels (Fig. 21.3). The lesions contain granulomatous inflammatory tissue and the yeast cells. The fungus does not cause disseminated infection. 6. DIAGNOSIS The diagnosis of these infections is based primarily on clinical presentation, histopathology, and culture. Culture is not useful in the diagnosis of lobomycosis as L. loboi has not yet been cultured. The tissue forms for these infections are distinctive, and can be used to a limited degree for presumptive identification purposes (see Fig. 3.2, Chapter 3). Chains of yeast cells with a narrow connection between each yeast cell are characteristic of L. loboi (9). The architecture of the sclerotia in mycetoma patients can be associated with particular known agents (Fig. 21.4). The structure is especially useful for distinguishing the granules of aerobic actinomycetes composed of 0.5 to 1.0 μm gram-positive filaments from sclerotia of true fungi having hyphal cells. The muriform cells and hyphae in the dermis of chromoblastomycosis cases do Fig. 21.4. Sclerotia of Madurella mycetomatis in a foot. Gridley Fungus Stain. Courtesy of Dr. L. Ajello. [Figure in color on CD-ROM]. 390 Michael B. Smith and Michael R. McGinnis not allow for the differentiation of the different etiologic agents, although they are virtually pathognomonic of the disease. In culture, the agents of chromoblastomycosis |
and mycetoma are easily identified. Madurella mycetomatis represents an interesting challenge because it is defined on the basis of being a typically sterile dematiaceous, slow growing mould isolated from a mycetoma. As mentioned, the fungus appears to be a complex of different phylogenetic members. 7. TREATMENT Chromoblastomycosis, lobomycosis, and mycetoma are chronic infections without a single treatment of choice. Currently used treatments often have low cure rates and high relapse rates, although these rates vary based on the etiologic agent, affected body area, and health status of the patient (Table 21.3). The dense fibrosis and lymphostasis that occurs in these infections can impede drug penetration into the infected area. Courses of therapy of 6 to 12 months (or more) are not uncommon. 7.1. Chromoblastomycosis Chromoblastomycosis is often treated with the triazole itraconazole, or terbinafine (3). These antifungal agents are used at high dosage concentrations, typically for 6 to 12 months. Of the azoles, itraconazole has been the most successful antifungal drug, . Table 21.3 Management of chromoblastomycosis, mycetoma, and lobomycosis Infection Management Chromoblastomycosis Surgery, electrodessication, and cryosurgery are effective in early stages. Local heat can reduce extension of lesions. Disabled or deformed limbs may require amputation. Combination therapy with terbinafine (500 mg daily) plus itraconazole (50–100 mg daily) or terbinafine alone has been successful. Mycetoma Historically, surgery has been used for treatment of mycetoma. Mycetomas are well encapsulated, and care must be exercised to avoid rupturing. There are some reports of successful treatment with ketoconazole and itraconazole. The dose for both is 400 mg daily with treatment continuing for a few to many years. A good clinical response has been seen in some patients with ketoconazole, itraconazole, voriconazole, and terbinafine. Lobomycosis Wide surgical excision of the affected area. Relapse is common. Electrodessication is useful in early stages of the disease. Clofazimine at 300 mg/d has been used with good results in some patients. Antifungal drugs such as ketoconazole, itraconazole, amphotericin B, and 5-fluorocytosine are generally ineffective, although there is one report of successful treatment with itraconazole. Modified from Lupi et al.(4) 21. Subcutaneous Fungal Infections 391 given as long-term continuous or pulse therapy. In one large study, the cure rate was 42% with a mean treatment period of 7.2 months (2). In contrast, a large clinical study with terbinafine had a cure rate of 74.2% after 12 months therapy (10). Overall cure rates for both drugs range from 40% to 70% (3). These two drugs can be used alone to treat small to medium-sized lesions, or they can be combined with localized heat and cryosurgery in one to several sessions. Combined antifungal drug therapy with itraconazole and terbinafine may be a useful approach to consider. Superficial forms of chromoblastomycosis tend to respond well to antifungal drugs, whereas verrucous or tumorlike forms often do not respond well. These patients often require both chemotherapy and thermotherapy. Because of the high possibility of secondary bacterial infections, prophylactic antibacterial therapy is often given. If surgery is to be used, it is recommended that antifungal agents be given systematically both before the procedure and afterwards. 7.2. Mycetoma It is important to distinguish mycetoma caused by aerobic actinomycetes from cases caused by fungi because their treatment is different. An actinomycetoma is amenable to therapy with antibacterial agents. A fungal mycetoma is more difficult to manage and may require aggressive surgery up to and including amputation in advanced disease. Surgical management may be appropriate after antifungal agents have been used. A good clinical response has been seen in some patients treated with ketoconazole, itraconazole, voriconazole, and terbinafine. The Mycetoma Research Center (Khartoum, Sudan; www.mycetoma.org) recom- mends both ketoconazole and itraconazole for first line use. Treatment duration depends upon the severity of the infection and the general health status of the patient. Therapy may be needed for extended periods (years). Itraconazole treatment has been shown to have a high degree of success with a low recurrence rate. Surgery is used to completely excise small, well-encapsulated lesions, or to reduce of the amount of infected tissue. Effective surgery may require aggressive excision, which may lead to permanent disability. Surgery without effective chemotherapeutic treatment has a high rate of recurrence; up to 90% in advanced cases. Surgery is the treatment of choice for early cases, typically well-encapsulated lesions without bone involvement. 7.3. Lobomycosis Lobomycosis is an infection that involves the dermis and subcutaneous tissue that is frequently managed with surgical excision (with wide margins). Surgical excision is curative in most instances (4). Itraconazole may have value in treating this mycosis, but the use of this antifungal agent has been reported in only one patient. REFERENCES 1. Pang KR, Wu JJ, Huang DB, Tyring SK. Subcutaneous fungal infections. Dermatol Ther 2004;17:523–531. 2. Queiroz-Telles F, McGinnis MR, Salkin I, Graybill JR. Subcutaneous mycoses. Infect Dis Clin North Am 2003;17:59–85. 3. Bonifaz A, Saul A, Paredes-Solis V, Araiza J, Fierro-Arias L. Treatment of chromoblasto- mycosis with terbinafine: experience with four cases. J Dermatol Treat 2005;16:47–51. 392 Michael B. Smith and Michael R. McGinnis 4. Lupi O, Tyring SK, McGinnis MR. Tropical dermatology: fungal tropical diseases. J Am Acad Dermatol 2005;53:931–951. 5. Surash S, Tyagi A, De Hoog GS, Zeng JS, Barton RC, Hobson RP. Cerebral phaeohy- phomycosis caused by Fonsecaea monophora. Med Mycol 2005;43:465–472. 6. de Hoog GS, Adelmann D, Ahmed AO, van Belkum A. Phylogeny and typification of Madurella mycetomatis, with a comparison of other agents of eumycetoma. Mycoses 2004;47:121–130. 7. McGinnis MR. Chromoblastomycosis and phaeohyphomycosis: new concepts, diagnosis, and mycology. J Am Acad Dermatol 1983;8:1–16. 8. McGinnis MR. Mycetoma. Dermatol Clin 1996; 14:97–104. 9. Haubold EM, Cooper CR, Wen JW, McGinnis MR, Cowan DF. Comparative morphology of Lacazia loboi (syn. Loboa loboi) in dolphins and humans. Med Mycol 2000;38:9–14. 10. Esterre P, Inzan CK, Ramarcel ER, et al. Treatment of chromomycosis with terbinafine: preliminary results of an open pilot study. Br J Dermatol 1996;134 (Suppl 46):33–36. Instructive Cases INTRODUCTION The instructive cases that follow are provided to allow the reader a review of many of the important concepts presented in this book. They may also provide the clinician preparing for subspecialty boards practice cases and questions. Each case is presented as an unknown to test knowledge obtained from reading this book or to emphasize an important or complex topic in the field of medical mycology. INSTRUCTIVE CASE 1 A 57-year-old patient with acute myelogenous leukemia (AML) and a 3-week history of neutropenia develops fever that is unresponsive to broad-spectrum antimicrobial therapy (piperacillin–tazobactam and levofloxacin). High-resolution computed tomog- raphy (HRCT) of the lungs revealed several pleural-based nodular lesions. The patient was empirically started on amphotericin B deoxycholate 1 mg/kg every 24 hours. After 5 days of therapy, the patient’s serum potassium begins to drop but the serum creatinine and blood urea nitrogen (BUN) remain stable. Questions 1. The decrease in serum potassium is most likely due to what amphotericin B toxicity? A. Afferent arteriole constriction in the kidney B. Distal tubular damage in the kidney C. Suppression of erythropoietin synthesis D. Damage of the pancreatic islet cells 2. Which of the following approaches should be used if the patient’s serum creatinine doubles and he begins to develop azotemia? A. Change amphotericin B dosing to every other day. B. Change amphotericin B to continuous infusion dosing. C. Change amphotericin B to a lipid amphotericin B formulation. D. Begin administering 500 ml of normal saline before and after infusion. INSTRUCTIVE CASE 2 While riding his dirt bike on a vacant lot in southeastern Michigan, a 22-year-old man abraded his right leg on the dirt while rounding a corner. He was taken to the emergency room, where the large abrasion was cleaned and débrided. Because there was extensive loss of skin, a skin graft was placed several weeks later. About 2 weeks after the graft was placed, he noted that a few “bumps” had developed in the grafted area. These “bumps” became larger, broke open, and began to discharge what he 393 394 Instructive Cases Fig. IC.1. Poor healing and nodular disease about lower extremity scar prior to antifungal therapy (patient later proven to have sporotrichosis). [Figure in color on CD-ROM]. described as a thin fluid with some pink discoloration. Similar lesions then developed in the thigh proximal to the original injury and graft. Several different antimicrobial agents were prescribed (including those with Staphylococcus aureus coverage), but the lesions did not respond, and in fact new lesions appeared. When the patient was seen by an infectious diseases consultant, the graft was beginning to break down and multiple nodules of various sizes were noted in the original abraded area that had been grafted and proximal to the graft into the upper thigh (Fig. IC.1). Some were ulcerated and weeping serosanguinous fluid, and others were crusted. The nodules were not tender to palpation. The patient felt well and specifically denied having chills or fever. Biopsies were taken of several nodules for culture and histopathological exami- nation. The tissue sections showed granulomas, but no organisms were seen. Within 1 week, the cultures held at 25 C showed growth of an off-white mould that on micro- scopic examination showed tiny conidia arranged “bouquet-like” on thin hyphae. The diagnosis of sporotrichosis was made and the patient was begun on an experimental protocol using fluconazole, 400 mg daily. The lesions began to resolve by 2 months (Fig. IC.2), and were finally all resolved by 5 months. Therapy was continued for a total of 6 months. No recurrences were noted. The patient was admonished to change hobbies from dirt bike riding to something less dangerous, but he chose to ignore that medical advice. INSTRUCTIVE CASE 3 A 29-year-old African American man was in his usual state of good health until September, at which time he presented with a nonproductive cough, shortness of breath, back pain, chills, fever, headache, and body aches. Evaluation led to a diagnosis of pneumonia with right hilar and peritracheal adenopathy. He was originally treated Instructive Cases 395 Fig. IC.2. Improvement of sporotrichosis after 2 months of fluconazole therapy. [Figure in color on CD-ROM]. with antimicrobial agents for community-acquired pneumonia without resolution of symptoms. Approximately 2 weeks later, three skin lesions consistent with granulomas were noted. A punch biopsy performed approximately 1 month after his original presentation revealed granulomatous inflammation with endosporulating spherules, thus confirming the diagnosis of cutaneous coccidioidomycosis. Since the patient had complained of intermittent headache, and there was now a diagnosis of pulmonary and cutaneous coccidioidomycosis, it was essential to rule out coccidioidal meningitis. A lumbar puncture was done, which was normal. At this time, the patient was also noted to have subcutaneous nodules in the left preauricular and right submandibular areas as well as over the right humerus and left knee. The patient was started on oral fluconazole, 800 mg daily. Subsequently the patient developed multiple draining lesions over the anterior chest. A bone scan was positive for disease of multiple ribs and the manubrium as well as the left parietal skull, right frontal skull, right superior orbit, and left hind foot. The positive areas on bone scan were confirmed with plain radiographs, as osteomyelitis. Culture of fluid from the draining sternal lesion grew Coccidioides immitis. Given the extensive dissemination to bone and skin in this patient, therapy was intensified to a lipid preparation of amphotericin B at a dose of 5 mg/kg three times per week for approximately 3 months. At the same time, aggressive surgical débridement of the draining thoracic lesions was undertaken, in an attempt to reduce the fungal burden. The patient’s original complement fixation titer at presentation was less than 1:2. It subsequently rose to 1:16 and eventually to ≥1:512, and stayed at that level despite therapy. The patient continues to have draining lesions of the anterior chest despite repeat surgeries and 396 Instructive Cases several courses of amphotericin, and is now being treated with voriconazole 4 mg/kg twice daily. Lifelong therapy is anticipated. INSTRUCTIVE CASE 4 A-27 year-old male agricultural worker seen in consultation to evaluate an illness of 2 months duration characterized by the presence of rapidly enlarging lymph nodes in both cervical chains, accompanied by pain, and high fever, especially at nights. One of the nodes had drained spontaneously, producing yellow-tinged purulent material. The patient also experienced productive cough without dyspnea. On examination, he was pale, looked frail, and had enlarged cervical, axillary, and inguinal lymph |
nodes (Fig. IC.3). His spleen and liver were normal by physical examination. Lung auscultation did not reveal altered breath sounds and chest radiographs were normal. A human immunodeficiency (HIV) test was negative, hemoglobin was 8.8 g/dl, and white blood cell count was 25,700 cells/l. A direct KOH examination of the discharge from the ruptured nodule showed abundant yeast cells, some with the characteristic multiple budding of Paracoccidioides brasiliensis; this fungus was also isolated in culture. Serology with paracoccidioidin proved reactive with one band of precipitate and a complement fixation (CF) titer of 1:1024. INSTRUCTIVE CASE 5 A 70-year-old man with hypereosinophilic syndrome, non-insulin dependent diabetes mellitus (NIDDM), and long-term prednisone therapy (25 mg/day × 25 years) was admitted with fatigue, weakness, and shortness of breath. Physician examination was Fig. IC.3. Paracoccidioidomycosis. Hypertrophied cervical lymph node about to rupture. Scarring lesions of a similar process can be seen above. [Figure in color on CD-ROM]. Instructive Cases 397 remarkable for a temperature of 99˚F, an area of erythema over the left thigh that was tender to palpation and an effusion in the right knee. On admission, he had a hemoglobin of 5.9 g/dl, WBC 8600/mm3, a serum creatinine 2.7 mg/dl, and a glucose 382 mg/dl. Aspiration of the right knee revealed a WBC of 40,500/mm3 with 96% neutrophils, Gram stain revealed gram-negative bacilli and budding yeasts. The patient was initially started on imipenem, vancomycin, tobramycin, and fluconazole (FLZ) 400 mg/day. Within 72 hours, the blood cultures drawn on admission were found to be positive for yeast, later identified as Trichosporon asahii (beigelii). In addition, joint fluid cultures were also positive forTrichosporon asahii and Pseudomonas aeruginosa. Biopsy and culture of the left thigh area of erythema also grew P. aeruginosa and T. asahii. After 5 days of fluconazole treatment, the blood cultures were still positive and caspofungin (Cancidas) 50 mg IV qd was added to the fluconazole. He also underwent arthroscopic flushing of the right knee. Ten days after admission, the patient had negative blood cultures and had responded well to antimicrobial therapy. He was subsequently transferred to a rehabilitation unit in good condition. In vitro susceptibilities (MICs) of T. asahii revealed fluconazole 8 g/mg, itraconazole 0.5 g/ml, and caspofungin 2 g/ml. INSTRUCTIVE CASE 6 A 42-year-old black man presented to the University Hospital emergency department with a 1-week history of fever, chills, cough productive of white sputum, night sweats, and malaise. He denied hemoptysis. Risk factors for HIV infection included a blood transfusion in 1985 and heterosexual promiscuity. He denied intravenous drug abuse or homosexual activity. Over the last 8 months he had lost 80 pounds. Past medical history was significant for a prior appendectomy, a perirectal abscess that was surgically drained, and a chronic perirectal fistula for 5 years. There was also a history of venereal disease including primary syphilis associated with a chancre and a positive rapid plasma reagin (RPR) of 1:32. There was no documented therapy and the patient was lost to follow-up. On admission, the patient was afebrile but cachectic. He was in no acute respiratory distress. On physical examination, he was noted to have dry crusting lesions on the tip of his nose and both cheeks of the face (Fig. IC.4). Similar lesions were noted over the shins and a small draining abscess was noted over the medial aspect of the left foot. Oral hairy leukoplakia was present on the tongue and he was noted to have prominent generalized lymphadenopathy. No rales, rhonchi, or wheezes were heard on auscultation of chest. A fistula-in-ano was also noted. Chest radiograph revealed bilateral reticulonodular infiltrates, which were more prominent in the upper lobes. The patient was lymphopenic with an absolute lymphocyte count of 410 cells/l. The serum RPR of 1:32 was positive. Skin scrapings of the lesions were performed and wet preparations of the specimens revealed characteristic thick-walled multinucleated yeast forms compatible with Blastomyces dermatitidis (Fig. IC.5). Subsequently, the patient underwent bronchoscopy with bronchoalveolar lavage (BAL), and cytology prepara- tions of the washings also revealed B. dermatitidis. Cultures of skin scrapings and BAL washings grew this organism. An enzyme-linked immunoassay (ELISA) and Western 398 Instructive Cases Fig. IC.4. Multiple cutaneous lesions in a patient with end-stage AIDS. Similar lesions were noted on both cheeks of the face and both shins. [Figure in color on CD-ROM]. Blot confirmed HIV infection and total CD4+ T lymphocyte count was 8 cells/l. A computed tomography (CT) scan of the head was performed because of complaints of headache. This study revealed multiple enhancing brain lesions (Fig. IC.6). Owing to concerns of a second opportunistic pathogen, i.e., Toxoplasma gondii, the brain lesions were aspirated. Wet preparations again revealed characteristic yeast forms of B. dermatitidis. The patient was begun on highly active antiretroviral therapy (HAART) and ampho- tericin B deoxycholate therapy at an initial dose of 1 mg/kg per day. Intravenous penicillin, 18 million units per day in divided doses, was administered for 21 days because of concerns of neurosyphilis. During the remainder of the hospitalization, the patient was carefully monitored for evidence of renal insufficiency and 500 ml of normal saline were infused prior to each amphotericin B infusion. During this Instructive Cases 399 Fig. IC.5. Wet preparation of skin scrapings. This figure shows the characteristic yeast forms in a wet preparation of skin scrapings. Scraping of the edges of the verrucous and ulcerative lesions yield the best diagnostic results. [Figure in color on CD-ROM]. Fig. IC.6. CNS blastomycosis in an AIDS patient. Diagnosis may require aspiration of the abscesses if no active pulmonary or cutaneous disease is present. 400 Instructive Cases hospitalization, he received a total of 1600 milligrams of amphotericin B. Serial CT scans documented progressive improvement of brain abscesses and oral fluconazole was substituted after the full course of amphotericin B and the patient was discharged to home. He completed 8 more weeks of fluconazole therapy at a dose of 800 mg/day. Although the brain abscesses had resolved on CT scan, a maintenance suppressive dose of fluconazole of 400 mg per day was initiated. HAART was continued and a clinical response was documented by a falling HIV viral load and rising CD4+ T lymphocyte count. INSTRUCTIVE CASE 7 A 45-year-old construction worker presented with a 3-week history of fever, chills, myalgias, headache, and dyspnea. Examination was unremarkable and laboratory tests revealed a white blood cell count of 3300 cells/l and a platelet count of 94,000 cells/l. Aspartate transaminase (AST) was 87 units/l, alkaline phosphatase 337 units/l. Angiotensin converting enzyme was elevated at 213 units/l. Chest and abdomen CT showed small nodules in the lungs, enlarged mediastinal lymph nodes, small pleural effusions, and splenomegaly. Transbronchial biopsies of the subcarinal lymph node and right lower lobe and bone marrow biopsy showed noncaseating granuloma. Histopathology was negative for fungi and cultures were negative after 1 week of incubation. Prednisone 60 mg daily was prescribed for presumed sarcoidosis, which resulted in resolution of fever and improvement of dyspnea. The prednisone dosage was tapered over 4 weeks to 10 mg daily, which was maintained. Two months later the patient complained of recurrent fever, 10-pound weight loss, and worsening dyspnea. Chest CT showed more extensive diffuse interstitial infil- trates and increasing splenomegaly. Hemoglobin was 9.3 g/dl, white blood cell count 2500 cells/l, and a platelet count of 89,000 cells/l. Alkaline phosphatase was 987 units/l. Cultures from the lung tissue, lymph nodes, and bone marrow performed during the earlier admission were negative after 4 weeks of incubation. Bronchoscopy was performed and cytology revealed small yeastlike structures resembling Histoplasma capsulatum, which was later confirmed by culture. Treatment was started with ampho- tericin B, and the patient noted progressive improvement. INSTRUCTIVE CASE 8 A 52-year-old man who underwent renal transplantation 2 years earlier presented with a slightly painful mass above his right knee. This mass began as a small lesion 4 weeks ago and has been slowly enlarging since first noted by the patient. He denies fever, chills, night sweats, or trauma to the area. He is taking tacrolimus and prednisone as his immunosup- pressive regimen. The patient was afebrile with stable vital signs, but his physical exami- nation was significant for a 1.5 cm firm nodule above his right knee, which was slightly tender, but without erythema or drainage. Routine laboratory studies are unremarkable. Surgery was consulted for excision of the nodule, which was performed without complications. Pathology showed chronic inflammation and pigmented fungal elements suggestive of phaeohyphomycosis; margins were clear of infection (Fig. IC.7). Culture of the specimen grew Exophiala jeanselmei. He was given itraconazole for 3 months, and no further lesions appeared. Instructive Cases 401 Fig. IC.7. Histopathological examination of subcutaneous nodule showing granulomatous changes and dark-walled fungal elements. Phaeohyphomycosis. [Figure in color on CD-ROM]. INSTRUCTIVE CASE 9 A 34-year-old African American man, who has been followed for approximately 10 years, originally presented with back pain and tenderness over the right sternoclavicular joint. Coccidioidomycosis was suspected and a serology was obtained with an initial complement fixation (CF) titer of 1:256. A whole body bone scan showed focal areas of increased uptake involving the left frontal bone, the right sternoclavicular joint, the left sacroiliac joint, and the cervicothoracic region. These findings were confirmed with plain radiograph and CT scans. Approximately 1 month after original presentation, an incision and drainage of the proximal clavicle and the posterior superior spine of the iliac crest was performed. Cultures were positive for Coccidioides immitis. Three months after presentation, a cervical corpectomy of C5 and C6 with autograft fusion and anterior cervical plating was performed. Five months after presentation, decompression of the spinal cord with corpectomy at T3 and T4 with autologous rib grafting was performed. Medical therapy initially consisted of amphotericin B, which was infused over a period of approximately 4 months, three times per week. Follow-up therapy with high- dose oral fluconazole (1000 mg daily as a single dose) was administered. As the patient improved clinically, CF titers steadily came down, and the dose was deescalated to 400 mg of fluconazole daily. With approximately 10 years of follow-up, no further evidence of disease has been discerned. Complement fixation titers in the recent past have remained positive at 1:2. It has been recommended that he remain on therapy for life. 402 Instructive Cases INSTRUCTIVE CASE 10 A 68-year-old man with a long standing history of hairy cell leukemia was admitted to receive treatment with the experimental immunotoxin BL-22. He had been pancytopenic for several months and had been receiving antifungal prophylaxis with itraconazole. On admission, antifungal prophylaxis was changed to fluconazole. On day 2 the patient developed fever. At that time, his absolute neutrophil count was 76 cells/l. Blood cultures recovered a highly susceptible E. coli and the patient defer- vesced promptly on ceftazidime. On day 7 a new fever developed and meropenem, tobramycin, and caspofungin were started. A chest CT was obtained which showed a 3 cm right upper lobe (RUL) nodule (Fig. IC.8). A bronchoalveolar lavage (BAL) was performed, and voriconazole and levofloxacin were added. Bacterial, fungal, and viral cultures, as well as Gram, calcofluor white, acid-fast, modified-acid-fast, and Gomori methenamine silver (GMS) stains were negative. Polymerase chain reaction (PCR) for Pneumocystis, Chlamydophila, Mycoplasma, and Legionella was also negative. The patient continued to have fever up to 40oC without new symptoms or hypotension. On day 11 he complained of chest pain and dry cough. A repeat CT showed marked enlargement of the nodule with development of a halo sign and abutting of the fissure (Fig. IC.9). A fine-needle aspirate of the mass showed broad, ribbonlike, nonseptate hyphae (Fig. IC-10). Voriconazole was discontinued and liposomal amphotericin B 7.5 mg/kg per day was started. The patient developed hemoptysis and a repeat CT showed further progression of the mass, but with apparent localization in the RUL Fig. IC.8. CT scan of chest revealing small pulmonary nodule in the right upper lung. Instructive Cases 403 Fig. IC.9. CT scan of chest revealing progression of pulmonary nodule in right upper lung field to involve the right pleural surface. Fig. IC.10. Calcofluor staining of fine-needle aspiration demonstrates broad nonseptate hyphae consistent with zygomycosis. [Figure in color on CD-ROM]. 404 Instructive Cases Fig. IC.11. CT scan of chest with severe advancement of locally progressive disease in the right upper lung fields. (Fig. IC.11). An emergent right upper lobectomy was performed on day 18 (Fig. IC.12). An angioinvasive |
mould was readily seen on the GMS stain (Fig. IC.13), and was later identified as Rhizomucor pusillus. On the day of the surgery, granulocyte transfusions were initiated. Hemoptysis and fever resolved 2 days after the surgery. Despite local control of the fungal disease, the patient never recovered his white blood cell counts and over the next 6 weeks developed several complications in the ICU, including herpes simplex pneumonia. At his own request, care was withdrawn. Fig. IC.12. Gross pathology of right upper lobectomy. [Figure in color on CD-ROM]. Instructive Cases 405 Fig. IC.13. Zygomycosis with hyphae invading blood vessel, infiltrating vascular wall, and causing subsequent thrombosis. GMS. [Figure in color on CD-ROM]. INSTRUCTIVE CASE 11 A 48-year-old male rural worker was referred for consultation to evaluate the presence of painful, ulcerated lesions in the external region of his right foot (Fig. IC.14). This process had gone on for 18 months, and multiple local and systemic treatments had been given without success. The patient looked well and had no other symptoms. Physical examination also revealed the presence of an oral mucosal ulceration and of hypertrophied cervical lymph nodes. Lung auscultation found fine rales and the chest radiograph showed interstitial infiltrates in the central fields with fibrous zones and basal bullae (Fig. IC.15). Direct KOH examination of ulcer exudate revealed multiple budding yeasts consistent with Paracoccidioides brasiliensis; cultures later grew the fungus. The patient’s serum gave a band of precipitate and a CF titer of 1:32 with paracoccidioidin. INSTRUCTIVE CASE 12 A 40-year-female with acute myelogenous leukemia now 115 days following matched allogeneic donor hematopoietic stem cell transplantation complicated by a suspected Aspergillus pneumonia presents to the clinic with increasing complaints of nausea, stomach cramping, and rash on the hands spreading up her arms. By laboratory examination, she is noted to have an alanine transaminase (ALT) of 85 units/l, AST 75 units/l, and total bilirubin of 2.1 mg/dl. Her current medica- tions include tacrolimus 5 mg twice daily (recent level 8 ng/ml), voriconazole 200 mg twice daily, levofloxacin 500 mg daily, valacyclovir 500 mg twice daily, metoprolol 25 mg twice daily and benzonatate (tesselon) pearls. She is 406 Instructive Cases Fig. IC.14. Paracoccidioidomycosis. Multiple ulcerated lesions in the right foot with crusting, exudation, and hemorrhagic dots. The borders are granulomatous and show some scarring. [Figure in color on CD-ROM]. admitted to the hospital for suspected graft versus host disease exacerbation. The primary service wishes to continue voriconazole therapy as this patient has a history of poorly tolerating lipid amphotericin B formulations, but they are concerned about the possibility of drug-induced hepatitis caused by voriconazole. Questions 1. Which of the following approaches should be recommended to the primary team to manage the suspected drug-induced hepatotoxicity? A. Discontinue voriconazole and switch to itraconazole. B. Discontinue voriconazole and switch to lipid amphotericin B with premedications. C. Continue voriconazole and lower dose by 50%. D. Continue voriconazole and closely monitor the patient. INSTRUCTIVE CASE 13 A 44-year-old male field worker was in his usual state of health until 2 weeks prior to admission, when he developed headache, low-grade fever, nausea, and vomiting. His family noted decreased mentation and brought him to the emergency room. Initial examination revealed a disheveled gentleman with lethargy and disorientation. A CT scan of the head was performed without contrast and was normal. A lumbar puncture revealed 450 white cells/l, of which 76% were lymphocytes, 10% monocytes, 10% neutrophils, and 4% eosinophils. The protein was 176 mg/dl and the glucose was 27 Instructive Cases 407 Fig. IC.15. Paracoccidioidomycosis. Bilateral interstitial infiltrates in central fields, basal bullae, and fibrous areas. The apices appear free of disease. mg/dl. Studies were sent to rule out cryptococcosis, tuberculosis, and coccidioidomy- cosis. Coccidioidal antibody in the serum was 1:256 and in the cerebrospinal fluid (CSF) was 1:2. Other fungal and mycobacterial laboratory evalutions were negative. Magnetic resonance imaging (MRI) of the brain was done to evaluate for meningeal involvement or vasculitic complications of coccidioidomycosis. This study revealed basilar cisternal enhancement. A bone scan was ordered and was negative for bony involvement. An HIV test was performed and confirmed as positive. The HIV RNA PCR was 330,000 copies/ml with an absolute CD4+ T lymphocyte count of 61 cells/l. The patient was placed on high-dose oral fluconazole (1000 mg daily as a single dose). Highly active antiretroviral therapy (HAART) was also initiated and patient was discharged to be followed in clinic. Patient and family were counseled regarding strict adherence to fluconazole and HAART therapy, both of which needed to be life-long. He also underwent lumbar punctures monthly, CSF was sent for cell count, glucose, protein, complement fixation (CF) titers, and fungal culture. Serum for fluconazole level was sent when the possibility of noncompliance was considered. The patient followed up with appointments inconsistently. Four months after admission, he was brought to the Emergency Room with confusion, lethargy, nausea, vomiting, and ataxic gait. Evaluation again showed encephalopathy. CT scan now demonstrated 408 Instructive Cases ventriculomegaly. Lumbar puncture revealed an elevated opening pressure of 250 mmH2O, white cell count 50 cells/l (85% lymphocytes, 11% neutrophils), protein 153 mg/dl, and glucose of 10 mg/dl. HAART therapy and high dose fluconazole were reinitiated. Neurosurgical consultation was obtained and the patient was taken to the operating room for ventriculoperitoneal shunting. The patient’s symptoms improved significantly after shunting, but his clinical course was complicated by hyponatremia secondary to syndrome of inappropriate antidiuretic hormone secretion (SIADH), which was managed with fluid restriction. He gradually improved and was discharged with close follow-up at clinic. INSTRUCTIVE CASE 14 A 22-year-old female with a failing 4-year-old renal allograft received several doses of OKT3 and high doses of corticosteroids in an attempt to reverse the acute rejection of the transplanted kidney. Three months after this increased immunosuppressive trial and still receiving her normal immunosuppressive regimen of tacrolimus, mycophenolate, and prednisone, she presented with a several week course of headaches, nausea, and vomiting. Her temperature was 37.2oC, and although mental status was normal, she had bilateral clonus and papilledema on physical examination. Her laboratory results showed a normal complete blood count and a serum creatinine of 4 mg/dl. An MRI of her brain demonstrated basilar inflammation and lumbar puncture (LP) revealed a white blood cell count of 100 cells/l with 80% lymphocytes. CSF glucose was 43 mg/dl and protein 79 mg/dl. India ink was positive for encapsulated yeasts, CSF cryptococcal polysaccharide antigen test was 1:256, and culture grew Cryptococcus neoformans. Her opening pressure was 400 mm H2O. She was started on 5 mg/kg per day of AmBisome for 20 days and flucytosine at 25 mg/kg per day for 14 days, and then placed on 200 mg/day of fluconazole. The patient’s symptoms did not worsen and she was reevaluated at 2 weeks with a repeat LP. That LP found an opening pressure of 140 mm H2O, and India ink and culture were negative. CSF antigen was 1:256 and CSF white cell count was 28 cells/l. Case Continued Patient did relatively well on her suppressive fluconazole therapy at 200 mg/day for 4 months (dosed for reduced renal function), when she developed severe headaches and an MRI scan showed diffuse supra- and infratentorial leptomeningeal enhancement. At that time CSF cryptococcal polysaccharide antigen was 1:16, white blood cell count was 100 cells/l, and cultures negative. After 2 weeks of AmBisome at 5 mg/kg per day and no improvement, the patient was continued on fluconazole and a 6 week dexamethasone taper was begun with immediate improvement in symptoms. Tacrolimus and mycophenolate were stopped and the patient was started on dialysis. One week after stopping the 6-week taper of corticosteroids, her headaches returned and a 4-month steroid taper was begun. She improved and was eventually weaned off corticosteroids and now has received suppressive fluconazole for 1 to 2 years and is doing well awaiting a new transplant. Instructive Cases 409 INSTRUCTIVE CASE 15 A 47-year-old black man presented to the University Hospital emergency department complaining of pleuritic chest pain for 2 weeks before admission. He subsequently developed severe dyspnea on exertion, fever and chills, and productive cough with hemoptysis. Over the 24 hours preceding admission, pleuritic chest pain, which was initially only on the left side, became bilateral and he presented to the Emergency Department for evaluation. Past medical history was pertinent for hyperthyroidism and cigarette smoking. He denied any risk factors for HIV infection. In the emergency department, he was in moderate respiratory distress but was afebrile. Chest radiograph revealed diffuse bilateral miliary infiltrates with a masslike lesion in the lung field. White blood count was 14,400 cells/l with a left shift. Arterial blood gases on room air revealed a pH of 7.41, po2 of 33 mm Hg, and pco2 of 46 mm Hg. The patient was admitted to the hospital and placed in respiratory isolation. Differ- ential diagnosis included severe community-acquired pneumonia, atypical pneumonia, miliary tuberculosis, and fungal disease. He was placed on supplemental oxygen, intravenous azithromycin and ceftriaxone, and a four-drug antituberculous treatment regimen. Multiple sputum samples were remarkable only for many polymorphonuclear leukocytes. Direct fluorescent antibody (DFA) and urinary antigen tests were negative for Legionella. Likewise, fungal and acid-fast stains of sputum samples were negative. An ELISA for HIV was also negative. On hospital day 3 the patient became febrile, a chest radiograph revealed worsening bilateral pulmonary infiltrates, and intravenous trimethoprim–sulfamethoxazole was added to the existing antibacterial regimen as empiric therapy for possible Pneumocystis pneumonia. On the fifth hospital day he complained of increasing dyspnea and was noted as having a respiratory rate of 60 breaths per minute. Arterial blood gases on 100% oxygen by face mask revealed a pH of 7.45, po2 of 102 mm Hg, and pco2 of 42 mm Hg. The patient was transferred to intensive care unit, where he was intubated and placed on mechanical ventilation. Chest radiographs revealed bilateral pulmonary infiltrates (Fig. IC.16) with acute lung injury. Cytology samples obtained via the endotracheal tube at the time of intubation revealed numerous broad-based budding yeast forms compatible with Blastomyces species (Fig. IC.17). The patient received intravenous amphotericin B immediately at a dose of 0.7 mg/kg per day, but was rapidly increased to 1 mg/kg per day. During the remainder of his hospitalization, the patient became increasingly difficult to oxygenate, developed hypotension requiring pressers, progressed to multiorgan failure, and died with pulseless electrical activity. INSTRUCTIVE CASE 16 An 18-year-old female presented with chest discomfort. Dilated veins were noted over the chest and upper abdomen. A chest radiograph that showed right hilar enlargement, which was calcified on CT. The right pulmonary artery was narrowed and the superior vena cava was occluded. Ventilation–perfusion lung scan that showed reduced blood flow to the right lung. Pulmonary function tests showed normal air flow and lung capacity. Tuberculin skin test was negative. Histoplasma immunodiffusion 410 Instructive Cases Fig. IC.16. Diffuse pulmonary infiltrates in a patient with ARDS. Patients presenting with this syndrome have a mortality rate greater than 50%. tests showed an M band and the Histoplasma complement fixation test showed titers of 1:32 to the yeast and 1:16 to the mycelial antigen. Mediastinal biopsy showed chronic inflammatory cells, granuloma, and fibrosis. Culture of the mediastinal biopsy tissue was negative for fungus. Fig. IC.17. Cytology preparation of endotracheal tube specimen revealing large, thick- walled yeasts consistent with Blastomyces dermatitidis. [Figure in color on CD-ROM]. Instructive Cases 411 Question Is surgery indicated to correct the obstruction of the pulmonary artery or of the superior vena cava, and should the patient receive a course of antifungal therapy? INSTRUCTIVE CASE 17 A 41-year-old male rural worker and heavy smoker consulted because of 3 months of dry cough, severe progressive dyspnea, weight loss, asthenia, adynamia, and anorexia. He looked emaciated and experienced difficulties in breathing even at rest. Respiratory rate was noted to be 36 breaths per minute with accessory muscles utilization. On auscultation rales, rhonchi and hypoventilation were noticed. The chest radiograph (Fig. IC.18) revealed the presence of a diffuse reticulonodular infiltrates predominating in both central fields with fibrous lines. Follow-up CT of chest documented widespread fibrosis (Fig. IC.19). Arterial blood gas analysis revealed pH 7.44, po2 37 mm Hg, pco2 23 mm Hg, O2 saturation 81%, and bicarbonate 16 mEq/l. A bronchoalveolar lavage fluid sample was examined for acid-fast bacilli with negative results, but multiple budding cells corresponding toParacoccidioides brasiliensis were seen on direct examination and |
recovered in culture later on. Serologic tests with paracoccidioidin were reactive with one band of precipitate and a titer of 1:1024 in the complement fixation (CF) test. Fig. IC.18. Paracoccidioidomycosis. Lung fibrosis involving specially the central field with apices appearing free. Bilateral basal bullae and pleural adhesions are seen in both lower fields. 412 Instructive Cases Fig. IC.19. Par acoccidioidomycosis. High-resolution CT showing widespread fibrosis with honeybee aspect, bullae formation. and pleural thickening in both lower lung fields. INSTRUCTIVE CASE 18 A 51-year-old man was newly assigned to a construction project in Kern County. Two weeks after he began his assignment, he developed cough, shortness of breath, fever, chills, and night sweats. His physician obtained a chest radiograph, which showed consolidation in the anterior left upper lobe and lingula, associated with adenopathy of the lateral aortic, left hilar, and pericarinal nodes. He was diagnosed with community- acquired pneumonia and prescribed antibiotics. One week later, he had not improved and went to the local emergency room, where he was found to be febrile but not hypoxic. His white blood count was 9400 cells/l, with an absolute eosinophil count of 959 cells/l. He was prescribed oral levofloxacin 750 mg daily and discharged to home. Serum for Coccidioides serology was collected at this visit and reported 5 days later: EIA IgM positive; immunodiffusion (ID) IgG negative, ID IgM positive; and CF titer (IgG) 1:2. These results were suggestive of early disease, but were not communicated to the patient and possibly misinterpreted as negative. Almost 3 weeks after initial presentation, the patient developed increasing shortness of breath, became hypoxic, somewhat confused, with high fevers, and drenching sweats. He presented to our emergency room. His subsequent Coccidioides CF titer was 1:64. Since he was hypoxic with a po2 of less than 70 mm Hg, he was started on a lipid amphotericin preparation at 5 mg/kg daily as well as oral prednisone at a dose of 40 mg twice daily for 5 days, followed by 40 mg once daily for 5 days, then 20 mg once daily for 11 days. He improved and was discharged home with continuation of amphotericin for 6 weeks, three times per week, and then switched to oral fluconazole at 800 mg per day. He was followed monthly. He developed a painful left shoulder while on therapy. Bone scan was negative and the shoulder pain was considered to be fluconazole Instructive Cases 413 shoulder syndrome. Therapy was changed to Itraconazole, 200 mg twice daily. He complained of headache, which eventuated in a lumbar puncture which proved to be unremarkable. He gradually improved clinically and radiologically. His CF titers decreased to 1:2, and remained at this level for three readings, at which point the itraconazole was discontinued almost exactly 1 year from initial presentation. He was followed every 3 months for 1 year with serology after completing treatment with no rise in CF titer or clinical evidence of recurrence. Instructive Cases Discussion INSTRUCTIVE CASE 1 Answers: 1. B, 2. C Discussion Question 1 Amphotericin B-induced nephrotoxicity occurs primarily through two mechanisms: (1) constriction of afferent arterioles leading to direct decreases in glomerular filtration rate (GFR; glomerular toxicity) and (2) direct damage to the distal tubules (tubular toxicity; answer B), which in turn can lead to glomerular feedback that further constricts the afferent arterioles. Tubular toxicity of amphotericin B is essentially limited to the distal tubules and most commonly evident as hypokalemia (answer B). It occurs in the majority of patients receiving amphotericin B and may require up to 15 mmol of supple- mental potassium per hour. Amphotericin B-induced hypokalemia is not associated with increased plasma aldosterone or renin levels and appears to result from increased perme- ability of the distal tubular cells due to direct toxic effects of amphotericin B. Although lipid amphotericin B formulations reduce distal tubular toxicity, they do not eliminate this side effect. Distal tubular toxicity (decreases in serum potassium and magnesium) frequently precede decreases in glomerular filtration rate (increases in serum creatinine, BUN) during amphotericin B therapy, particularly in patients receiving lipid ampho- tericin B formulations. Afferent arteriole constriction (answer A) would be more specif- ically associated with decreases in glomerular filtration (decrease in serum creatinine). Suppression of erythropoietin synthesis (answer C) is a more chronic effect of ampho- tericin B and manifests primarily as normochromic, normocytic anemia. Amphotericin B has not been shown to directly damage pancreatic islet cells (answer D). Question 2 Nephrotoxicity is the dose-limiting toxicity of amphotericin B therapy. Although all of the answers are potential approaches that have been applied to prevent the development of nephrotoxicity during amphotericin B therapy, switching to a lipid amphotericin B formulation (answer C) is advocated by most infectious diseases experts once glomerular toxicity has developed during amphotericin B therapy. Alternative daily dosing of amphotericin B (answer A) is no longer recommended and there is no evidence that this method is less nephrotoxic than daily dosing. Administering amphotericin B by continuous infusion (answer B) has been shown in two small prospective trials to reduce infusion-related and nephrotoxic side effects; however, this dosing method is not practical and the efficacy of amphotericin B administered by continuous infusion has not been well explored. Saline loading (answer D) can reduce 415 416 Instructive Cases Discussion tubular-glomerular feedback and delay the onset of glomerular toxicity; however, it will not reverse glomerular toxicity once it has developed. Instructive case 1 contributed by R. E. Lewis and A. W. Fothergill INSTRUCTIVE CASE 2 Discussion This case is somewhat unusual in that the site of inoculation of Sporothrix schenckii had received a skin graft, and thus the lesions were atypical. They arose in the grafted area and contributed to loss of portions of the graft. The lack of response to antistaphy- lococcal antibiotics was a clue that this was not a typical bacterial infection. The possibility of sporotrichosis was raised by the infectious diseases consultant because of the proximal spread of nodules and the exposure to soil during the original accident. Biopsy confirmed this suspicion when the cultures yielded a mould. The fact that no organisms were seen on the biopsy is not unusual. Fluconazole was used because an experimental protocol was available at the time, and the patient had no insurance and was unable to purchase other antifungal agents. The response to fluconazole was adequate, but slower than usually noted with itraconazole, which is the treatment of choice for sporotrichosis. Instructive case 2 contributed by C.A. Kauffman INSTRUCTIVE CASE 3 Discussion This case demonstrates how an apparently uncomplicated coccidioidal pneumonia can evolve into complex multifocal disseminated disease. Such cases frequently neces- sitate multiple therapy modalities. Instructive case 3 contributed by R. H. Johnson and S. Baqi INSTRUCTIVE CASE 4 Discussion This is an example of the juvenile type paracoccidioidomycosis. Instructive case 4 contributed by A. Restrepo, A. M. Tobón, and C. A. Agudelo INSTRUCTIVE CASE 5 Discussion This case is instructive for several reasons. The patient’s past medical history is significant for a long history of immunosuppression due to long-term corticosteroid use and his diabetes mellitus. Both of these conditions predispose the patient to an increased risk of fungal infections because of alterations in cell-mediated immunity. The case demonstrates the increasing incidence and the capacity of previously nonpathogenic yeast to produce invasive infection. In addition, the presentation of the patient with Instructive Cases Discussion 417 nonspecific signs and symptoms is not uncommonly seen in many invasive fungal infections. In fact, the problem with the diagnosis of invasive fungal infections is that there are no “classic or pathognomonic manifestations.” This makes the diagnosis difficult to establish and creates a delay in the initiation of appropriate antifungal therapy. In this case, the aspiration of the infected knee demonstrated several organisms (gram-negative bacilli and yeast), thus assisting with the diagnosis. However, Candida, not Trichosporon would have been the more common cause of infection in this patient. It is not until the laboratory identifies the organisms that the true diagnosis is estab- lished. Although there are no clinical trials establishing the best antifungal agent for Trichosporon species, the azoles (fluconazole, voriconazole) have been shown to have in vitro activity. In this case, in vitro susceptibility results demonstrated that both fluconazole and itraconazole had good activity. Although there are no established breakpoints for the echinocandins, the minimum inhibitory concentration (MIC) of 2.0 g/ml for caspofungin appears to be within the “standard” ranges described for clinical activity against Candida species. Further, although there are some in vitro and animal studies demonstrating additive or synergistic activity with the combination of echinocandins and azoles, the use of combination antifungal therapy has not been demonstrated to be any better than monotherapy. Instructive case 5 contributed by J. A. Vazquez INSTRUCTIVE CASE 6 Discussion Blastomycosis in AIDS 1. Blastomycosis in acquired immunodeficiency syndrome (AIDS) patients is more likely to be multiorgan disease, with CNS involvement being noted in up to 40% of patients. Routine magnetic resonance imaging (MRI) or CT scans should definitely be performed in any patient with severe end-stage AIDS whether or not the patient has neurologic signs or symptoms. 2. Wet preparations of skin lesions allowed a presumptive clinical diagnosis and early initiation of amphotericin B therapy in this patient. 3. Central nervous system disease in end-stage AIDS patients should be treated with a full course of amphotericin B, e.g., 1.5 to 2.5 grams. 4. Fluconazole at high doses (800 mg/day) may be a reasonable substitute in patients intolerant to amphotericin B or as step down therapy in patients who have responded to initial treatment with amphotericin B. 5. In immunosuppressed patients, chronic suppressive therapy with fluconazole should be considered. Instructive case 6 contributed by S. W. Chapman and D. C. Sullivan INSTRUCTIVE CASE 7 Discussion This case represents acute pulmonary histoplasmosis misdiagnosed as sarcoidosis. It illustrates the importance of thorough testing to exclude histoplasmosis before 418 Instructive Cases Discussion beginning immunosuppressive treatment for sarcoidosis. While there are clinical features that help to distinguish sarcoidosis from histoplasmosis, differentiation requires laboratory testing to exclude histoplasmosis. Administration of corticos- teroids resulted in transient clinical improvement, followed by progression with worsening pulmonary disease and progressive dissemination. Failure to demonstrate yeast resembling H. capsulatum on the initial bronchoscopy resulted in a mistaken diagnosis of sarcoidosis. Of note is that cytology and culture of respiratory secre- tions are often negative in acute histoplasmosis, and cannot be used to exclude the diagnosis. Additional testing should include serology for antibodies to H. capsulatum, and tests for Histoplasma antigen in urine and respiratory secretions. Serology is often negative during the first month after exposure, but positive thereafter. Antigen may be detected in the urine or bronchoscopy specimen of 75% of cases during the acute illness and before antibodies have appeared. Corticosteroids for sarcoidosis should not be given without thorough evaluation to exclude histoplasmosis. Of note is that fungal cultures require up to 4 weeks of incubation for isolation of H. capsulatum, during which corticosteroids should be withheld except in severe cases. Tests for antigen and antibody, and cytology on bronchial washing or bronchoalveolar lavage, should be performed and may provide early evidence for histoplasmosis. Of note, these tests do not exclude the diagnosis. If lung or other tissues are biopsied, they also should be cultured and examined for fungus by histopathology. If cytology, histopathology, antigen testing, and serology are negative and corticos- teroids are required for severe sarcoidosis, itraconazole may be given while waiting for culture results in selected patients in whom the diagnosis of histoplasmosis is suspected based on epidemiologic grounds. Instructive case 7 contributed by L. J. Wheat and N. G. Conger INSTRUCTIVE CASE 8 Discussion Subcutaneous phaeohyphomycosis is among the most common manifestations of disease due to dematiaceous fungi. It is seen in both immunocompetent and immuno- compromised individuals and is not usually associated with dissemination, although the risk of dissemination is higher in immunosuppressed patients. Complete excision alone has been reported as a successful therapy, particularly in immunocompetent patients. In immunocompromised patients, antifungal therapy is often given after surgical excision to reduce the risk of dissemination. However, itraconazole and voriconazole both have significant interactions with immunosuppressive agents such as tacrolimus and sirolimus, and combined use of these drugs requires close monitoring and commonly adjustment of the immunosuppressive agents. Instructive case 8 contributed by S. G. Revankar Instructive Cases Discussion 419 INSTRUCTIVE CASE 9 |
Discussion This case illustrates the importance of surgery in the management of complex axial and nonaxial osteomyelitis secondary to coccidioidomycosis. Instructive case 9 contributed by R. H. Johnson and S. Baqi INSTRUCTIVE CASE 10 Discussion Nodular infiltrates in the neutropenic patient are often caused by pathogenic fungi. Identification is critical for proper management. This case illustrates that pulmonary zygomycosis in a neutropenic host may be associated with fever but a paucity of other findings on initial presentation. A BAL is often negative and more invasive procedures, such as a fine needle aspiration, may be necessary to establish a diagnosis. During neutropenia, pulmonary zygomycosis may progress rapidly, despite amphotericin B therapy. Surgery has an important role in these patients, as it may be the only way of controlling this angioinvasive infection. Granulocyte transfusions may have a role to gain time until neutropenia resolves. Ultimately, however, recovery from these infec- tions is often contingent on recovery of bone marrow function. In the case presented, control of the pulmonary infection was achieved with combined medical and surgical intervention. Unfortunately, the patient ultimately succumbed to complications of his hairy cell leukemia. Instructive case 10 contributed by C. Antachopoulos, J. C. Gea-Banacloche, and T. J. Walsh INSTRUCTIVE CASE 11 Discussion This patient’s case is an example of chronic, adult multifocal paracoccidioidomy- cosis. Instructive case 11 contributed by A. Restrepo, A. M. Tobón, and C. A. Agudelo INSTRUCTIVE CASE 12 Answer: D Discussion Unpredictable, low-frequency idiosyncratic liver failure with azoles occurs on a background of a much higher frequency of mild asymptomatic liver injury. Mild liver injury is exacerbated by concomitant medications or dominated by other disease states (as in this case with the graft versus host disease that often affects the liver). Because mild liver injury is generally reversible and transient, immediate discontinuation of voriconazole is not necessary (answer A or B). In clinical trials, voriconazole was continued in the majority of patients with elevated serum transaminases until they 420 Instructive Cases Discussion reached greater than 3 times the upper limit of normal (which has not been reached in this patient). Because the patient will likely receive corticosteroids for the graft versus host disease reactivation, continuation of voriconazole and monitoring of liver function tests with the initiation of steroid therapy (answer D) would be the most reasonable approach. Although reduction of the voriconazole dose is an option (answer C), reducing antifungal intensity in an immunocompromised patient with active graft versus host disease and receiving steroids is undesirable. Many clinicians would poten- tially add a second antifungal agent in this patient if she had other signs of infection (i.e., pulmonary nodules in lung). Instructive case 12 contributed by R. E. Lewis and A. W. Fothergill INSTRUCTIVE CASE 13 Discussion This case demonstrates the presentation and evaluation of coccidioidal meningitis and the management of two common complications, hydrocephalus, and SIADH. Instructive case 13 contributed by R. H. Johnson and S. Baqi INSTRUCTIVE CASE 14 Discussion For induction therapy, she had received combination therapy with amphotericin B in a lipid formulation and a reduced dose of flucytosine because of kidney dysfunction. Her clinical response did not require repeated lumbar punctures to control raised intracranial pressure because symptoms did not worsen and actually improved. Her response to antifungal combination therapy was appropriate with a negative CSF culture at the end of 2 weeks of induction therapy. Discussion In this case, the patient developed cryptococcal meningitis after receiving severe therapeutic immunosuppression in an attempt to save her renal transplant from rejection. She initially responded to potent antifungal combination therapy, which was adjusted to renal dysfunction. Initially she did well on this suppressive therapy, but as she completely lost kidney function and immunosuppressive therapy was reduced she again developed meningeal symptoms and signs, but the work-up did not reveal evidence of an ongoing viable yeast infection. It was then decided that this may represent immune reconstitution inflammatory syndrome (IRIS) and she was started on corticosteroids, which improved and eventually resolved her symptoms. This case illustrates the dynamic relationship between the immune system and cryptococcosis. Although there are standardized, well-studied antifungal treatment regimens, there are clearly times when clinical judgment must be used regarding management. Currently management of increased intracranial pressure and IRIS is performed without the luxury of guidance from robust evidence-based studies. Instructive case 14 contributed by M. Chayakulkeeree and J. R. Perfect Instructive Cases Discussion 421 INSTRUCTIVE CASE 15 Discussion Blastomycosis Presenting with Acute Respiratory Distress Syndrome (ARDS) 1. Patients presenting with severe pulmonary disease, whether miliary or ARDS, have a high rate of mortality (> 50%). All patients presenting in this fashion should be initially treated with amphotericin B. 2. Life-threatening pulmonary disease may be seen in nonimmunocompromised patients. Hence, blastomycosis must be considered in any patient living in or with recent travel to the endemic area and who presents with severe or over whelming pneumonia. 3. Most patients who die do so within the first week of therapy, emphasizing that ampho- tericin B therapy should be initiated as soon as possible after diagnosis. 4. Cytology may have a higher diagnostic yield than expectorated sputum examined under wet preparation. Instructive case 15 contributed by S. W. Chapman and D. C. Sullivan INSTRUCTIVE CASE 16 Discussion Fibrosing mediastinitis (FM) results from excessive scarring around the hilar and mediastinal lymph nodes. This scar tissue extends from the lymph nodes to invade important nearby structures, such as the pulmonary arteries or veins, bronchial arteries, vena cava, trachea, main stem and lobar or segmental bronchi, esophagus, pericardium, and even the heart. FM represents a scarring response to a prior episode of histoplas- mosis rather than an active and progressive infection. The severity of the illness depends on the extent of the scarring, and the specific structures that are involved. In many cases, the consequences are mild and nonpro- gressive, causing minimal or no limitation to function, and requiring no consideration for therapy. In others, symptoms may be more severe, or even disabling, prompting consideration of the treatment options. In patients with extensive involvement in both lungs, the illness is progressive and eventually fatal in nearly half of cases. Medical treatment with antifungal drugs that are used for treatment of other types of histoplasmosis is not effective in patients with fibrosing mediastinitis, because the manifestations of FM are caused by the scar tissue, not by active infection, and scar tissue is not affected by any medical treatments. Minimally invasive procedures to open the blockages are useful in some cases. These procedures are relatively safe and sometimes effective, although the long-term results are not fully understood. The largest experience has been with stenting of the occluded blood vessels. Stenting of obstructed airways is not believed to be as useful, and there is little experience with this procedure. Stenting or dilatation of obstructed vessels is not always successful because the fibrotic tissue may be as hard as stone, preventing passage of a small wire past the blockage. Active bleeding may be stopped by embolization of the involved blood vessel. Surgical correction of the obstructed blood vessel or airway also is not often possible, and carries a high operative mortality. Thus, surgery should be reserved for severe cases 422 Instructive Cases Discussion and only then after less risky procedures are tried. The operative mortality is at least 20% overall, but can range from 50% to 75% in patients who undergo total removal of the involved lung (pneumonectomy). Of note is that the operative mortality may be even higher if the surgeon is not experienced with treatment of fibrosing mediastinitis. The reason that the mortality is so high is that the scar tissue is like cement, encasing the blood vessels and airways and obliterating tissue planes. Vital structures are often damaged while attempting to remove the scar tissue, causing bleeding or airway leaks. Death is caused by uncontrollable bleeding, respiratory failure, infection, and other post-operative complications. The indications for surgery are not well described, but at a minimum should include patient limitations severe enough to justify the risk of the known operative mortality. Surgical indications might include severe and recurrent bleeding not responsive to embolization, recurrent pneumonias that are not preventable by antibiotic prophylaxis, or respiratory failure. Surgery should be conducted only by surgeons who are experienced with fibrosing mediastinitis. Instructive case 16 contributed by L. J. Wheat and N. G. Conger INSTRUCTIVE CASE 17 Discussion This patient’s case is an example of the chronic unifocal pulmonary paracoccid- ioidomycosis. Instructive case 17 contributed by A. Restrepo, A. M. Tobón, and C. A. Agudelo INSTRUCTIVE CASE 18 Discussion This case demonstrates the evolution of pneumonia from mild to severe over a time course characteristic of coccidioidomycosis (but not community-acquired pneumonia). The management of coccidioidomycosis with respiratory failure is also noted. Instructive case 18 contributed by R. H. Johnson and S. Baqi Index Absidia, 25, 46, 117 Aspergillus niger, 183 Absidia corymbifera, 228 Aspergillus terreus, 117, 183 Acervulus, 24 Azole, 109–111, 124–128, 192–193 Acremonium, 208–209 Acremonium falciforme, 208 Basidiobolaceae, 228 Acremonium kiliense, 208 Basidiobolales, 228 Acremonium recifei, 208 Basidiobolus, 29, 228, 234 Acremonium strictum, 208 Basidiobolus ranarum, 234 Actinomycetes, 391 Basidiocarps, 25 Actinomycetoma, 391 Basidiomycetes, 25 Actinomycoticmycetoma, 386 Basidiomycota, 16, 25 Adiaspiromycosis, 48, 50 Basidiospore, 22, 25, 255–258 Aflatoxin, 185 Basidium, 257 Ajellomyces, 277, 333 Beauveria, 201, 210 Ajellomyces dermatitidis, 277 Bifonazole, 374, 378, 379 Alcian blue stain, 266 Biofilms, 202 Aleurioconidia, 46 Bipolaris, 216, 217, 219 Allylamine, 111, 370, 374 Bipolaris hawaiiensis, 29 Alternaria Black piedra, 218, 356 Alternaria alternate, 217 Blastic, 29 Amphotericin B, 109, 117–124, Blastoconidia (sing., blastoconidium), 20, 42, 140, 190–192, 238 164, 331 Anamorph, 16, 22 Blastomyces, 19, 21, 277–279, 286–287 Ancylistaceae, 228 Blastomyces dermatitidis, 19, 21, 277–279, Anidulafungin, 128, 129 286–287 Annellides, 29 Blastomycoma, 287 Annelloconidia, 206 Blastomycosis, 70–72, 277–290 Anthropophilic, 356 Blastoschizomyces, 175–176 Apophysomyces, 227, 228 Blastoschizomyces capitatus, 175 Apophysomyces elegans, 228 Butenafine, 374 Appressed endospores, 44 Butoconazole, 155 Arabinitol, 59 Arthroconidia (sing., arthroconidium), 42, 44, 164, Calcofluor white, 18, 174, 317, 405 296–298, 303, 370 Candida Asci (sing., ascus), 21–23 Candida albicans, 137–139, 152–153 Ascomata (sing., ascoma), 22, 23 Candida dubliniensis, 138 Ascomycete, 20–22, 384 Candida glabrata, 137–138, 152–153 Ascomycota, 22–25 Candida guilliermondii, 129, 137–138 Ascospore, 22, 169 Candida kefyr, 138 Aseptate (syn., nonseptate), 50, 228, 237 Candida krusei, 138, 152–153 Aspergilloma, 82, 86, 91, 185–186 Candida lusitaniae, 117, 137, 152–153 Aspergillosis, 48, 60–63, 181–194 Candida parapsilosis, 129, 138, 152–153 Allergic bronchopulmonary aspergillosis (ABPA), Candida tropicalis, 138–139, 152–153 89, 185 Candidemia, 148–149 Aspergillus, 29 Candidiasis, 55–59, 137–159 Aspergillus flavus, 183 Esophageal candidiasis, 142–144 Aspergillus fumigatus, 183 Hepatosplenic (chronic disseminated) candidiasis, 150 423 424 Index Oropharyngeal candidiasis, 141–142 Curvularia, 216–220, 385 Vulvovaginal candidiasis, 144–145 Curvularia lunata, 385 Candiduria, 145–147, 156 Cyclodextran, 126 Caspofungin, 128–128, 193 Cycloheximide, 18–20, 206, 256, 270 Chaetomium, 216 Chaetothyriales, 384 Dalmau, 20 Charcot-Leyden crystals, 217 Deferoxamine, 7, 229–231, 237 Cheilitis, 142 Dematiaceous, 29, 215 Cheilosis, 142 Dermatomycoses, 17 Chitinase, 72 Dermatophyte, 355–356 Chitosan, 111 Dermatophytoma, 366, 378 Chlamydoconidia, 20, 46 Dermatophytosis, 355–379 Chlormidazole, 105 Dikaryons, 25 Chorioretinitis, 149, 166 Disjunctor, 24 Chromoblastomycosis, 47, 215, 383–391 Dothideales, 386 Chromogenicmedia, 19 Chromomycosis, see chromoblastomycosis Echinocandin, 111, 128–129, 193 Ciclopirox, 153, 374, 375, 377–379 Econazole, 153, 374 Cilofungin, 107 Ecthyma gangrenosum, 204 Cladophialophora Endonyx, 366, 367 Cladophialophora bantiana, 216, 219 Endophthalmitis, 149, 151, 166, 202, 206–208, 210, Cladophialophora carrionii, 386 264, 303 Cladosporium Endospore, 37, 44, 296–297 Cladosporium carrionii (currently Cladophialophora Endosporulation, 297 carrionii) Enolase, 55 Cleistothecia (sing., cleistothecium), 22 Entomophthorales, 25, 29, 227, 229, 230, 234 Clotrimazole, 153, 155, 162, 374, 378 Entomophthoramycosis, 234, 383 Coccidioides Epidermophyton, 355, 356 Coccidioides immitis, 296–297 Epitheliomatoushyperplasia, 387 Coccidioides posadasii, 296–297 Ergosterol. 108–111 Coccidioidin, 73, 333, 335 Erythema multiforme, 299 Coccidioidomycosis, 72–76, 295–312 Erythema nodosum, 298, 299 Coelomycetes, 20, 29 Eschar, 235, 236 Cokeromyces Euascomycetes, 296 Cokeromyces recurvatus, 23 Eumycetoma (syn., eumycotic mycetoma), see mycetoma Colletotrichum, 24 Eurotiales, 386 Columellae, 28 Excysted, 246 Conidiobolus Exoantigen, 305, 324 Conidiobolus coronatus, 234 Exophiala Conidiobolus incongruus, 23 Exophiala dermatitidis, 385 Conidiogenesis, 21, 22, 29 Exophiala spinifera, 385 Conidioma, 32 Conidiomata, 24 Favic, 365 Conidiophore, 206 Filobasidiella Cryptococcaceae, 171 Filobasidiella bacillospora (anamorph Cryptococcus Cryptococcemia, 265 gattii), 256 Cryptococcoma, 82, 262 Filobasidiella neoformans, 256 Cryptococcosis, 63–67, 255–273 Fluconazole, 125–126 Cryptococcuria, 265 Flucytosine (5-fluorocytosine, 5-FC), 112, 129–130 Cryptococcus, 256–257 Fluoropyrimidine, 129–130 Cryptococcus gattii, 4, 256–258 Folliculitis, 141, 153, 174 Cryptococcus grubii, 256–258 Fonsecaea, 384, 385 Cryptococcus neoformans, 18, 37, 256–258, 265–270 Fonsecaea compacta, 385 Cunninghamella Fonsecaea monophora, 385 |
Cunninghamella bertholletiae, 228 Fonsecaea pedrosoi, 385, 386 Cunninghamellaceae, 228 Fumagillin, 185 Index 425 Fungemia, 140, 142, 158, 166, 170, 172, 175, 201–204, Intracystic, 246 206, 207, 210 Intrathecalamphotericin B, 311 Fungi Imperfecti (mitosporic fungi), 23, 29 Itraconazole, 125–126, 192 Funguria, see candiduria Fusariosis, 202–204 Keratinocytes, 370 Fusarium, 202–204 Keratitis, 202–204, 206–208, 218, 222 Fusarium moniliforme, 202 Kerion, 363–365 Fusarium oxysporum, 202 Ketoacidosis, 7, 187, 229, 230, 237 Fusarium proliferatum, 202 Ketoconazole, 287 Fusarium solani, 202 Lacazia Galactomannan, 60, 188, 194 Lacazia loboi (formerly Loboa loboi), 37, 386 Geniculate, 24 Lasiodiplodia, 216 Geophilic, 356, 358, 364 Loboa loboi (currently Lacazia loboi) Geotrichum capitatum (currently Blastoschizomyces Lobomycosis, 383–391 captitatus) Lysis centrifugation, 16, 174 Glucan, 55, 111 Glucuronoxylomannan, 259, 267, 268 Madura foot, see Mycetoma, 383–391 Gomori methenamine silver stain, 18 Madurella Granuloma, 5, 44, 82, 86, 90, 94, 97, 99, 140, 141, 151, Madurella grisea, 386 259, 263, 285, 303, 305, 322, 323, 325, 326, 336, Madurella mycetomatis, 386, 390 338, 350, 386, 389 Malassezia, 173–175, 358, 378 Granulomatous, 5, 44, 86, 97, 99, 140, 151, 259, 305, Malassezia equi, 356 320, 323, 325–327, 335, 350, 386 Malassezia furfur, 89, 173–175, 356 Graphium, 22 Malassezia globosa, 173, 356 Graphium eumorphum, 22 Malassezia japonica, 356 Griseofulvin, 355, 370, 374, 375, 377, 378 Malassezia nana, 356 Gymnothecia (sing., gymnothecium), 22 Malassezia obtusa, 356 Malassezia pachydermatis, 173, 174, 356 Hansenula (currently Pichia), 175 Malassezia restricta, 173, 356 Hemiascomycetes, 23 Malassezia slooffiae, 173, 356 Herpotrichiellaceae, 384 Malassezia sympodialis, 173, 356 Histoplasma Malassezia yamatoensis, 356 Histoplasma capsulatum, 16, 19, 21, 317–319, 324 Malbranchea, 29 Histoplasma duboisii, 317, 327 Mannan, 55, 59 Histoplasmin, 69 Mannitol, 60 Histoplasmosis, 67–70, 317–327 Mediastinal lymphadenopathy, 93, 94, 319, 323 Holomorph, 22 Mediastinitis, fibrosing, 94, 322, 323, 325 Hortaea Meningoencephalitis, 206, 262, 264 Hortaea werneckii, 218 Mesomycetozoa, 383 Hyalohyphomycetes, 44, 201 Micafungin, 107, 128, 129, 167, 190, 193, 289 Hyalohyphomycosis (pl., hyalohyphomycoses), 201–210 Miconazole, 124, 153, 171, 175, 374, 375, 379 Hydroxyitraconazole, 126 Microabscesses, 82, 149, 150, 283, 383, 387 Hymenomycetes, 23 Microascaceae, 22 Hyperendemic, 278, 319 Microascales, 386 Hyperhidrosis, 357, 361 Microascus Hyperkeratotic, 362, 366, 387 Microascus cinereus, 21, 22 Hypha (pl., hyphae), 42 Microconidia, 202, 318 Hyphomycetes, 29, 44 Microfoci, 278, 318 Hypocreales, 386 Microniche, 333 Hyponychium, 366 Microsporum Microsporum audouinii, 357, 364, 370 Microsporum canis, 356, 357, 364, 365, 370 Imidazole, 110, 176, 218 Microsporum ferrugineum, 365, 370 Immunodiffusion, 54, 287, 305, 306, 325, 337 Microsporum gypseum, 364 Intertrigo, 141 Miliary, 88, 91, 282, 299, 308, 319 426 Index Mitosporic fungi, 16, 22, 29 Phaeoid, 20, 22, 29, 215 Moniliaceous, 209, 263 Phialides, 29 Mortierella, 228 Phialoconidia, 23 Mortierellaceae, 228 Phialophora Mucicarmine stain, 37, 266 Phialophora Americana, 23 Mucor Phialophora verrucosa, 385 Mucor circinelloide, 226 Phoma, 23 Mucor hiemalis, 226 Pichia, 175 Mucor racemosus, 226 Piedra Mucor ramosissimus, 226 Black piedra, 218, 356 Mucor rouxianus, 226 White piedra, 164, 165, 356 Mucoraceae, 226 Piedraia Mucorales, 25, 225, 227, 228, 232, 235 Piedraia hortae, 218 Mucormycosis, see zygomycosis Pityriasis versicolor, 357–358 Multiseptate, 200 Pityrosporum, 356 Muriform, 47, 385, 387 Pleosporales, 386 Mycetoma, 47, 99, 176, 202, 206, 208, 215, 383–391 Pneumocystis, 245–253 Mycobioticmedium, 19 Pneumocystis carinii, 246, 247 Mycotoxicoses (sing., mycotoxicosis), 15 Pneumocystis jirovecii, 246 Myospherulosis, 44 Pneumocystosis, 245–253 Myxotrichum Polyene, 109 Myxotrichum deflexum, 26 Posaconazole, 125, 127, 192–193, 239 Prostatitis, 303 Protoctista, 15, 383 Naftifine, 372 Protoctistan, 383 Nonseptate, see aseptate Prototheca, 44 Nystatin, 153–155 Protothecosis, 50 Pseudallescheria Ochratoxin, 185 Pseudallescheria boydii, 22, 46, 83, 99, 204, 386 Ochroconis, 216 Pseudallescheriasis, 93 Olamine, 374 Pseudohyphae, 20, 42, 47, 137, 151, 164, 383 Onychocola, 218 Pseudomembranes, 142 Onycholysis, 365, 367 Pycnidium, 32 Onychomycosis, 355, 365–367 Pyrimidine, 112, 129–130 Onygenaceae, 333 Pythiosis, 50 Onygenales, 333, 386 Osteolytic, 99, 265, 285 Ramichloridium Ostiole, 32 Ramichloridium mackenzei, 216 Otomycosis, 165, 186 Ravuconazole, 189, 192, 207 Rhinocerebralzygomycosis, 230 Paecilomyces, 207–208 Rhinocladiella aquaspersa, 385 Paecilomyces javanicus, 207 Rhinosporidiosis, 383 Paecilomyces lilacinus, 117, 207, 208 Rhinosporidium Paecilomyces marquandii, 207 Rhinosporidium seeberi, 37, 40 Paecilomyes varioti, 207, 208 Rhizomucor Paracoccidioides Rhizomucor pusillus, 228 Paracoccidioides brasiliensis, 331–334, 337 Rhizopus Paracoccidioidin, 333, 335, 398, 409 Rhizopus arrhizus, 46 Paracoccidioidomycosis, 76–77, 331–352 Rhizopus arrhizus (currently Rhizopus oryzae), 46 Paronychia, 141 Rhizopus microsporus var rhizopodiformis, 23 Penicilliosis, 209–210 Rhizopus oryzae, 227, 230, 238 Penicillium Rhodotorula, 171–173 Penicillium marneffei, 209–210 Rhodotorula aurantiaca, 171 Perithecia (sing., perithecium), 22 Rhodotorula glutinis, 171 Perlèche, 142 Rhodotorula minuta, 171 Phaeohyphomycosis, 215–222 Rhodotorula mucilaginosa, 171 Index 427 Rhodotorula pallida, 171 Sulconazole, 374 Rhodotorula pilimanae, 171 Synanamorph, 20, 22 Rhodotorula rubra (currently Rhodotorula Syncephalastraceae, 228 mucilaginosa) Syncephalastrum Sabouraud dextrose agar, 20 Syncephalastrum racemosum, 228 Saccharomyces, 167–171 Saccharomyces boulardii, 167 Saccharomyces carlsbergensis, 167 Teleomorph, 16, 22 Saccharomyces cerevisiae, 111, 167, 169–171 Terbinafine, 111, 155, 207, 221, 222, 351, 364, 374, Saccharomyces fragilis, 167 375, 377–379, 390–391 Saksenaceae, 228 Thallic, 22, 29 Saksenaea Thamnidaceae, 228 Saksenaea vasiformis, 228 Tinea saprobic (saprophytic), 18 Tinea barbae, 355 Scedosporium, 204–207 Tinea capitis, 357 Scedosporium apiospermum, 22, 117, 125, Tinea corporis, 355–358, 363–364, 374 204–207, 386 Tinea cruris, 357, 358, 363 Scedosporium inflatum (currently Scedosporium Tinea faciei, 355, 357, 363, 375 prolificans) Tinea imbricata, 363, 375 Scedosporium prolificans, 117, 125, 204–207 Tinea manuum, 355, 357, 362, 374 Schizophyllum Tinea nigra, 218, 356 Schizophyllum commune, 25 Tinea pedis, 355, 357, 361, 362, 374, 378 Sclerotia (sing., sclerotium), 383, 384, 387, 389 Tinea unguium, 355, 357 Tinea versicolor (archaic term for pityriasis versicolor) Scopulariopsis, 210 Tokelau (tinea imbricata), 363 Scopulariopsis acremonium, 210 Transepithelial elimination, 386 Scopulariopsis brevicaulis, 210 Triazole, see Azole (triazoles include fluconazole, Scopulariopsis brumptii, 210 itraconazole, posaconazole, ravuconazole, and Scopulariopsis cinereus, 21, 22 voriconazole) Scopulariopsis cirrosus, 29 Trichophyton Scopulariopsis fusca, 210 Trichophyton concentricum, 363 Scopulariopsis koningii, 210 Trichophyton mentagrophytes, 356–358 Scytalidium Trichophyton rubrum, 356, 361, 362 Scytalidium dimidiatum, 32 Trichophyton schoenleinii, 364, 365, 370 Seborrheic dermatitis, 358, 368–369, 378–379 Trichophyton soudanense, 367 Septation, 42 Trichophyton tonsurans, 356, 357, 364, 365, 370 Sordariales, 386 Trichophyton verrucosum, 363, 364 Spherulin, 73 Trichophyton violaceum, 357, 365, 367 Spicules, 25 Trichosporon, 163–167 Splendore-Hoeppli phenomenon, 350 Trichosporon asahii, 163, 398 Sporangiole, 28, 29 Trichosporon asteroides, 164 Sporangiophore, 228 Trichosporon beigelii, 163, 268, 398 Sporangiospore, 228–230, 233, 240 Trichosporon capitatum (currently Blastoschizomyces Sporobolomyces, 176 capitatus) Sporobolomyces holsaticus, 176 Trichosporon cutaneum, 164 Sporobolomyces roseus, 176 Trichosporon inkin, 163, 164 Sporobolomyces salmonicolor, 176 Trichosporon mucoides, 163 Sporobolomycetaceae, 176 Trichosporon ovoides, 164 Sporomiella, 23 Trichosporonosis, 64, 165–167 Sporothrix Trophic forms, 246 Sporothrix schenckii, 21, 44, 343–346, 350 Trophocytes, 39 Sporotrichosis, 343–352 Trophozoites, 246, 250 Sporozoites, 246, 250 Stenella Stenella araguata, 218 Urease, 20, 257, 259 Straminipila, 15 Urediniomycetes, 23 Subungual onychomycosis, 365, 366, 369 Ustilaginomycetes, 23 428 Index Verrucous lesions, 283, 301, 346, 387, 389, 391 Zoophilic, 356, 358, 363, 364 Voriconazole, 125–127, 192 Zygomycetes, 21, 23, 25, 42, 46, 227–230, 234–237 Zygomycosis, 227–240 Zygomycota, 16, 21, 25–29 Wangiella, 216 Zygospore, 228 |
biological and medical physics, biomedical engineering biological and medical physics, biomedical engineering The fields of biological and medical physics and biomedical engineering are broad, multidisciplinary and dynamic. They lie at the crossroads of frontier research in physics, biology, chemistry, and medicine. The Biological and Medical Physics, Biomedical Engineering Series is intended to be comprehensive, covering a broad range of topics important to the study of the physical, chemical and biological sciences. Its goal is to provide scientists and engineers with textbooks, monographs, and reference works to address the growing need for information. Books in the series emphasize established and emergent areas of science including molecular, membrane, and mathematical biophysics; photosynthetic energy harvesting and conversion; information processing; physical principles of genetics; sensory communications; automata networks, neural networks, and cellu- lar automata. Equally important will be coverage of applied aspects of biological and medical physics and biomedical engineering such as molecular electronic components and devices, biosensors, medicine, imag- ing, physical principles of renewable energy production, advanced prostheses, and environmental control and engineering. Editor-in-Chief: Elias Greenbaum, Oak Ridge National Laboratory, Oak Ridge, Tennessee, USA Editorial Board: Masuo Aizawa, Department of Bioengineering, Judith Herzfeld, Department of Chemistry, Tokyo Institute of Technology, Yokohama, Japan Brandeis University, Waltham, Massachusetts, USA Olaf S. Andersen, Department of Physiology, Mark S. Humayun, Doheny Eye Institute, Biophysics & Molecular Medicine, Los Angeles, California, USA Cornell University, New York, USA Pierre Joliot, Institute de Biologie Robert H. Austin, Department of Physics, Physico-Chimique, Fondation Edmond Princeton University, Princeton, New Jersey, USA de Rothschild, Paris, France James Barber, Department of Biochemistry, Lajos Keszthelyi, Institute of Biophysics, Hungarian Imperial College of Science, Technology Academy of Sciences, Szeged, Hungary and Medicine, London, England Robert S. Knox, Department of Physics Howard C. Berg, Department of Molecular and Astronomy, University of Rochester, Rochester, and Cellular Biology, Harvard University, New York, USA Cambridge, Massachusetts, USA Aaron Lewis, Department of Applied Physics, Victor Bloomfield, Department of Biochemistry, Hebrew University, Jerusalem, Israel University of Minnesota, St. Paul, Minnesota, USA Stuart M. Lindsay, Department of Physics Robert Callender, Department of Biochemistry, and Astronomy, Arizona State University, Albert Einstein College of Medicine, Tempe, Arizona, USA Bronx, New York, USA David Mauzerall, Rockefeller University, Britton Chance, Department of Biochemistry/ New York, New York, USA Biophysics, University of Pennsylvania, Eugenie V. Mielczarek, Department of Physics Philadelphia, Pennsylvania, USA and Astronomy, George Mason University, Fairfax, Steven Chu, Lawrence Berkeley National Virginia, USA Laboratory, Berkeley, California, USA Markolf Niemz, Medical Faculty Mannheim, Louis J. DeFelice, Department of Pharmacology, University of Heidelberg, Mannheim, Germany Vanderbilt University, Nashville, Tennessee, USA V. Adrian Parsegian, Physical Science Laboratory, Johann Deisenhofer, Howard Hughes Medical National Institutes of Health, Bethesda, Institute, The University of Texas, Dallas, Maryland, USA Texas, USA Linda S. Powers, University of Arizona, George Feher, Department of Physics, Tucson, Arizona, USA University of California, San Diego, La Jolla, Earl W. Prohofsky, Department of Physics, California, USA Purdue University, West Lafayette, Indiana, USA Hans Frauenfelder, Andrew Rubin, Department of Biophysics, Moscow Los Alamos National Laboratory, State University, Moscow, Russia Los Alamos, New Mexico, USA Michael Seibert, National Renewable Energy Ivar Giaever, Rensselaer Polytechnic Institute, Laboratory, Golden, Colorado, USA Troy, New York, USA David Thomas, Department of Biochemistry, Sol M. Gruner, Cornell University, University of Minnesota Medical School, Ithaca, New York, USA Minneapolis, Minnesota, USA Lorenzo Pavesi Philippe M. Fauchet (Eds.) Biophotonics With 185 Figures 123 Professor Lorenzo Pavesi University of Trento, Department of Physics, Laboratory of Nanoscience Via Sommarive 14, 38050 Povo, Italy E-mail: pavesi@science.unitn.it Philippe M. Fauchet University of Rochester, Department of Electrical and Computer Engineering 160 Trustee Road, Rochester, NY 14627-0231, USA E-mail: fauchet@ece.rochester.edu ISBN 978-3-540-76779-4 e-ISBN 978-3-540-76782-4 Biological and Medical Physics, Biomedical Engineering ISSN 1618-7210 Library of Congress Control Number: 2008920259 ©c 2008 Springer-Verlag Berlin Heidelberg This work is subject to copyright. All rights are reserved, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broad- casting, reproduction on microfilm or in any other way, and storage in data banks. Duplication of this publication or parts thereof is permitted only under the provisions of the German Copyright Law of September 9, 1965, in its current version, and permission for use must always be obtained from Springer-Verlag. Violations are liable to prosecution under the German Copyright Law. The use of general descriptive names, registered names, trademarks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. Cover: eStudio Calamar Steinen Printed on acid-free paper 9 8 7 6 5 4 3 2 1 springer.com To Anna To Melanie Preface This book is the brainchild of the fourth Optoelectronic and Photonic Winter School on “Biophotonics” which took place from 25th February 2007 to 3rd March 2007 in Sardagna, a small village on the mountains around Trento in Italy. This school, held every two years, have been promoted to trace the very fast developing technologies and the tremendous progress in photonics which have occurred and will occur in the near future. It is a common view that its current explosive development will lead to deep paradigm shifts in the near future. Identifying the plausible scenario for the future evolution of photonics presents an opportunity for constructive actions and scouting killer technologies. This book gathers together distinguished authors to give an overview of the latest developments in biophotonics. Optical technologies have demonstrated a long tradition in the fields of life science and health care. The microscope has opened for us a view into the world of cells and bacteria and has evolved into a modern powerful tool for basic biological research on cell processes. Modern surgical microscopes have become key tools in neurosurgery, as well as in ophthalmology and surgery. Image-guided systems make use of computer tomography and MRI data in navigated surgery. Endoscopes have opened an easy access to the inner parts of the human body in minimal invasive surgery. Optical methods for gene sequencing and biochips open new routes for diagnosis and treatment of cancer. Fluorescence methods have replaced radioactive methods in screening processes in drug development. The role of optical technologies as the key enabling factor in health care and life sciences will grow tremendously in future. It is therefore a good time to write a book on biophotonics with the aim to introduce students and young researchers to this field and make a point of the importance of this science and technology. In particular, each chapter consists of a review, an introduction to the subject and a presentation of the current state of the art. The first three chapters of the book are by Bassi, Giacometti and cowork- ers on photosynthesis, on the application of photosynthesis to biofuel produc- tion and on the role of carotenoids in photoprotection. Then, the chapter by VIII Preface Diaspro and coworkers introduces the readers to non-linear microscopy. An- other kind of microscopy, spectral self-interference microscopy, is presented in the chapter by Ünlü and coworkers. The following chapter, also by Ünlü and coworkers, deals with resonant cavity biosensors for sensing and imaging. A different way to engineer biosensors is the use of optical microcavities: these are reviewed in the chapter by Fauchet and coworkers. The success and application of optical coherence tomography are described in the chapter by Boppart. Other coherent laser techniques for medical diag- nostics are described in the chapter by Kemper and von Bally. Two chapters follow on the use of fluorescence labelling for sensing and quantitative analysis. Caiolfa and coworkers detail both the principle and applications of luminescence probes in quantitative biology. Ligler reviews fluorescent-based optical biosensors. The next chapter, by Seitz, discusses optical biochips and their applications to lab-on-a-chip. An example of the merging of microelectronics with biology is given in the chapter by Charbon on the use of CMOS camera for bioimaging applications. Going back to basic optics, the chapter by Chiou and coworkers shows the use of optical forces in trapping and manipulating bio-objects, such as red blood cells or proteins. Optics can also be used in surgery. Pini and coworkers discuss the application of lasers in ophthalmology and vascular surgery in their chapter. The final two chapters are examples of how photonics is used in common clinical practice. In the chapter by Pentland, the photobiology of the skin and the phototherapy are discussed, while in the chapter by Wilson the use of light in therapy is introduced and a few examples are given in detail. We thank all the authors who presented very interesting state-of-the art lectures and chapters. Last but not least, we express our gratitude also to all those who have contributed to the success of the fourth Optoelectronic and Photonics Winter School on Biophotonics: the organizing committee (L. Pavesi, P. Fauchet, M. Anderle, L. Gonzo, R. Antolini, M. Ferrari, S. Iannotta, G. Righini), the staff of the University of Trento and all the students, whose participation was lively and stimulating. This year the winter school was also the first edition of the AIV school on nanotechnology. We acknowledge the generous contribution by the Società Italiana di Scienze e Tecnologie through its president Mariano Anderle. Further support came from the University of Trento, the Department of Physics, FBK-irst, LaserOptronic and Spectra- Physics, Crisel-Instruments and LOT Oriel group Europe. Particular thanks are also due to Alessandro Pitanti and his brother for their help in the editing the Latex files. Trento, Rochester Lorenzo Pavesi May 2008 Philippe M. Fauchet Contents 1 Light Conversion in Photosynthetic Organisms S. Frigerio, R. Bassi, and G.M. Giacometti . . . . . . . . . . . . . . . . . . . . . . . . . 1 1.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 1.2 Chloroplast Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 1.3 Pigments and Light Absorption . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 1.4 Photosynthetic Apparatus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 1.4.1 Photosystem II . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 1.4.2 Photosystem I . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 1.4.3 Cytochrome b6f . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 1.4.4 ATP Synthase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 1.5 Cyclic Phosphorylation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 1.6 Photoinhibition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . |
. 10 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 2 Exploiting Photosynthesis for Biofuel Production C. Govoni, T. Morosinotto, G. Giuliano, and R. Bassi . . . . . . . . . . . . . . . 15 2.1 Biological Production of Vehicle Traction Fuels: Bioethanol and Biodiesel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 2.1.1 Bioethanol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 2.1.2 Biodiesel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18 2.1.3 Biofuels Still Present Limitations Preventing Their Massive Utilization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18 2.2 Hydrogen Biological Production by Fermentative Processes . . . . . . . 19 2.2.1 Hydrogen Production by Bacterial Fermentation . . . . . . . . . 20 2.3 Hydrogen Production by Photosynthetic Organisms . . . . . . . . . . . . . 21 2.3.1 Cyanobacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22 2.3.2 Eukaryotic Algae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 2.4 Challenges in Algal Hydrogen Production . . . . . . . . . . . . . . . . . . . . . . 23 2.4.1 Oxygen Sensitivity of Hydrogen Production . . . . . . . . . . . . . . 23 2.4.2 Optimization of Light Harvesting in Bioreactors . . . . . . . . . . 25 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27 X Contents 3 In Between Photosynthesis and Photoinhibition: The Fundamental Role of Carotenoids and Carotenoid-Binding Proteins in Photoprotection G. Bonente, L. Dall’Osto, and R. Bassi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29 3.1 When Light Becomes Dangerous for a Photosynthetic Organism . . 29 3.2 Acclimation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31 3.3 State 1–State 2 Transitions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32 3.4 Carotenoids Play a Fundamental Role in Many Photoprotection Mechanisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33 3.5 Analysis of Xanthophyll Function In Vivo . . . . . . . . . . . . . . . . . . . . . . 36 3.6 Nonphotochemical Quenching . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38 3.7 Feedback Deexcitation of Singlet-Excited Chlorophylls: qE . . . . . . . 39 3.8 ∆pH - Independent Energy Thermal Dissipation (qI) . . . . . . . . . . . . 40 3.9 Chlorophyll Triplet Quenching . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41 3.10 Scavenging of Reactive Oxygen Species . . . . . . . . . . . . . . . . . . . . . . . . 41 3.11 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44 4 Non-Linear Microscopy D. Mazza, P. Bianchini, V. Caorsi, F. Cella, P.P. Mondal, E. Ronzitti, I. Testa, G. Vicidomini, and A. Diaspro . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47 4.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47 4.2 Chronological Notes on MPE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48 4.3 Principles of Confocal and Two-Photon Fluorescence Microscopy . . 49 4.3.1 Fluorescence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49 4.3.2 Confocal Principles and Laser Scanning Microscopy . . . . . . . 50 4.3.3 Point Spread Function of a Confocal Microscope . . . . . . . . . 52 4.4 Two-Photon Excitation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55 4.5 Two-Photon Optical Sectioning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59 4.6 Two-Photon Optical Setup . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60 4.7 Second Harmonic Generation (SHG) Imaging . . . . . . . . . . . . . . . . . . . 63 4.8 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66 5 Applications of Optical Resonance to Biological Sensing and Imaging: I. Spectral Self-Interference Microscopy M.S. Ünlü, A. Yalc. in, M. Doǧan, L. Moiseev, A. Swan, B.B. Goldberg, and C.R. Cantor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71 5.1 High-Resolution Fluorescence Imaging . . . . . . . . . . . . . . . . . . . . . . . . . 71 5.2 Self-Interference Imaging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71 5.3 Physical Model of SSFM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73 5.3.1 Classical Dipole Emission Model . . . . . . . . . . . . . . . . . . . . . . . 73 5.4 Acquisition and Data Processing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75 5.4.1 Microscope Setup . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75 5.4.2 Fitting Algorithm . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76 Contents XI 5.5 Experimental Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77 5.5.1 Monolayers of Fluorophores on Silicon Oxide Surfaces: Fluorescein, Quantum Dots, Lipid Films . . . . . . . . . . . . . . . . 77 5.5.2 Conformation of Surface-Immobilized DNA . . . . . . . . . . . . . . 79 5.6 SSFM in 4Pi Configuration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82 5.7 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84 References . . . . . . . . . . |
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85 6 Applications of Optical Resonance to Biological Sensing and Imaging: II. Resonant Cavity Biosensors M.S. Ünlü, E. Özkumur, D.A. Bergstein, A. Yalc. in, M.F. Ruane, and B.B. Goldberg . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87 6.1 Multianalyte Sensing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87 6.2 Resonant Cavity Imaging Biosensor . . . . . . . . . . . . . . . . . . . . . . . . . . . 88 6.2.1 Detection Principle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88 6.2.2 Experimental Setup, Data Acquisition, and Processing . . . . 90 6.2.3 Experimental Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91 6.2.4 Spectral Reflectivity Imaging Biosensor . . . . . . . . . . . . . . . . . 92 6.3 Optical Sensing of Biomolecules Using Microring Resonators . . . . . 94 6.3.1 Basics on Microring Resonators . . . . . . . . . . . . . . . . . . . . . . . . 94 6.3.2 Setup and Data Acquisition . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95 6.3.3 Data Analysis and Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . 96 6.4 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98 7 Biodetection Using Silicon Photonic Crystal Microcavities P.M. Fauchet, B.L. Miller, L.A. DeLouise, M.R. Lee, and H. Ouyang . . 101 7.1 Photonic Crystals: A Short Introduction . . . . . . . . . . . . . . . . . . . . . . . 101 7.1.1 Electromagnetic Theory . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101 7.1.2 One-Dimensional and Two-Dimensional PhC . . . . . . . . . . . . . 103 7.1.3 Microcavities: Breaking the Periodicity . . . . . . . . . . . . . . . . . . 105 7.1.4 Computational Algorithms . . . . . . . . . . . . . . . . . . . . . . . . . . . . 106 7.2 One-Dimensional PhC Biosensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107 7.2.1 Preparation and Selected Properties of Porous Silicon . . . . . 107 7.2.2 Sensing Principle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109 7.2.3 One-Dimensional Biosensor Design and Performance . . . . . 111 7.2.4 Fabrication of One-Dimensional PhC Biosensors . . . . . . . . . . 112 7.3 Selected Biosensing Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114 7.3.1 DNA Detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114 7.3.2 Bacteria Detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114 7.3.3 Protein Detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115 7.3.4 IgG Detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117 7.4 Two-Dimensional PhC Biosensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118 7.4.1 Sample Preparation and Measurement . . . . . . . . . . . . . . . . . . 118 7.4.2 Sensing Principle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119 7.4.3 Selected Biosensing Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120 XII Contents 7.5 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 124 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 124 8 Optical Coherence Tomography with Applications in Cancer Imaging S.A. Boppart . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127 8.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127 8.2 Principles of Operation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 128 8.3 Optical Sources for Optical Coherence Tomography . . . . . . . . . . . . . 133 8.4 Fourier-Domain Optical Coherence Tomography . . . . . . . . . . . . . . . . 133 8.5 Beam Delivery Instruments for Optical Coherence Tomography . . . 135 8.6 Spectroscopic Optical Coherence Tomography . . . . . . . . . . . . . . . . . . 136 8.7 Applications to Cancer Imaging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 138 8.7.1 Cellular Imaging for Tumor Cell Biology . . . . . . . . . . . . . . . . 138 8.7.2 Translational Breast Cancer Imaging . . . . . . . . . . . . . . . . . . . . 140 8.8 Optical Coherence Tomography Contrast Agents . . . . . . . . . . . . . . . . 141 8.9 Molecular Imaging using Optical Coherence Tomography . . . . . . . . . 145 8.10 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149 9 Coherent Laser Measurement Techniques for Medical Diagnostics B. Kemper and G. von Bally . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151 9.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151 9.2 Electronic Speckle Pattern Interferometry (ESPI) . . . . . . . . . . . . . . . 152 9.2.1 Double Exposure Subtraction ESPI . . . . . . . . . . . . . . . . . . . . . 152 9.2.2 Spatial Phase Shifting (SPS) ESPI . . . . . . . . . . . . . . . . . . . . . 153 9.3 Endoscopic Electronic Speckle Pattern Interferometry (ESPI) . . . . . 156 9.3.1 Proximal Endoscopic ESPI . . . . . . . . . . . . . . . . . . . . . . . . . . . . 156 9.3.2 Distal Endoscopic ESPI . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 158 9.4 Microscopic (Speckle) Interferometry . . . . . . . . . . . . . . . . . . . . . . . . . . 161 9.5 Digital Holographic Microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . |
. . . 164 9.5.1 Principle and Measurement Setup . . . . . . . . . . . . . . . . . . . . . . 164 9.5.2 Nondiffractive Reconstruction . . . . . . . . . . . . . . . . . . . . . . . . . . 166 9.5.3 Resolution and Numerical Focus . . . . . . . . . . . . . . . . . . . . . . . . 170 9.5.4 Digital Holographic Phase Contrast Microscopy of Living Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 171 9.6 Discussion and Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 173 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 173 10 Biomarkers and Luminescent Probes in Quantitative Biology M. Zamai, G. Malengo, and V.R. Caiolfa . . . . . . . . . . . . . . . . . . . . . . . . . . . 177 10.1 Fluorophores and Genetic Dyes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 177 10.1.1 Small Organic Dyes and Quantum Dots . . . . . . . . . . . . . . . . . 177 10.1.2 Fluorescent Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 178 Contents XIII 10.2 Microspectroscopy in Quantitative Biology: Where and How . . . . . . 183 10.2.1 Fluorescence Correlation Spectroscopy . . . . . . . . . . . . . . . . . . 183 10.2.2 Fluorescence Lifetime Imaging (FLIM) . . . . . . . . . . . . . . . . . . 188 10.2.3 Glossary of Molecular Biology . . . . . . . . . . . . . . . . . . . . . . . . . . 194 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 195 11 Fluorescence-Based Optical Biosensors F.S. Ligler . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 199 11.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 199 11.2 Biological Recognition Molecules and Assay Formats . . . . . . . . . . . . 200 11.3 Displacement Immunosensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 203 11.4 Fiber Optic Biosensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 204 11.4.1 Fiber Optics for Biosensor Applications . . . . . . . . . . . . . . . . . 205 11.4.2 Optrode Biosensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 207 11.4.3 Evanescent Fiber Optic Biosensors . . . . . . . . . . . . . . . . . . . . . . 207 11.5 Bead-Based Biosensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 209 11.6 Planar Biosensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 210 11.7 Critical Issues and Future Opportunities . . . . . . . . . . . . . . . . . . . . . . . 212 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 214 12 Optical Biochips P. Seitz . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 217 12.1 Taxonomy of Optical Biochips . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 217 12.1.1 Basic Architecture of Optical Biochips . . . . . . . . . . . . . . . . . . 217 12.2 Analyte Classes for Optical Biochips . . . . . . . . . . . . . . . . . . . . . . . . . . 220 12.2.1 DNA (DNA Fragments, mRNA, cDNA) . . . . . . . . . . . . . . . . . 220 12.2.2 Proteins (Antigens) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 221 12.2.3 Specific Organic Molecules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 221 12.2.4 Cell Gene Products (cDNA, Proteins) . . . . . . . . . . . . . . . . . . . 221 12.2.5 Tissue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 222 12.3 Optical Effects for Biochemical Sensors . . . . . . . . . . . . . . . . . . . . . . . . 222 12.3.1 Spectral Absorption . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 222 12.3.2 Phase Shift . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 223 12.3.3 Fluorescence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 223 12.3.4 Luminescence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 223 12.3.5 Raman Scattering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 224 12.3.6 Nonlinear Optical (NLO) Effects . . . . . . . . . . . . . . . . . . . . . . . 224 12.4 Preferred Sensing Principles for Optical Biochips . . . . . . . . . . . . . . . . 224 12.4.1 Evanescent Wave Sensing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 225 12.4.2 Fluorescence Sensing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 228 12.5 Readout Methods for Evanescent Wave Sensors . . . . . . . . . . . . . . . . . 229 12.5.1 Angular Scanning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 229 12.5.2 Wavelength Tuning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 230 12.5.3 Grating Coupler Chirping . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 230 12.6 Substrates for Optical Biochips . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 230 12.7 Realization Example of an Optical Biosensor/Biochip: WIOS . . . . . 231 XIV Contents 12.8 Outlook: Lab-on-a-Chip Using Organic Semiconductors . . . . . . . . . . 232 12.8.1 Basics of Organic Semiconductors . . . . . . . . . . . . . . . . . . . . . . 233 12.8.2 Organic LEDs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 233 12.8.3 Organic Lasers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 234 12.8.4 Organic Photodetectors and Image Sensors . . . . . . . . . . . . |
. . 234 12.8.5 Organic Photovoltaic Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 234 12.8.6 Organic Field Effect Transistors and Circuits . . . . . . . . . . . . 235 12.8.7 Monolithic Photonic Microsystems Using Organic Semiconductors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 235 12.9 Conclusions and Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 236 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 236 13 CMOS Single-Photon Systems for Bioimaging Applications E. Charbon . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 239 13.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 239 13.2 Spectroscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 240 13.3 Lifetime Imaging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 242 13.4 Time-of-Flight in Bio- and Medical Imaging . . . . . . . . . . . . . . . . . . . . 244 13.5 System Considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 245 13.6 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 247 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 248 14 Optical Trapping and Manipulation for Biomedical Applications A. Chiou, M.-T. Wei, Y.-Q. Chen, T.-Y. Tseng, S.-L. Liu, A. Karmenyan, and C.-H. Lin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 249 14.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 249 14.2 Theoretical Models for the Calculation of Optical Forces . . . . . . . . . 252 14.2.1 The Ray-Optics (RO) Model . . . . . . . . . . . . . . . . . . . . . . . . . . . 252 14.2.2 Electromagnetic (EM) Model . . . . . . . . . . . . . . . . . . . . . . . . . . 255 14.3 Experimental Measurements of Optical Forces . . . . . . . . . . . . . . . . . . 255 14.3.1 Axial Optical Force as a Function of Position along the Optical Axis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 255 14.3.2 Transverse Trapping Force Measured by Viscous Drag . . . . . 257 14.3.3 Three-Dimensional Optical Force Field Probed by Particle Brownian Motion . . . . . . . . . . . . . . . . . . . . . . . . . . 257 14.3.4 Optical Forced Oscillation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 261 14.4 Potential Biomedical Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 265 14.4.1 Optical Forced Oscillation for the Measurement of Protein–Protein Interactions . . . . . . . . . . . . . . . . . . . . . . . . . 266 14.4.2 Protein–DNA Interaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 267 14.4.3 Optical Trapping and Stretching of Red Blood Cells . . . . . . 269 14.5 Summary and Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 271 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 271 Contents XV 15 Laser Tissue Welding in Minimally Invasive Surgery and Microsurgery R. Pini, F. Rossi, P. Matteini, and F. Ratto . . . . . . . . . . . . . . . . . . . . . . . . 275 15.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 275 15.2 Laser Welding in Ophthalmology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 281 15.2.1 Laser Welding of the Cornea . . . . . . . . . . . . . . . . . . . . . . . . . . . 281 15.2.2 Combing Femtosecond Laser Microsculpturing of the Cornea with Laser Welding . . . . . . . . . . . . . . . . . . . . . . 285 15.2.3 Laser Closure of Capsular Tissue . . . . . . . . . . . . . . . . . . . . . . . 287 15.3 Applications in Microvascular Surgery . . . . . . . . . . . . . . . . . . . . . . . . . 289 15.4 Potentials in Other Surgical Fields . . . . . . . . . . . . . . . . . . . . . . . . . . . . 291 15.4.1 Laser Welding of the Gastrointestinal Tract . . . . . . . . . . . . . . 291 15.4.2 Laser Welding in Gynaecology . . . . . . . . . . . . . . . . . . . . . . . . . 291 15.4.3 Laser Welding in Neurosurgery . . . . . . . . . . . . . . . . . . . . . . . . . 292 15.4.4 Laser Welding in Orthopaedic Surgery . . . . . . . . . . . . . . . . . . 292 15.4.5 Laser Welding of the Skin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 292 15.4.6 Laser Welding in Urology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 293 15.5 Perspectives of Nanostructured Chromophores for Laser Welding . . 293 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 296 16 Photobiology of the Skin A.P. Pentland . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 301 16.1 Basics of Skin Structure: Cell Types, Skin Structures, and Their Function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 301 16.2 Effects of Light Exposure on Skin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 304 16.3 Sun Protection and Sunscreens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 309 16.4 Phototherapy: Use of Light for Treatment for Skin Disease . . . . . . . 311 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 313 17 Advanced Photodynamic Therapy B.C. Wilson . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 315 17.1 Introduction . . . . . . . . . . . . . . . . . . . . . . |
. . . . . . . . . . . . . . . . . . . . . . . . . . 315 17.2 Basic Principles and Features of “Standard PDT” . . . . . . . . . . . . . . . 316 17.3 Novel PDT Concepts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 319 17.3.1 Two-Photon PDT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 319 17.3.2 Metronomic PDT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 322 17.3.3 PDT Molecular Beacons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 323 17.3.4 Nanoparticle-Based PDT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 325 17.4 PDT Dosimetry Using Photonic Techniques . . . . . . . . . . . . . . . . . . . . 327 17.5 Biophotonic Techniques for Monitoring Response to PDT . . . . . . . . 330 17.6 Biophotonic Challenges and Opportunities in Clinical PDT. . . . . . . 331 17.7 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 333 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 333 Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 335 List of Contributors Gert von Bally Paolo Bianchini Center for Biomedical Optics LAMBS-MicroSCoBiO Research and Photonics, Center, Department of Physics, University of Münster, University of Genoa, Robert-Koch-Straße 45, Via Dodecaneso 33, 48129 Münster, 16146 Genoa, Italy Germany Ce.BOP@uni-muenster.de Giulia Bonente Laboratoire de Génétique et de Roberto Bassi Département de Biologie - Case 901, Biophysique des Plantes, Faculté des Sciences de Luminy, Faculté de Sciences de Luminy, Université Aix-Marseille II, LGBP, Université de Marseille, 163, Avenue de Luminy Marseille Cedex 9, 13288 Marseille Cedex 09, France France bonente@sci.univr.it and and Dipartimento Scientifico Dipartimento Scientifico e e Tecnologico, Tecnologico, Università di Verona, Università di Verona Strada le Grazie 15, Strada Le Grazie 15, 37134 Verona, 37134 Verona, Italy Italy Stephen A. Boppart bassi@sci.univr.it Beckman Institute for Advanced David A. Bergstein Science and Technology, Department of Electrical Departments of Electrical and Computer Engineering, and Computer Engineering, Boston University, Bioengineering, and Medicine 8 St. Mary’s St., University of Illinois at 02215 Boston, MA, USA Urbana-Champaign bdave@bu.edu Mills Breast Cancer Institute XVIII List of Contributors Carle Foundation Hospital Yin-Quan Chen Urbana, IL 61801 Institute of Biophotonics boppart@uiuc.edu Engineering, National Yang-Ming University Taipei, Taiwan, ROC Valeria R. Caiolfa Arthur Chiou Department of Molecular Biology Institute of Biophotonics and Functional Genomics, Engineering, National Yang-Ming San Raffaele Scientific Institute University Taipei, Taiwan, ROC Milano, Italy aechiou@ym.edu.tw valeria.caiolfa@hsr.it and Luca Dall’Osto IIT Network Research, Dipartimento Scientifico e Unit of Molecular Neuroscience, Tecnologico, Università di Verona, San Raffaele Scientific Institute, Strada le Grazie 15, Milano, Italy 37134 Verona, Italy Charles R. Cantor dallosto@sci.univr.it Department of Biomedical Lisa A. DeLouis e Engineering, 44 Cummington St., Center for Future Health, 02215 Boston, MA, USA University of Rochester, crcantor@bu.edu Rochester, NY, USA and and Center for Advanced Biotechnology, Department of Dermatology, 36 Cummington St., University of Rochester 02215 Boston, MA, USA Rochester, NY, USA Valentina Caorsi Alberto Diaspro LAMBS-MicroSCoBiO Research LAMBS-MicroSCoBiO Research Center, Department of Physics, Center, Department of Physics, University of Genoa, University of Genoa, Via Dodecaneso 33, Via Dodecaneso 33, 16146 Genoa, Italy 16146 Genoa, Italy Francesca Cella LAMBS-MicroSCoBiO Research Mehmet Doǧan Center, Department of Physics, Department of Physics, University of Genoa, Boston University, Via Dodecaneso 33, 590 Commonwealth Ave., 16146 Genoa, Italy 02215 Boston, MA, USA mdogan@bu.edu Edoardo Charbon Philippe M. Fauchet Quantum Architecture Group Center for Future Health, (AQUA), EPFL, University of Rochester, 1015 Lausanne, Switzerland Rochester, NY, USA edoardo.charbon@epfl.ch and List of Contributors XIX Department of Electrical Department of Electrical and Computer Engineering, and Computer Engineering, University of Rochester, Boston University, Rochester, NY, USA 8 St. Mary’s St., and 02215 Boston, MA, USA Department of Biomedical and Engineering, Department of Biomedical University of Rochester, Engineering, 44 Cummington St., Rochester, NY, USA 02215 Boston, MA, USA and The Institute of Optics, Chiara Govoni University of Rochester, Dipartimento Scientifico e Rochester, NY, USA Tecnologico, Università di Verona, Strada Le Grazie 15, Sara Frigerio 37134 Verona, Italy Université Aix-Marseille II, LGBP, Faculté des Sciences de Luminy, Artashes Karmenyan Département de Biologie - Case 901, Institute of Biophotonics 163, Avenue de Luminy Engineering, National Yang-Ming 13288 Marseille Cedex 09, University Taipei, Taiwan, ROC France Björn Kemper Giorgio Giacometti Center for Biomedical Optics Dipartimento di Biologia, and Photonics, Università di Padova, University of Münster, Via Ugo Bassi 58 B, Robert-Koch-Straße 45, 35131 Padova, Italy 48129 Münster, Germany bkemper@uni-muenster.de Giovanni M. Giuliano Ente per le Nuove tecnologie, Mindy R. Lee l’Energia e l’Ambiente (ENEA), Center for Future Health, Unità Biotecnologie, University of Rochester, Centro Ricerche Casaccia, Rochester, NY, USA C.P. 2400, Roma 00100, and Italy The Institute of Optics, University of Rochester, Bennett B. Goldberg Rochester, NY, USA Department of Physics, Boston University, Frances S. Ligler 590 Commonwealth Ave., Center for Bio/Molecular Science 02215 Boston, MA, & Engineering, USA Naval Research Laboratory, goldberg@bu.edu Washington, DC 20375, USA and fligler@cbmse.nrl.navy.mil XX List of Contributors Chi-Hung Lin Lev Moiseev Institute of Biophotonics Center for Advanced Biotechnology Engineering, National Yang-Ming 36 Cummington St., 02215 Boston, University Taipei, Taiwan, ROC MA, USA leva1m@bu.edu Shang-Ling Liu Partha P. Mondal Institute of Biophotonics LAMBS-MicroSCoBiO Research Engineering, National Yang-Ming Center, Department of Physics, University Taipei, Taiwan, ROC University of Genoa, Via Dodecaneso 33, Gabriele Malengo 16146 Genoa, Italy Department of Molecular Biology and Functional Genomics, Tomas Morosinotto San Raffaele Scientific Institute, Dipartimento di Biologia, Università di Padova, Milano, Italy Via Ugo Bassi 58 B, 35131 Padova, Italy Paolo Matteini Istituto di Fisica Applicata, Huimin Ouyang Consiglio Nazionale delle Ricerche Department of Electrical Via Madonna del Piano 10, and Computer Engineering, 50019 Sesto Fiorentino, University of Rochester, Italy Rochester, NY, USA Emre Özkumur Davide Mazza Department of Electrical and LAMBS-MicroSCoBiO Research Computer Engineering, Center, Department of Physics, Boston University, University of Genoa, 8 St. Mary’s St., Via Dodecaneso 33, 02215 Boston, MA, USA 16146 Genoa, Italy eozkumur@bu.edu mazza@fisica.unige.it Alice P. Pentland Benjamin L. Miller Department of Dermatology, Center for Future Health, University of Rochester, University of Rochester, 601 Elmwood, Rochester, NY, USA Rochester, NY 14642, USA and Alice Pentland@urmc. Department of Biomedical rochester.edu Engineering, University of Rochester, Roberto Pini Rochester, NY, USA Istituto di Fisica Applicata, and Consiglio Nazionale delle Ricerche Department of Dermatology, Via Madonna del Piano 10, University of Rochester 50019 Sesto Fiorentino, Italy Rochester, NY, USA R.Pini@ifac.cnr.it List of Contributors XXI Fulvio Ratto Ilaria Testa Istituto di Fisica Applicata, LAMBS-MicroSCoBiO Research Consiglio Nazionale delle Ricerche, Center, Department of Physics, Via Madonna del Piano 10, University of Genoa, 50019 Sesto Fiorentino, Italy Via Dodecaneso 33, 16146 Genoa, Italy Emiliano Ronzitti LAMBS-MicroSCoBiO Research Te-Yu Tseng Center, Department of Physics, Institute of Biophotonics University of Genoa, Engineering, National Yang-Ming Via Dodecaneso 33, University, Taipei, Taiwan, ROC 16146 Genoa, Italy M. Selim Ünlü Francesca Rossi Department of Electrical Istituto di Fisica Applicata, and Computer Engineering, Consiglio Nazionale delle Ricerche Boston University, Via Madonna del Piano 10, 50019 Sesto Fiorentino, Italy 8 St. Mary’s St., 02215 Boston, MA, USA Michael F. Ruane Department of Electrical selim@bu.edu and Computer Engineering, and Boston University, Department of Physics, 8 St. Mary’s St., Boston University, 02215, Boston, MA, USA 590 Commonwealth Ave., mfr@bu.edu 02215 Boston, MA, USA and Peter Seitz Department of Biomedical Swiss Center for Electronics Engineering, and Microtechnology, 44 Cummington St., CSEM SA, 02215 Boston, MA, Zurich Switzerland, USA and Institute for Microtechnology, Giuseppe Vicidomini University of Neuchâtel, LAMBS-MicroSCoBiO Research Neuchâtel, Switzerland Center, Department of Physics, peter.seitz@csem.ch University of Genoa, Via Dodecaneso 33, Anna Swan 16146 Genoa, Department of Electrical Italy and Computer Engineering, Boston University, Ming-Tzo Wei 8 St. Mary’s St., Institute of Biophotonics 02215 Boston, MA, USA Engineering, National Yang-Ming swan@bu.edu University, Taipei, Taiwan, ROC XXII List of Contributors Brian C. Wilson 8 St. Mary’s St., Division of Biophysics and 02215 Boston, MA, USA Bioimaging, Ontario Cancer ayca@bu.edu Institute, Toronto, ON, Canada Moreno Zamai and Department of Molecular Biology Department of Medical Biophysics, and Functional Genomics, University of Toronto, Toronto, San Raffaele Scientific Institute, ON, Canada, M5G 2M9 Milano, Italy wilson@uhnres.utoronto.ca and IIT Network Research, Ayça Yalc.in Unit of Molecular Neuroscience, Department of Electrical San Raffaele Scientific Institute, and Computer Engineering, Milano, Italy Boston University, moreno.zamai@hsr.it 1 Light Conversion in Photosynthetic Organisms S. Frigerio, R. Bassi, and G.M. Giacometti 1.1 Introduction The sun is the ultimate source of free energy that drives all of the processes in living cells. The radiant energy of the sun is captured and converted to chemical energy by photosynthesis. The flux of carbon through the biosphere begins with photosynthesis. Photosynthetic organisms produce carbohydrates and molecular oxygen from carbon dioxide and water: 6H2O + 6CO2 + light → (CH2O)6 pedice + 6O2 (1.1) The carbohydrates produced by photosynthesis serve as the energy source for other non-photosynthetic (heterotrophic) organisms. In this process, carbo- hydrates are recycled to carbon dioxide and water by the combined action of cellular catabolic processes. The fixation of carbon dioxide into sugars requires free energy in the form of ATP and reducing power in the form of NADPH. The light reactions of photosynthesis respond to this need: the visible component of solar radiation is captured and its energy is converted into ATP and NADPH through a complex series of redox reactions and membrane-mediated energy conversions. The following reactions, referred to as dark phase of photosynthesis, as, in principle, they do not need solar radiation to be carried out if NADPH and ATP are provided, drive the reduction of CO2 to the carbohydrate GAP (glyceraldehyde-3-phosphate). The series of reactions are altogether indicated as the Calvin-Benson cycle [1]. The enzyme directly responsible for the addition of CO2 to ribulose 1,5 - bisphosphate is RubisCO [2]. The whole carboxylation cycle requires three CO2 molecules to generate a molecule of GAP, which is then used for the synthesis of more complex sugars or other compounds. The energy required for this cycle is nine molecules of ATP and six of NADPH, as summarized in the following equation: 3CO2 + 9ATP + 6NADPH → GAP + 9ADP + 8Pi + 6NADP+ (1.2) 2 S. Frigerio et al. To carry out these reactions, in particular those of the light phase, plants need a specific apparatus, which is in part shared by other photosynthetic organisms like cyanobacteria and green algae. The photosynthetic apparatus is located within the cells in dedicated organelles called chloroplasts. The origins of chloroplasts is most probably due to an event of endosymbiosis of a cyanobacterium by an eukaryotic cell [3]. 1.2 Chloroplast Structure Chloroplasts are characterized by two different membrane systems: a double membrane envelope, which encloses a soluble fraction called stroma; within the stroma are located thylakoids, an extended and morphologically complex membrane system carrying the |
photosynthetic apparatus. This inner membra- nous system defines an internal space called thylakoid lumen. The membrane can be divided in two main regions: the grana region, where the membranes form stacked structures of flat vesicles and the stroma lamellae, which create connections between stacked vesicles, insuring continuity to the lumenal space (Fig. 1.1). Thylakoid membranes are composed principally of glycerol-lipids, in particular monogalactosyldiacyl-glycerol and digalactosyldiacyl-glycerol, which distinguish photosynthetic membranes from all the others [4]. The prokaryotic origin of chloroplast is supported by the presence of a circular genome, which encodes almost 90–120 sequences, while many others (∼3,400) or more proteins, predicted as chloroplastic, are encoded by nuclear genes [5]. Fig. 1.1. Chloroplast structure. Localization in a typical mesophyll cell, schematic representation and electron micrograph, showing thylakoid membranes divided in grana and stroma lamellae 1 Light Conversion in Photosynthetic Organisms 3 1.3 Pigments and Light Absorption The capacity of plants to absorb light covers a wide range of wavelengths, between 350 and 700 nm, which include almost 60% of incident sunlight spectrum. All the pigments involved in light absorption, which are grouped in to two big classes, chlorophylls and carotenoids, are located within the thylakoids. Chlorophylls, named a or b, because of two different chemical forms, are organo-metallic compounds in which a substituted porphyrin ring structure coordinates a magnesium atom in its center. A phytol chain, neces- sary for their localization within the lipid membrane, constitutes one of the lateral chains of the porphyrin. These pigments strongly absorb red and blue light, while they scatter green light, thus giving reason for plant leafs color [6]. The absorption of a photon drives chlorophylls to an excited state, con- verting electromagnetic energy into electronic excitation; red light determines excitation to the first singlet excited state, while blue light causes excitation to the second singlet state (Fig. 1.2). The photosynthetic apparatus makes the job of converting such molecular excitation into different forms of stable free energy. Electronic excitation of molecules decays to ground state in the nanosecond time scale, and the reac- tions that convert the excitation energy must be fast enough to successfully compete with decay. The higher singlet-excited states, as well as the excited vibrational states, decay by internal conversion to the lowest vibrational state of the lowest ex- cited singlet, in the time range of femtosecond, which is much faster than the Blue 2nd excited singlet state Energy Absorption Red 1st excited singlet state Fluorescence Ground state Fig. 1.2. Light absorption and energy decay scheme Absorption of blue light Relaxation Relaxation Absorption of red light Relaxation Fluorescence 4 S. Frigerio et al. rate of any subsequent process; therefore, the system equilibrate at the first singlet-excited state before anything else can happen. Relaxation to ground state may occur through different pathways: (1) internal conversion, where the excitation energy is dissipated as heat; (2) fluorescence emission, where the energy is emitted as a photon; (3) energy transfer, where excitation energy is transferred to a nearby acceptor molecule (Forster energy transfer) [7]. In the photosynthetic apparatus, the most im- portant is the latter: reiterated energy transfer between neighbor chlorophyll molecules allows excitation energy to reach a particular site (reaction center), where a redox reaction takes place, converting the excitation into chemical energy. For efficient conversion of light energy into chemical energy, a highly effi- cient energy transfer through neighboring pigments is essential. However, the ideal conditions for high efficiency in energy transfer is not always satisfied in the photosynthetic apparatus; if the energy transfer rate is not competing successfully with the other relaxation mechanisms, the excitation energy is lost and a fluorescence emission or heat dissipation takes place. Another important phenomenon must be taken into account. The lowest singlet-excited state of chlorophylls can also undergo intersystem crossing to the triplet state. This excited state cannot transfer its energy to the reaction center and therefore cannot contribute to photochemistry. Worse than that, chlorophyll triplet state can easily activate molecular oxygen to its singlet state, producing a highly reactive and dangerous molecule. The second class of pigments is represented by carotenoids, linear poly- enes [8] involved in facing the problem of singlet oxygen production, besides contributing to absorb light and transfer it to the nearby chlorophylls: they can quench chlorophyll triplet-excited states and also scavenge singlet oxy- gen. In both cases, this leads to the dissipation of the absorbed photon but it prevents oxidative damage to the membrane and photosynthetic apparatus components. 1.4 Photosynthetic Apparatus Light absorption and energy conversion take place in specific and highly or- ganized structures embedded in the thylakoid membrane. All together they constitute the photosynthetic apparatus. There are four different complexes: two Photosystems (PSI and PSII), the cytochrome b6f, and the F-ATPase. These are composed of several protein subunits, cofactors, and pigments, while additional electron carriers can move within the lipid bilayer of the mem- brane or in the aqueous medium (Fig. 1.3). In the last years, high resolution structures for the four complexes, even though from different species, have become available, thus allowing a deeper comprehension of photosynthetic mechanisms [9–13]. 1 Light Conversion in Photosynthetic Organisms 5 Fig. 1.3. Photosynthetic apparatus: Both the photosystems, cytochrome b6f, and F-ATPase are shown, in addition to movable subunits as plastoquinone (PQ), plas- tocyanin (PC), and ferredoxin (Fdx) As pointed out earlier, the task of the photosynthetic apparatus is that of providing reducing power and ATP to drive CO2 fixation. The reducing power in the form of NADPH is generated through a redox reaction in which water is the ultimate electron donor: 2NADP+ + 2H2O + light → 2NADPH + O2 + 2H+ (1.3) The energy of a single photon is not sufficient to drive an electron from water to NADP+, and two photons must be used to make the job. For this reason, organisms using water as electron donor, i.e., oxygenic organisms such as higher plants, algae and cyanobacteria, make use of two photosystems that can operate in series. Photosystem II is able to extract electrons from water and brings them to plastoquinone, a quinone molecule freely diffusible in the membrane lipid moiety. Photosystem I makes use of another photon to bring the electron at the level of NADP+. The connection between the two photosystems is operated by cytochrome b6f complex, which transfers electrons from the plastoquinone pool to plasto- cyanine, a soluble Cu-protein freely diffusible in the thylakoids lumen. This electron transfer is in favor of potential gradient, and there is no need of light energy to drive it. Actually, the role of this complex is not limited to simply catalyze an electron transfer, as this would lead to a significant loss of energy. On the contrary, cytochrome b6f, using the electron transfer energy, acts as a proton pump, which transfers protons against gradient from the stromal to the lumenal side of the membrane. The action of cytochrome b6f is all the way analogue to that of complex III (or cytochrome b1c) of the mitochondrial 6 S. Frigerio et al. − PSI 1.5 P700* A −1.0 0 PSII A1 F P680* X Pha FB −0.5 FA Fd PQ Fd-NADP+ 2NADP + A oxidoreductase 0 PQpool PQB 2H+ Cytochrome bf complex Plastocyanin 2NADPH Oxygen- P700 +0.5 evolving complex 8H + Light 2H2O Z +1.0 P680 O2 + 4H + Light +1.5 Fig. 1.4. Electron transport chain respiratory chain, and plays an essential role in building up the proton gradient necessary to drive ADP phosphorylation by ATP synthase (Fig. 1.4). Photo- systems are composed of proteins and pigments, chlorophylls and carotenoids, responsible for light harvesting; PSI and PSII are different in several aspects, in particular for the supramolecular organization, but it is possible to identify the same two functional moieties in both: (a) a core complex, responsible for charge separation and the first steps of the electron transport and (b) an an- tenna complex responsible of increasing the light harvesting and transferring of absorbed energy to the reaction center. Both the core complexes are composed of protein encoded by psa and psb genes, respectively, for PSI and PSII, which bind pigments and surround the so called “special chlorophyll pair,” where the charge separation actually occurs. The core complex is surrounded by antenna proteins, encoded by the multi- genic family of light-harvesting complexes (lhc), which primarily increase the light-harvesting capacity, but are also involved in regulation and photopro- tection [14]. PSI antenna complex is composed of Lhca1-4 as major subunits and Lhca5-6 as minor [15]; on the contrary, the major component of PSII antenna complex is a heterotrimer of Lhcb1-3 subunits, while remaining Lhcb proteins, Lhcb4-6, are altogether classified as minor antennas and they are generally found as monomers [16]. 1.4.1 Photosystem II Photosystem II (PSII), which is mainly located in grana thylakoids, is a light driven water-plastoquinone reductase. It performs the thermodynami- cally most demanding and most dangerous reaction: splitting water into its elementary components, molecular oxygen and protons. Reduction potential (V) 1 Light Conversion in Photosynthetic Organisms 7 The reaction center (RCII) of PSII is composed of the heterodimer con- stituted by the two proteins D1 (PsbA) and D2 (PsbD), which coordinate all the essential cofactors for its electron transport chain [9]. When the central chlorophylls special pair (P680) is excited by a photon absorbed by the antenna apparatus, a charge separation occurs as an electron is transferred from chlorophyll to pheophytin, in one of the fastest electron transfer reaction (2–20 ps) ever observed in nature. The cation P+ 680 that is formed on this chlorophyll pair is one of the most oxidizing centers that can be formed in a living cell. The subsequent electron transfer is to QA,, a plas- toquinone molecule tightly bound to the D2 protein near the stromal surface. This electron transfer is slower than the previous one but still fast enough (∼400 ps) to compete with recombination. This step involves a significant loss of energy that contributes to stabilize the charge separation. Because of its very high oxidation potential, P+ 680 is able to extract an electron from a tyrosine residue of the D1 protein (Tyr161, also named TyrZ) in about 20 ns; the electron hole at the donor side of PSII ends its run at the Mn cluster, near the lumenal surface of the membrane. In the mean time, in the acceptor side at the other membrane surface, the electron standing on QA finds its way to QB , a plastoquinone loosely bound on the D1 protein, also thanks to the assistance of a non-heme iron, sitting in between the two. This is the last reaction in the electron transfer chain within PSII reaction center and its rate is the limiting step (200–300 µs). A second turnover, driven by a further excitation from the antenna, brings a second electron to QB with the very same reaction sequence. When QB receives the second electron, two protons are up-taken from the stroma and the plastoquinol leaves its binding site on D1. This is then reoccupied by an oxidized plastoquinone from the membrane pool. A two-photons-two-electrons gated mechanism brings about the reduction of a plastoquinone molecule at the acceptor side of the complex. In a four-photons-four-electrons gated mechanism, two more photons and two more electrons injected in the transport chain are required to reduce a second PQ and oxidize two water molecules with the evolution of one oxygen molecule. The so-called “oxygen evolving complex” (OEC) sitting in the lu- menal side of PSII, often indicated also as the “water splitting enzyme,” can be considered as the heart of the PSII activity, and most of the today research efforts are directed to elucidate the detailed mechanism for its activity [17]. 1.4.2 Photosystem I In photosystem I (PSI), which is located almost exclusively in stroma lamellae, the excitation of the chlorophyll special pair P700 in the reaction center of PSI (RCI) initiates a series of electron transfer reactions that culminate in reduction of NADP+ to NADPH. RCI is activated by the absorption of red light that promotes it to an excited state (P700*) at about −0.6 V with a jump of about 1 V with respect to the ground state P700 (E˚’= +0.4 V). 8 S. Frigerio et al. This generates a reductant strong enough to reduce ferredoxin, a soluble iron- sulfur protein able to donate electrons to NADP+ by the action of the stromal soluble enzyme ferredoxin-NADP+ reductase. P+ |
700 formed is rereduced by an electron carried by the reduced plastocyanine (Fig. 1.4). Therefore, PSI which is situated mainly in the nonstacked, stroma lamellae regions of the thylakoid membrane, functions as a plastocyanine–ferredoxin reductase. The core of the RCI is composed of two major subunits, PsaA and PsaB, which contain all the pigments of the complex (including most of the antenna chlorophylls and carotenoids) and all the electron carriers. Upon absorption of a photon, charge separation occurs and an electron is transferred to a primary acceptor Ao (chlorophyll a monomer) from which it proceeds to the intermediate acceptor A1 (phylloquinone) and subsequently to the second intermediate acceptor A2 also indicated as Fx. This is a (4Fe–4S) cluster, as well as the subsequent electron acceptors FA and FB, which transfer the electron to the ferredoxin bound in its docking site on the stromal surface of the PSI complex. P700, Ao, A1, and FX are associated with the PSI intrinsic core subunits PsaA and PsaB, while the terminal electron acceptors FA and FB are bound to an extrinsic low molecular mass subunit PsaC [11]. It should be noticed that the electron transfer kinetics within RCI is very fast and not completely resolved yet. 1.4.3 Cytochrome b6f The electron transfer bridge between the two photosystems is realized by the cytochrome b6f complex. This is assembled as a functional dimer with a twofold symmetry axis, each monomer being composed of four subunits: the cytochrome b6 carrying two b-type heme groups classified as high and low potential, the cytochrome f carrying a single c-type heme group, the Rieske iron-sulphur protein carrying a (Fe2-S2) cluster and the subunit IV [13]. The reduced plastoquinoles, freely diffusing inside the lipid bilayer, bind to the complex and electrons are transferred through cytochrome b6f to the acceptor, plastocyanin. The transfer of two electrons from plastoquinol to the single electron carrier plastocyanin (summarized in the reaction below) QH2 + 2plastocyanin(Cu2+) ⇀↽ Q + 2plastocyanin(Cu+) + 2H+ (1.4) is realized through a complex series of redox reactions, which involves the semiquinone form Q−, named “Q cycle,” with the effect of transferring 4 H+ instead of only two from the stroma to the lumen. This increases the efficiency of converting reduction potential energy into transmembrane proton gradient. Reduced plastocyanin moves in the lumen and reaches the PSI where it will donate its electron to P+ 700 (see earlier). 1.4.4 ATP Synthase The last complex in the photosynthetic apparatus is the ATP synthase (F- ATPase). This kind of enzyme is ubiquitous in energy-transducing membranes, 1 Light Conversion in Photosynthetic Organisms 9 and the structure is highly conserved [12]. It presents a transmembrane do- main and a stromal portion. F-ATPase in the chloroplast synthesizes ATP, using the proton-motive force generate by H+ accumulation in thylakoid lu- men. The diffusion of protons through a channel (Fo) determines the rotation of F-ATPase inner subunits inducing cyclic conformational changes in the F1 subunits, which provide the energy for ADP phosphorylation and release of ATP in the stroma; four protons are needed for the synthesis of one molecule of ATP [18]. The equilibrium along the electron transport chain, between reduced and oxidized species, is crucial for plant life. Excess energy absorbed by chloro- phylls drives the formation of extremely dangerous reactive oxygen species (ROS), which can oxidize all the biological component of a cell. For this rea- son, absorbed energy can be redistributed among photosystems. When excess light is absorbed by PSII, thanks to a phosphorylation process, a population of LHCII trimers moves through the membrane and attach to PSI, thus in- creasing PSI light absorption capacity [19]. This phenomenon is called “state 1–state 2 transition.” 1.5 Cyclic Phosphorylation High electron transport rates drive the formation of reduced ferredoxin (Fd) in excess with respect to the ferredoxin-NADP+ reductase activity. To avoid over-reduction of the electron transport chain and block of proton pumping and ATP synthesis, a mechanism, alternative to liner electron transport and involving PSI, can be activated, the so-called cyclic phosphorylation [20]. In this case, reduced ferredoxin can diffuse in the stroma back to the cytochrome b6f and transfer electrons to the heme exposed in this region, through the action of a Fd-NADP-reductase [21]. Translocation of the elec- tron through cytochrome to plastocyanin and then again to PSI leads to the generation of protons in the lumen, which can be used for ATP synthesis, even without NADPH production. This alternative pathway exists in parallel with linear electron transport; the latter is limited by the oxidation rate of PSI acceptors, thus leading, under constant light intensity, to reach a kind of steady-state. Before the attainment of this steady-state, if PSII is inhibited, few plastocyanin molecules move to PSI, so all the electrons that reach Ferredoxin will be used for NADP+ reduc- tion, thus inhibiting cyclic electron flow [22]. With the increase of excitation pressure, the oxidation of Fd becomes the limiting step, so the cyclic phos- phorylation is activated. In addition, the dark phase of photosynthesis is also involved in this alternative pathway, as it has been recently observed that in dark-adapted plants cyclic electron transport is activated first, as a conse- quence of a scarce NADPH requirement due to Calvin cycle inhibition [23]. The two alternative electron flows can exist simultaneously because they are structurally separated [24]. Because PSI and PSII are differentially located 10 S. Frigerio et al. within thylakoids, the position of the cytochrome b6f complex assumes a dif- ferent role: the population located near to PSII participates to linear electron flow, while the population close to PSI is involved in cyclic electron transport. This separation is driven to the extreme level in the case of C4 plants, like maize, where PSII, thus the linear electron flow, is located in mesophyll, while PSI, thus cyclic electron flow, is located in bundle sheath [25]. The size of the populations located close to PSII vs. PSI can be regulated by phosphorylation events [26]. 1.6 Photoinhibition In describing the function of PSII, the attention has been focused on the reac- tion center, where the excitation energy coming from the antenna apparatus is converted into chemical energy through a series of redox reactions. The composition of the reaction center and its electron transfer cofactors has been briefly discussed, but PSII core is much more complex and contains a number of other protein subunits as always happens with enzymatic complexes that must be finely regulated. In fact, a PSII core monomer contains at least 16 integral subunits, 3 lume- nal subunits, 36 chlorophylls a (two loosely attached), 7 all-trans carotenoids, 2 heme groups (b and c type) belonging to the cytochrome b559, 1 non-heme iron, 2 quinones, 2 pheophitins, and it is organized in functional dimers char- acterized by a pseudo twofold symmetry [9]. Such a great complexity is partially justified by the difficult task the PSII core must accomplish of splitting water bringing its electrons at the level of plastoquinone in the membrane, but it also strongly suggests a need for a fine regulation of its function. There are, indeed, several good reasons for PSII activity to be regulated: the electron transport turnover depends on the frequency of P680 excitation, which, in turn, depends on the light fluence reaching the light harvesting apparatus. This must be large enough to ensure good photosynthetic activity even when the environment light intensity is very dim. But the photon fluence can vary very quickly by several orders of magnitude. Thus, a plant, which is tuned for optimal electron transport in dim light, will find itself overexcited when exposed to full sunlight. Over excitation of PSII brings oxidative damage to the protein-pigment complexes, thus impairing the photosynthetic activity. This is a well-known phenomenon called “photoinhibition” and represents the most important limiting factor to crop productivity [27]. Therefore, a regulation is needed at the level of the excitation transfer from the antenna to the reaction center. Indeed several sophisticated control mechanisms have been set up by evolution for this process. Nevertheless, at a light fluence typical of midday full sun, a significant amount of photoinhibition takes place, which severely limits energy conversion. 1 Light Conversion in Photosynthetic Organisms 11 Although some kind of photoinhibition has been shown to take place also at the level of PSI [28], the main phenomenon regards PSII, where an inactivation mechanism inherent to the same electron transfer activity takes place, with low quantum yield, even at low light irradiance [29]. At increasing light intensities, inactivation of PSII occurs with higher frequency and in extreme cases may bring to irreversible oxidative damage of the complex. In the last decades, considerable efforts were directed to the elucidation of the molecular mechanisms of this important phenomenon. It is long known that the D1 protein (PsbA) of the PSII reaction center is rapidly turned over and that its turnover rate increases with the light intensity [30]. Why is that so? It has been hypothesized that D1 is the protein subunit, which is preferentially damaged when the reaction center is over excited, and it needs to be frequently substituted to maintain the reaction center active. In fact, the water splitting photochemistry of PSII produces various radicals and active oxygen species, which cause irreversible damage to PSII. However, damaged PSII reaction centers do not usually accumulate in the thylakoid membrane because of a rapid and efficient repair mechanism. It has been calculated that this repair mechanism is at least as important as that of DNA, so that the green kingdom in the biosphere could not survive in its absence. It is commonly believed that the design of PSII allows protection for most of its protein and pigment components, with the oxidative damage be- ing mainly targeted to a single subunit, the reaction center D1 protein. Re- pair of PSII via turnover of the D1 protein is a complex process (Fig. 1.5) STACKED THYLAKOID MEMBRANES LIGHT INACTIVATION DAMAGE P P LHC D1 D2 D2 D1 LHC P P P P P P LHC D2 D2 LHC LHC D1 D1 D1 D2 LHC b559 b559 b559 b559 b559 43 47 47 43 1. 47 43 43 47 47 43 33 16 33 16 2. 33 16 33 16 33 16 23 23 23 23 23 PHOSPHORYLATION MONOMERIZATION MIGRATION DIMERIZATION MIGRATION 7. psbA D1 mRNA 3. D2 D1 D2 DEPHOSPHORYLATION P P P OF CP43. D2 AND D1 D2 D1 b559 b559 47 43 D1 PROCESSING b559 47 43 D1 PROTEOLYSIS 47 43 33 16 D1 FRAGMENTS 4. 23 6. 33 16 23 5. 33 16 SYNTHESIS AND INSERTION OF D1 23 LIGATION OF COFACTORS STROMA-EXPOSED THYLAKOID MEMBRANES Fig. 1.5. Repair cycle of photosystem II 12 S. Frigerio et al. that involves reversible phosphorylation of PSII proteins and changes in the oligomeric structure of the complex [31]. This is combined with the shuttling of the complex between grana and stroma-exposed thylakoid domains, partial PSII disassembly, and highly specific proteolysis of the damaged D1 protein. Replacement of degraded D1 protein with a new copy requires a complex co- ordination between its degradation, resynthesis, insertion, and assembly into the PSII core. Although enhanced during higher irradiances, turnover of D1 occurs at all light intensities, and can be easily monitored in pulse-chase ex- periments, an efficient tool to study the phenomenon. It is now clear that most of the regulation of gene expression required for PSII repair cycle is exerted at the translational and posttranslational levels by the redox conditions in the thylakoid membrane and in the stroma. D1 protein together with D2 constitutes the scaffold for all the electron transfer cofactors within RCII. It is therefore not surprising that its sacrificial high turnover produces profound effects on the photosynthetic activity as a whole. There are intrinsic difficulties in understanding the rational of the pro- posed model: why should the D1 subunit be preferentially damaged by excess irradiation? How the specific proteases (belonging to the FtsH family) can actually recognize the damaged protein, considering that oxidative damage is probably a random process with no specific targets? The common view proposes a damage-induced conformational change on the D1 protein that would trigger its proteolytic degradation and some experimental evidences are brought about to support this view [32]. Let us take into consideration the main mechanism for oxidative damage inside the PSII core. When the frequency of excitation of P680 is higher |
than the rate of utilization of the reduction equivalents by PSI or, ultimately by CO2 fixing activity (Calvin-Benson cycle), an over-reduction of the plasto- quinone pool is produced and the electrons coming from RCII cannot find the physiological exit from the complex. In these conditions, i.e., when the elec- tron acceptors are fully reduced and cannot receive further electrons, charge recombination at P680 produces its triplet form T P680 that is able to activate oxygen molecules to their singlet state 1O2. Normally carotenoids take care to avoid oxygen activation in the antenna by scavenging the chlorophyll triplet states by thermal dissipation. In the reac- tion center, there are actually carotene molecules, but none of them are close enough to P680 for the chlorophyll triplet to be transferred to the carotene and dissipated. If they were, they would immediately become oxidized by P+ 680. Singlet oxygen is the main responsible of direct or indirect oxidative damage to both pigments and proteins of PSII but it is not completely clear why the action of activated form of oxygen should be limited in their target to the D1 subunit. A possible alternative model comes from experiments on PSII from cyanobacteria [33]. In this paper, it is shown that, under conditions of strong over-reduction of the PSII electron acceptors, massive chlorophyll bleaching 1 Light Conversion in Photosynthetic Organisms 13 from singlet oxygen is observed in a mutant that is not able to quickly degrade the D1 protein. On the contrary, in a mutant strain in which degradation of D1 takes place quickly, chlorophylls bleaching is very little, if any. On the basis of these and other experimental evidences, it is proposed that triggering of D1 degradation is not necessarily associated to an oxidative damage, which makes it a substrate for the proteolytic activity but, provided that the D1 subunit is maintained in the right conformation, the protein is simply turned over and replaced by the so-called “repair cycle” at a rate which is regulated by the redox state of the chloroplast. When the excitation supply exceeds the rate at which carbon dioxide can be fixed by the Calvin-Benson cycle, the reduction level increases and the DegP-FtsH proteases are activated to degrade D1. This mechanism ensure a protection to PSII before the oxidative damage by activated species of oxygen takes place. It is like the case of a prudent car driver who decides to substitute its car tires after a given number of kilometers rather than waiting for them to blow up. 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Andersson, Biochim. Biophys. Acta. 1019, 269–275 (1990) 33. E. Bergantino, A. Brunetta, E. Touloupakis, A. Segalla, I. Szabo, G.M. Giacometti, J. Biol. Chem. 278, 41820–41829 (2003) 2 Exploiting Photosynthesis for Biofuel Production C. Govoni, T. Morosinotto, G. Giuliano, and R. Bassi During the recent “Energy Outlook and Modeling Conference” of March 2006 in Washington DC, it was estimated the world current energy consumption in 100 “Quads,” where one Quad corresponds to about 25 million of oil equiv- alent tons (MTep, http://www.eia.doe.gov/oiaf/aeo/conf/). Nowadays, over 85% of world energy demands are met by the combustion of fossil fuels: coal, oil, and natural gas. Current oil reserves are estimated to be about 1,277 billions of barrels and, assuming a stable consumption, they would be suf- ficient for next 42 years. However, in the middle-term oil utilization is ex- pected to rise of 1.6% every year thus making reserves exhaustion even faster (http://www.eia.doe.gov/oiaf/aeo/conf/pdf/petak.pdf). It is thus clear that to sustain our lifestyle to find alternative and renewable sources of energy is a striking and urgent need. Besides the problem of fossil reserves depletion, the massive combustion of fossil fuels in the past decades also had a high environmental impact. In fact, this leads to releases of large amounts of carbon dioxide and other pollu- tants in the atmosphere. However, these emissions are reassimilated by natural processes, which cannot keep the pace of the present CO2 production rates. In fact, every year the forests are able to fix about 1 billion of tons of carbon in organic matter and further 2 billion of tons are fixed in the ocean every year by the sea photosynthesis, but the CO2 emissions caused by the human activity are about 6 billion of tons. So the balance is positive and every year 3 billion of tons increase the global level of CO2 in the atmosphere (Fig. 2.1). The alteration of this equilibrium caused an accumulation of carbon diox- ide in the atmosphere, which is estimated to have risen of about 15% since 1750. The main consequence of the accumulation of CO2 in the atmosphere has been proposed to be the “greenhouse effect” some gases like carbon diox- ide, in fact, are able to retain the infrared radiation into the atmosphere and cause a global temperature increase of the planet. The correlation between CO2 and temperature increase is clear, although a cause/effect relationship has not been clearly established, yet. 16 C. Govoni et al. Atmospheric net carbon Increase 3-4 Gt / y Net Terrestrial Fossil Fuels Uptake: 0-1 Release: 6 Net Ocean Uptake: 2 Photosynthesis Plant Biomass Photosynthesis Respiration Microbial Decomposition Soil Deep ocean Rocks Sediments Fig. 2.1. Simplified global carbon cycle. Biogeothermal carbon cycle is schematized, distinguishing between terrestrial and aquatic processes. Photosynthesis, which fixes atmospheric CO2 is indicated in black, while processes responsible of carbon dioxide release (respiration, microbial decomposition, and fossil fuels combustion) are indi- cated in italic. The net uptake activity of terrestrial and ocean activities are circled. Plants biomass, soil, rock, deep ocean, and marine sediments are indicated in white and are sites of long-term carbon storage. This temperature increase has two major consequences. The first is deser- tification of subtropical areas, which caused a massive reduction of forests. As trees are photosynthetic organisms, which fix the atmospheric carbon dioxide into biomass, their massive reduction is causing further enhancement of car- bon dioxide accumulation. Although a transitory effect, even more important is that desertification implies oxidation to CO2 of the organic mass contained in the soil (1–2%). The second effect consists into the thawing out of the polar areas. In these regions, a large amount of CO2 has been fixed as frozen organic matter in the underground during several million years. Because of the global warming, this organic matter is being thawed out and mineralized by bacteria and fungi causing a further CO2 evolution. The public conscience that the search of energy sources alternative to fossil fuels is fundamental for future prospects is generally increasing, and research efforts are devoted to this field in several countries. The attention is focused on renewable resources, such as sun and wind, which can be considered as no exhaustible. Furthermore, their massive exploitation will drastically reduce chemical, radioactive, and thermal pollution, and they therefore stand out as a viable source of clean and limitless energy. 2 Exploiting Photosynthesis for Biofuel Production 17 Among the possible renewable energy sources, an interesting emerging option is the exploitation of higher plants or algae and their capacity to harvest solar light and convert it into chemical energy. This option is particularly interesting over other methods of solar radiation exploitation because these organisms are able to convert CO2 into biomass, through the photosynthesis, and thus their utilization will also contribute to reduce carbon dioxide levels. A further advantage is that, unlike fossil fuels, photosynthetic organisms are rather uniformly distributed over earth surface. Thus, their exploitation would not require large plants, and energy will be produced locally in small bio-power plants. This different spatial distribution of power plants has alone a positive effect on energy efficiencies: in fact, as a relevant percentage of energy is lost only during its transport from production to utilization, the reduction in distance would reduce significantly energy production needs. Possible applications of plants or algae for energy production are described, with particular attention on their possible exploitation for biofuels and bio- hydrogen production. 2.1 Biological Production of Vehicle Traction Fuels: Bioethanol and Biodiesel Any fuel derived recently from living organisms or from their metabolic by- product is defined as a biofuel. Unlike other natural resources like oil, coal, and nuclear fuels, biofuels are a renewable energy source. They present the advantage of a cleaner combustion in comparison to conventional fuels, and thus have a lower impact on atmospheric CO2 levels. Liquid biofuels are mainly developed for vehicle traction. At present the most promising options are bioethanol and biodiesel. 2.1.1 Bioethanol Bioethanol can be utilized as a fuel in combustion engines in different ways: (1) as hydrous ethanol (95% by volume), containing small percentage of water, it can be used as a gasoline substitute in cars with modified engines. (2) As anhydrous (or dehydrated) ethanol, free from water or at least 99% pure, it can be blended with conventional fuels in ratios between 5 and 85% (E85). As a 5% component, it can be used in all recently produced engines without modification. Higher blends require modified engines installed in the so-called flexible fuel vehicles. (3) Finally, bioethanol is also used to manufacture ethyl- tertiary-butyl-ether (ETBE), a fuel additive for conventional gasoline. Bioethanol is produced from starch plants (like grain, corn, and tubers- like cassava), sugar plants (sugar beet or sugar cane), and – although not yet on a large scale – from cellulose. Carbohydrates present in these plants are used as a substrate for the microbial |
fermentation, and ethanol is subsequently 18 C. Govoni et al. enriched by distillation/rectification and dehydration. The CO2 liberated dur- ing its combustion was first fixed from the atmosphere by the crops during their growth, and thus the use of bioethanol in place/addition of fossil fuels would then reduce the net release of greenhouse gases in the atmosphere. The potential impact of this strategy is large if we consider that road transport ac- counts for 22% of all greenhouse gas emissions (http://:www.foodfen.org.uk). Bioethanol would have a positive environmental impact also because it is biodegradable and not toxic for living organisms as fossil fuels. A further advantage is that bioethanol can be easily integrated into the existing fuel distribution system. In fact, as mentioned, in quantities up to 5%, bioethanol can be blended with conventional fuel without the need of engine modifica- tions and adaptation of forecourts and of transportation system. Finally, its increased use would also have positive a socio-political impact, by reducing the dependence from the oil producing countries and boosting the rural economy. 2.1.2 Biodiesel Biodiesel is an alternative fuel, which can be produced from vegetal resources. It is made by the trans-esterification of vegetal lipids generating lipid methyl esters (the chemical name for biodiesel) and glycerine, which is a valuable by-product used in soaps and other products. In USA, biodiesel is mainly produced from soybean oil, while in Europe from rapeseed oil. In addition to these crops, biodiesel can be produced using some microalgae, which are able to accumulate large amount of triglycerides in their cells. Biodiesel contains no petroleum oil, but it can be blended at any level with petroleum diesel, and in this form it can be used in recent compression-ignition (diesel) engines with little or no modifications. Thus its introduction, even if massive, would not need modification in the fuel dis- tribution systems. It also shares other advantages with bioethanol. As it is produced from vegetal sources, its utilization would have no net impact on the concentration of carbon dioxide in the atmosphere. Furthermore, biodiesel is also biodegradable, nontoxic, and essentially free of sulphur and aromatics. 2.1.3 Biofuels Still Present Limitations Preventing Their Massive Utilization Although bioethanol and biodiesel present big advantages for the environment and they could rather easily substitute at least partially fossil fuels, there are still two major limitations to the massive use of biofuels: energetic yield and costs. First, the energetic yield is calculated as the ratio between all the energetic inputs (used for the cultivation of the crop, transport, and transformation) and all the outputs (fuel, but also all the by-products, as glycerine in the case of biodiesel). The energetic yield of biodiesel from soya, for example, is 2 Exploiting Photosynthesis for Biofuel Production 19 about 3.2, meaning that for every unit of energy employed in the production of biodiesel, 3.2 units are harvested at the end of the process. Performances with bioethanol from maize are even less encouraging being its energetic yield about 1 or even less [1, 2]. Second, considering the present oil price, biofuels are still not economically competitive. In fact, biodiesel is estimated to be economically convenient over the limit of 80$/barrel (and even higher for bioethanol). So, at least on a short term, biofuels introduction in the commercial networks needs to be financially supported by governments willing to stimulate their utilization. However, in a medium timespan perspective, when oil reserves will be exhausting and the oil price will consequently rise, biodiesel will be a major option for vehicle traction and investments will be paid back. 2.2 Hydrogen Biological Production by Fermentative Processes Hydrogen has some very interesting properties, which makes it the potential energy source of the future. It is easily converted into electricity, and it has very high energy content per weight unit. Moreover, its utilization has the lowest possible environmental impact. It produces electricity by reacting with oxygen and yielding water as the only waste. However, these advantages are effective only if hydrogen is produced by an efficient and clean process. Un- fortunately, this is currently not the case, and hydrogen is mainly derived from fossil fuels and its production generates considerable amounts of CO2 in addition to other air pollutants such as sulphur dioxide and nitrogen oxides (http://www.fao.org/docrep/w7241e/w7241e05.htm). Thus, hydrogen’s positive impact on the energy system depends on the development of new methods for its efficient production. One major option is the water electrolysis, which, however, requires an efficient, clean, and cheap method for electricity generation, which is at present not available. For this reason, other options are receiving an increasing attention and, among them, the biological H2 production. In fact, some living organisms are able to produce H2 in anaerobic conditions thanks to metabolic reactions coupled with fermentation, nitrogen fixation, or photosynthesis. This is a very promising approach, as it has the potential to provide cheap hydrogen without using fossil fuels and emitting pollutants. Living organisms can synthesize hydrogen from protons and electrons, us- ing special enzymes named hydrogenases. Hydrogenases fall into two major classes: Ni-Fe and Fe-Fe hydrogenase identified from the metal content in their active site [3]. They differ in the composition and in the structure of the metal center active site as well as in their polypeptide sequence, but also show some conserved properties, namely the presence of CN and/or CO lig- ands in the active site. In general, Fe-Fe hydrogenases were shown to be more 20 C. Govoni et al. efficient catalysers, and therefore they are more promising for practical ex- ploitations [4]. Both Fe-Fe and Ni-Fe hydrogenases have a strong sensitivity to O2 presence, probably because of its binding to the metallocatalytic sites. For this reason, all hydrogen producing organisms identified till very recently are active in anaerobic conditions only. Exceptions to this pattern are still under evaluation. Several organisms can produce hydrogen and differ for the source of reduc- ing power they use for this biosynthesis. Some bacterial strains can exploit organic compounds by fermentation, while other organisms, such as green algae, are able to use directly the solar radiation harvested by the photo- synthetic apparatus. However, we must be aware that, despite these strong potential advantages, several bottlenecks need to be resolved before practi- cal application can be economically sustainable and further research is still needed. 2.2.1 Hydrogen Production by Bacterial Fermentation Several bacteria are able to produce hydrogen from the fermentation of car- bohydrates in anaerobic conditions. As substrates for this reaction, several sources could be used. However, the use of human and industrial wastes is particularly interesting as it attains two objectives at the same time: process- ing the waste and produce environmental-friendly energy. Anaerobic bacteria use organic substances as source of electrons and en- ergy, converting them into hydrogen. The reactions involved in hydrogen pro- duction (2.1 and 2.2) are rapid and suitable for treating large quantities of wastewater in large scale fermenters. Glucose + 2H2O = 2Acetate + 2CO2 + 4H2 ∆G = −184.2 kJ (2.1) Glucose = Butyrate + 2CO2 + 2H2 ∆G = −257.1 kJ (2.2) The produced molecules, acetate and butyrate, still have a residual chemical energy, which remains unexploited. This is because the complete oxidation of carbohydrates to H2 and CO2 is not a spontaneous process as it has a ∆G of only −46 kJ mol−1 hydrogen. There are no known fermentation pathways that can achieve a conversion efficiency larger than 4 mol H2/mol hexose [5]. To achieve a further decomposition of the remaining organic substances, an external energy input is necessary. This can be derived, as recently described, from the application of an external small electric potential, which stimulates the fermentation of acetate to H2 in modified microbial fuel cells [6]. Another valuable option is to couple the first fermentation step by anaero- bic bacteria with the second one performed by photosynthetic sulphur bacte- ria, which use the residual organic acids to produce hydrogen (Fig. 2.2). This last step, which completes the fermentation of organic wastes to hydrogen, can be performed in these organisms, even if energetically unfavorable, as they employ light energy to drive the reaction. 2 Exploiting Photosynthesis for Biofuel Production 21 CO2 Gas Separator H2 H2 + CO2 BIOMASS CARBOHYDRATES FERMENTATION H2 + CO2 LIGHT Organic Acids LIGHT-DRIVEN FERMENTATION Fig. 2.2. Hydrogen production by dark and light fermentation of organic wastes. Carbohydrates from several sources such as human or industrial wastes are fermented from anaerobic bacteria into organic acids, carbon dioxide, and hydrogen. The former are decomposed by photosynthetic bacteria, using light energy to drive the reaction. Gases yields from both fermentation processes are separated by a gas separator, yielding pure hydrogen. Clearly, more research and development is required but, when developed, H2 fermentations from organic wastes would be relatively low cost comparable to methane fermentation (in the range of $4–8/MBTU1) as they could also use similar mixed-tank reactors, without need for the building of new installations. Thus, dark H2 fermentation of wastes is close to be competitive with fossil fuel- derived H2, providing a first approach to large scale biohydrogen production. 2.3 Hydrogen Production by Photosynthetic Organisms In addition to the mentioned sulphur bacteria, other photosynthetic microor- ganisms are able to produce hydrogen. However, they do not use light energy only to drive a fermentative reaction, but also to directly decompose water to hydrogen and oxygen. This possibility is clearly very attractive from the 1 British thermal unit. One Btu is equal to the amount of heat required to raise the temperature of one pound of liquid water by 1◦F at its maximum density, which occurs at a temperature of 39.1◦F. One Btu is equal to ∼251.9 calories or 1,055 J. 22 C. Govoni et al. point of view of environmental impact because this would be the perfect way to produce energy, using only two renewable resources, solar radiation and water. The photosynthetic microorganisms able to produce hydrogen are cyanobacteria and green algae. 2.3.1 Cyanobacteria Cyanobacteria can use two different enzymes to generate hydrogen gas [7]. The first is nitrogenase, which catalyzes the fixation of nitrogen into ammo- nia, producing hydrogen as a by-product. Hydrogen photo-evolution catalyzed by nitrogenases requires anaerobic conditions, as hydrogenases. As oxygen is a by-product of photosynthesis, cyanobacteria have developed two different strategies to achieve anaerobiosis: 1. Some evolved the capacity of building structures with reduced oxygen- permeability, called heterocysts, thus separating physically oxygen evolu- tion from nitrogenase activity [8]. 2. Nonheterocystous cyanobacteria instead separate temporally oxygen evo- lution from nitrogenase activity activating the latter only during dark periods. Whatever is the case, hydrogen production by nitrogenase enzyme is in- teresting but presents an important limitation. As the reaction is coupled to the nitrogen fixation, it is energetically demanding, making the whole process poorly efficient. The other hydrogen-metabolizing enzymes in cyanobacteria belong to the class of hydrogenases. One class, uptake hydrogenase (encoded by hupSL gene), however, catalyzes the oxidation of hydrogen molecules to produce pro- tons and electrons [9]. Uptake hydrogenase enzymes are found in the thylakoid membrane of heterocystis from filamentous cyanobacteria, and they transfer the electrons from hydrogen for to the respiratory chain. Nitrogenases of this type are not responsible for net hydrogen production in vivo, rather, allow for partial recuperation of energy invested in N2 fixation and employed for hydro- gen production. In the perspective of the production of hydrogen molecules, their activity is not desirable and should be inhibited. The second type of hydrogenases are reversible, or bi-directional hydroge- nases (encoded by hoxFUYH ) that can either consume or produce hydrogen. The biological role of these bidirectional or reversible hydrogenases is poorly understood and thought to be involved in the ions level control of the organ- ism. Reversible hydrogenases are associated with the cytoplasmic membrane and likely function as electron acceptor from both NADH and H2 [10]. If op- portunely regulated, these enzymes can be exploited for hydrogen production. 2 Exploiting Photosynthesis for Biofuel Production 23 2.3.2 Eukaryotic Algae Gaffron and coworkers over 60 years ago discovered that green algae, when illuminated after an anaerobic incubation in the dark, have the ability to produce H2 [11, 12]. This hydrogen production is directly coupled with wa- ter oxidation through Photosystem II (PSII) and the photosynthetic electron transport chain. Thus, H2 is synthesized directly from water, using sunlight as a source of energy, with a totally clean process, making very promising the perspective |
of exploiting green algae for a large scale H2 production. More- over, differently from the cyanobacteria, H2 production is achieved by Fe-Fe hydrogenases, thus in a more efficient way. 2.4 Challenges in Algal Hydrogen Production The exploitation of algae for hydrogen production is probably the method with the best perspectives in the long term among those that are analyzed here. However, it has two major limitations that prevent its large scale exploitation: (1) the great sensibility of the process to the oxygen presence; (2) the inefficient light energy distribution in the bioreactor. 2.4.1 Oxygen Sensitivity of Hydrogen Production As mentioned, hydrogen is always produced in anaerobic conditions because all hydrogenases are sensitive to oxygen. This limitation becomes deleterious when oxygenic photosynthetic organisms such as algae are discussed: in fact, they use water as electron donor to produce protons and oxygen. The latter is thus an unavoidable by-product of photosynthesis and even the incubation in anaerobic conditions is sufficient to avoid inhibition of hydrogen production. Different strategies are possible to obtain a significant level of hydrogen production by photosynthetic organisms: one is to separate temporally or spa- tially oxygen evolving process from the hydrogen production. Alternatively, modified hydrogenase enzymes, less sensitive to O2, can be searched. Some cyanobacteria, as mentioned, can maintain a state of controlled anaerobiosis in heterocystis; however, it is difficult to imagine modifying green algae to make them able to build such complex structures. In the case of algae, thus, the approach of a temporal separation of oxygen and hydrogen evolutions is more promising. In 2000, Melis et al. [13] demonstrated in Chlamydomonas reinhardtii that, when sulphur in the growth medium is limiting, rate of oxy- genic photosynthesis declines without affecting significantly the mitochondrial respiration. In fact, under illumination Photosystem II (PSII) polypeptides, in particular the D1 subunit, have a very fast turnover. Under sulphur de- privation, when the protein biosynthesis is inhibited, PSII is thus among the first protein complexes affected, causing the decrease of photosynthetic ac- tivity and oxygen evolution. On the contrary, the mitochondrial complexes, 24 C. Govoni et al. which have a lower turnover rate, can maintain longer their function. In sealed cultures, the imbalance between photosynthesis and respiration results in a net consumption of oxygen by the cells and the establishment of anaerobiosis, which stimulates the H2 production. Sulphur deprivation, however, is a stress condition for the cells and after a few hours H2 production yield decreases. Therefore, it is necessary to restore a normal level of sulphur in the culture to allow the cells to recover, re-establish photosynthesis and reconstitute energy reserves. However, the process is reversible and after a recovery delay the sulphur deprivation treatment can be repeated for further induction of hydrogen pro- duction. In fact, it has been demonstrated that phases of S deprivation and H2 production can be alternated to phases where sulphur is supplied and H2 is not produced. The maximum energy yield obtained by this method is ap- proximately 10%, so close to the best photovoltaic cells. The cost is lower, however, as solar cells need to be built while algal cells reproduce. However, the biological systems can reach maximum yields only in a transitory way, while in the long-term average yields are still too low (0.1–1%). In addition to the still insufficient yields, the implementation of this method of hydrogen production on a large scale system is complicated by the need of changing the growing medium to add or remove sulphur. For this reason, it is important to search more efficient methods to induce anaerobio- sis, as PSII is the only responsible of oxygen evolution, and the regulation of its expression is a relatively simple method to induce or suppress anaerobiosis. For this reason in the perspective of a large scale application, it would be more practical to have Chlamydomonas strains where one or more genes essential for the PSII assembly are under the control of an inducible and easily con- trolled promoter. If the presence or absence of PSII can be readily controlled in the culture, it would be possible to control the succession of anaerobiosis and aerobiosis. The oxygen sensitivity of the hydrogen production is mainly due to the sensitivity of the enzyme hydrogenase itself. In fact, these enzymes are inacti- vated by the presence of very low oxygen concentrations. The exact reason for this sensitivity is not fully understood at molecular detail and is the subject of intense research. However, as different hydrogenases have variable struc- tural organization but share a similar active site, the nature of this latter component is probably causing oxygen sensitivity. The most efficient method to obtain large scales hydrogen production would be to find an oxygen resistant hydrogenase. To this aim two differ- ent approaches are possible: first, to search for hydrogenase mutants with an higher selectivity for hydrogen. In particular, the idea of reducing the oxy- gen diffusion to the active site by modulating the molecular size of the gas channels within the molecule looks promising. The second possible approach is to find organisms that can synthesize hydrogen even in presence of oxygen and thus naturally have oxygen resistant hydrogenases. In this respect, it is interesting the finding that such organisms may actually exist in nature, as 2 Exploiting Photosynthesis for Biofuel Production 25 demonstrated by the example of Thermotoga neapolitana, which was shown to produce hydrogen even in the presence of 4–6% oxygen [14]. The gene encod- ing the hydrogenase of this organism has been isolated and sequence analysis suggested that its superior resistance could be due the fact that this enzyme is generally found as a trimer, while all the other hydrogenases known are monomers. Thanks to this larger molecular size, so that its active site can be buried and less accessible to oxygen molecules. However, this explanation alone is insufficient as a homologous organism, T. maritima has an hydro- genase with similar size but it is not equally resistant to oxygen. In a close future perspective, the study of this hydrogenase would suggest possible ways to further improve oxygen resistance, while the expression of this resistant hydrogenase in Chlamydomonas cells would allow understanding the possible impact of a oxygen-resistant hydrogenase on hydrogen yields. 2.4.2 Optimization of Light Harvesting in Bioreactors The first step in hydrogen production is light absorption by photosynthetic complexes. When algae grow in photobioreactors, this process has a poor effi- ciency because of the bad distribution of light. In fact, to have good produc- tion yields, biomass concentration need to be high, leading to increased optical density of the culture where light can only penetrate for a few millimeters, as shown in Fig. 2.3. As a consequence, the external cell layers are exposed to a very intense light leading to activation of photo-protective mechanisms to dissipate energy in excess, to avoid formation of oxygen reactive species and consequent cellular damages. This cells population, thus, is not efficient in the light utilization because absorbed energy is dissipated rather than used for metabolic reactions. On the contrary, cells located at more internal level in the culture are exposed to very low light, insufficient for sustaining intense metabolic activity. This unequal light distribution has another negative consequence: the con- nective fluxes in the medium cause algal cells to stir into the reactor where they can move from dark to strong light within few seconds. The fast variation in light intensity leads to an input of reducing power into the photosynthetic chain at very different rates, without allowing the time for acclimation of photosynthetic apparatus [15]. These conditions are highly stressful because, without the delay needed for the activation of photo-protective mechanisms, the energy absorbed in excess generates oxidative stress and leads to PSII photoinhibition. To overcome these problems and increase the productivity of photobiore- actors, two type of interventions are needed: 1. To reduce the antenna size to lower the optical density of the culture and allow for a better light distribution within the bioreactor without reducing the cellular density 2. To strengthen the algal mechanisms of oxidative stress resistance and of thermal energy dissipation 26 C. Govoni et al. WT strain Light Intensity Inc D r e e p a t s h in Heat g Dissipation Metabolic activity Strain with reduced antenna Light Intensity Inc D r e e p a t s Heat h in g Dissipation Metabolic activity Fig. 2.3. The limits of light distribution in photobioreactors yields. In photobiore- actors, a high cell concentration should be maintained to obtain a reasonable yield. This leads to unequal light distribution. In fact, more exposed cellular layers (left) absorb a very high light, which can be used only partially for metabolism. A large fraction is also dissipated thermally to avoid establishing oxidative stress in cells. Internal layers (right) are illuminated by a very poor light, insufficient to support intense metabolic activity. If a strain with reduced antenna is used in the photo- bioreactor, with an equal cellular concentration, the optical density of the culture is lower and light is more efficiently distributed. External cells absorb a lower propor- tion of available light, which thus reaches more internal layers supporting metabolic activity. Among the different mechanisms, it is important to increase the capacity to respond to fast alterations in light intensity. The only strategy attempted so far to increase the light distribution in pho- tobioreactors has been the use of mutants lacking all the antenna proteins, a class of proteins that are responsible of a large fraction of light harvesting in photosynthetic eukaryotes. With this approach, light distribution has been in- deed improved. However, cells suffered for strong photo-sensitivity and did not survive when exposed to full sunlight. This puzzling phenotype ( lower antenna size is expected to decrease the number of photons funnelled to RCII-reaction 2 Exploiting Photosynthesis for Biofuel Production 27 centre II- and thus reduce sensitivity) is explained by the observation that antenna proteins (named Lhc) are not only involved in light harvesting but also have a major role in photoprotection [16]. As a consequence, the deletion of all antenna proteins reduces the capacity for photoprotection to a level making insignificant the advantage obtained in terms of light distribution. However, among Lhc polypeptides, individual members proteins are spe- cialized in light harvesting, while others in photoprotection [17]. Thus, to obtain mutants optimized to bioreactor conditions, the first step consists in identifying members of Chlamydomonas Lhc protein family essential for pho- toprotection vs. those that can be deleted to lower the optical density. To this aim, we apply two different approaches: in vivo approach consists in the creation of a library of Chlamydomonas insertional mutants. In these mutants genes are randomly knocked out by the insertion of a resistance cassette. These mutants are screened for a smaller antenna size, by analyzing their fluorescence yield properties and Chl a/b ratio. Strains with a reduced antenna content, in fact, would have lower cell fluorescence yield and higher Chl a/b ratio, as Chl b is specifically bound to Lhc proteins. An in vitro approach is complementary: individual members of Lhc fam- ily are studied one by one. Each Lhc protein identified in the genome [18] is characterized by expressing in bacteria the corresponding polypeptides and refolded in vitro to obtain the native pigment protein complex. By this method, each protein will be characterized individually for its capacity of light harvesting and photoprotection. References 1. A.E. Farrell, R.J. Plevin, B.T. Turner, A.D. Jones, M. O’Hare, D.M. Kammen, Science 311, 506–508 (2006) 2. C.N. Hunter, J.D. Pennoyer, J.N. Sturgis, D. Farrelly, R.A. Niederman, Bio- chemistry 27, 3459–3467 (1988) 3. P.M. Vignais, B. Billoud, J. Meyer, FEMS Microbiol. Rev. 25, 455–501 (2001) 4. M.W. Adams, Biochim. Biophys. Acta 1020, 115–145 (1990) 5. R.K. Thauer, K. Jungmann, K. Decker, Bacteriol. Rev. 41, 100–180 (1977) 6. H. Liu, S. Grot, B.E. Logan, Environ. Sci Technol. 39, 4317–4320 (2005) 7. D. Dutta, D. De, S. Chaudhuri, S.K. Bhattacharya, Microb. Cell Fact. 4, 36 (2005) 8. B. Bergman, J.R. Gallon, A.N. Rai, L.J. Stal, FEMS Microbiol. Reviews 19, 139–185 (1997) 9. P. Tamagnini, E. Leitao, F. Oxelfelt, Biochem. Soc. Trans. 33, 67–69 (2005) 10. G. Boison, H. Bothe, A. Hansel, P. Lindblad, FEMS Microbiol. Letters 174, 159–165 (1999) 11. H. Gaffron, Nature 204–205 (1939) 12. H. Gaffron, J. Rubin, J. Gen. Physiol. 26, 219–240 (1942) 13. A. Melis, |
L. Zhang, M. Forestier, M.L. Ghirardi, M. Seibert, Plant Physiol. 122, 127–136 (2000) 28 C. Govoni et al. 14. S.A. Van Ooteghem, A. Jones, L.D. Van Der, B. Dong, D. Mahajan, Biotechnol. Lett. 26, 1223–1232 (2004) 15. A.V. Vener, P.J. van Kan, P.R. Rich, I.I. Ohad, B. Andersson, Proc. Natl. Acad. Sci. USA 94, 1585–1590 (1997) 16. D. Elrad, K.K. Niyogi, A.R. Grossman, Plant Cell 14, 1801–1816 (2002) 17. P. Horton, A. Ruban, J. Exp. Bot. 56, 365–373 (2005) 18. D. Elrad, A.R. Grossman, Curr. Genet. 45, 61–75 (2004) 3 In Between Photosynthesis and Photoinhibition: The Fundamental Role of Carotenoids and Carotenoid-Binding Proteins in Photoprotection G. Bonente, L. Dall’Osto, and R. Bassi 3.1 When Light Becomes Dangerous for a Photosynthetic Organism During operation of oxygenic photosynthesis, highly reactive molecules such as excited chlorophylls are placed in an environment, the chloroplast. This is probably the very spot where oxygen concentration is the highest on earth. There are two major sources of reactive oxygen species (ROS) in the pho- tosynthetic apparatus. The first source is electron transport, where electrons are extracted from water and transported to NADP+. The powerhouses of photosynthetic electron transport are the PSI and PSII reaction centers, which transfer one electron at time. Now, relatively stable oxygen forms are, respec- tively, the most oxidized one (O2) and the most reduced one (H2O). All the other intermediate redox states are highly reactive and are, in fact, called ROS. It can easily be understood that during a multistep electron transport chain, the occasions in which O2 can be exposed to reduction by a single electron thus yielding superoxide are easily produced. Moreover, in PSII, the site where electrons are extracted one by one from water, intermediate redox states may be produced. The second major source of ROS is the process of light energy absorp- tion by chlorophylls and transfer of the excitation energy between the many chlorophylls forming the antenna system. Plants are particularly prone to photo-oxidative damage, and for the same reasons they are effective at photo- synthesis, namely because the primary pigment, chlorophyll (Chl) is a very efficient sensitizer. In fact, chlorophyll has a long living singlet state (5 ns) thus allowing for intersystem crossing and formation of triplet chlorophyll-excited states, which can react with O2, a triplet in its ground state, to yield 1O∗ 2 . This reactive species as well as others deriving from its reaction with water and organic molecules cause oxidative damage to occur in proteins, lipid, and pigments, leading to photoinhibition of photosynthesis and, ultimately, to photobleaching of pigments. 30 G. Bonente et al. 1Chl* 1Chl* Pathway 1: Photochemistry S1 Reaction Center Pathway 2: Pathway 3: Fluorescence Heat (NPQ) T 3Chl* Pathway 4: Intersystem crossing 1Chl S0 Fig. 3.1. The singlet chlorophyll (S1) deexcitation pathways: Pathway 1, energy utilization in photosynthesis (photochemical quenching); pathway 2, the energy reemitted as fluorescence; pathway 3, thermal dissipation pathway (NPQ, nonpho- tochemical quenching); pathway 4, the singlet chlorophyll conversion into triplet (intersystem crossing) When does this happens and how? When a photon is absorbed by a chloro- phyll molecule, the molecule is promoted to its first singlet excited state. This excited state does not live long because it is usually dissipated through dif- ferent pathways (Fig. 3.1); the most important (for photosynthesis) is photo- chemistry, i.e., charge separation and electron transport. If this pathway is active, most of the energy is used through this way and Chl lifetime is below 1 ns. The second pathway is fluorescence: the photon is reemitted and Chl goes back to its ground state. This pathway is less important and fluorescence is usually very low (below 1%); however, it is very useful for scientists as it tells us in any moment the level of excited states in the photosynthetic system. The third path is heat dissipation, which can be extremely variable. Normally, it changes in a complementary way with respect to photochemistry. When the photochemistry is high, there is very little heat dissipation and the yield of the photosynthetic process is high. When some factor (water, temperature, CO2) limits photochemistry, then mechanisms are activated for unharmful dissipa- tion of the excitation energy into heat. The reason for this careful control is actually to keep as low as possible the number of excitons entering the last pathway of deexcitation: intersystem crossing (IC). Intersystem crossing is the conversion, through electronic spin inversion, of chlorophyll single-excited state into triplet-excited state. As light increases, chlorophyll-excited states concentration stays low as far as photochemistry keeps the pace. When photochemistry is limited, heat dissipation is activated, after saturation of heat dissipation pathway, no fur- ther prevention is available and the probability of intersystem crossing occur increases, followed by photodamage. Photodamage can be limited by another set of mechanisms collectively indicated as “ROS scavenging.” Singlet oxygen seems to be the main species produced in photoinhibited membranes, other reactive species are involved, as superoxide anion (O− 2 ), which is produced at both PSII oxygen evolving complex and PSI (Mehler reaction), hydrogen per- oxide (H2O2), and hydroxyl radical (.OH), species which are easily produced 3 Carotenoids Proteins in Photoprotection 31 when the electron transport carriers are over-reduced. A major target is mem- brane lipids, which undergo peroxidation. This proceeds by a free radical chain reaction mechanism. The level of lipid peroxidation, in fact, is one of the most valuable indicators for photooxidative damage. This article reviews several photoprotective processes that occur within chloroplasts of eukaryotic photosynthetic organisms, starting with medium and long-term response strategies to photooxidative stress, as state transi- tion and acclimation. Following, a particular emphasis will be laid on the molecular mechanisms, which prevents ROS formation or participate in ROS detoxification inside the chloroplast. In these mechanisms, carotenoids and carotenoids-binding proteins, which belong to the Lhc family, have a key role. 3.2 Acclimation When photosynthetic organisms are exposed to different light conditions with respect to those of growth, acclimation processes are activated, which consist into remodeling of the PSII composition. In case of acclimation to high light, e.g., we observe a strong decrease of LHCII trimeric complexes yielding a lower cross section for each PSII reaction center complex, while PSI complexes act as a pivot reference and do not change their composition [1]. An extreme example of such acclimation has been described in the green alga Dunaliella salina, where the chlorophyll antenna size of PSII has been reported to be as small as 60 chlorophyll molecules under high light conditions, while as large as 460 chlorophyll molecules under low light growth [2]. The light-harvesting antenna reduction seems to be a common strategy, across evolutionary distant organisms, adopted to optimize photosynthesis and avoid ROS production in stressing conditions. Besides the ratio between antenna and reaction center complexes, the stoichiometry of electron transport components with respect to light-harvesting components also changes to sustain an higher electron trans- port rate. Also, enzymes involved in metabolic sinks, such as the Calvin cycle, increase their relative abundance and activity [1]. The mechanism for stoichio- metric adaptation of PSII antenna size is posttranslational: accumulation of zeaxanthin induced by the excess light [3] binds to Lhc proteins into a spe- cific allosteric site called L2 [4], thus inducing a conformational change [5, 6], which leads to monomerization and degradation of LHCII but not of minor Lhcb components [7]. Increase in the stoichiometry of others electron trans- port components, such as cytochrome b6f complex, is likely to be controlled by transcriptional activation [8]. Although acclimation to constant light conditions has been studied to some extent, the resulting architecture of the photosynthetic apparatus can only reach a compromise condition reflecting the average conditions over a period of several days, as shown by the need of at least three days excess light period to elicit adaptative proteolysis of LHCII [9]. 32 G. Bonente et al. In fact, over a single day, light intensity and temperature change dramati- cally from dawn to midday and sunset, thus requiring regulation mechanisms operating over a short time-span (minutes, hours) and thus cannot be ac- tivated by the synthesis/degradation of photosynthetic components, whose everyday operation would be energetically unsustainable. These short-term processes will be described later. 3.3 State 1–State 2 Transitions State transitions are a protective mechanism, which acts through a reversible protein modification in a few minutes timescale [10,11] to balance the energy pressure between the two photosystems, physically displacing LHCII antenna complexes from PSII to PSI [12]. Higher plants and green algae oxygenic photosynthesis works through the synchronized action of two photosystems: PSII reaction center, whose maxi- mum absorption peak is at 680 nm, and PSI, where it is at 700 nm. The coordinated and energy collection by the two photosystems is a nec- essary condition for the proceeding of photosynthesis and the generation of a strongly reducing molecule, which is able to transfer its electron to NADP+. The kinetically limiting step in the electron transport chain between PSII and PSI involves the oxidation of liposoluble hydrogen carrier plastoquinone (PQH2), which once reduced at the Qb site PSII, diffuses in the thylakoid membrane to cytochrome b6f complex. In turn, cytochrome b6f is main- tained to an oxidized state by the activity of PSI, which transfers electrons to NADP+. Thus, over-excitation of PSII leads to over reduction of PQ, leaving PSII without a suitable electron acceptor. PSII undergoes charge recombi- nation and closure of energy traps thus increasing Chl lifetime and singlet oxygen production. To avoid this problem, the PQH2 acts as a signal activating chloroplastic protein kinase [13] to allow phosphorylation of LHCII antenna complex as- sociated to PSII (State I). Phosphorylated LHCII undergoes conformational change, disconnection from PSII, and migration to the stroma lamellae where it associates to PSI (State II). This increases light harvesting, and thus elec- tron transport capacity of PSI thus compensate for the uphill electron carriers over-reduction. Consequence is the oxidation of PQH2 to PQ and downregu- lation of the LHCII kinase. Dephosphorylation of LHCII and its reassociation to PSII reaction centers is ensured by chloroplastic phosphatases. This mecha- nism is photoprotective as it balances a potentially dangerous over-excitation on PSII, source of ROS, and it regulates light-harvesting activity depending on light intensity and light spectral quality changes consequent to rapid tran- sitions from shade to full sunlight. In fact, the absorption spectrum of PSI is red-shifted with respect to PSII. 3 Carotenoids Proteins in Photoprotection 33 In the green alga C. reinhardtii state transitions are the main photopro- tective strategy. This organism is able to shift from PSII to PSI up to 80% of its LHCII complexes, while in higher plants this parameter is not more than 15% [4]. 3.4 Carotenoids Play a Fundamental Role in Many Photoprotection Mechanisms The role of carotenoids in photoprotection of photosynthetic systems is ex- tremely important as shown by the early experiments of [15] with carotenoid- less bacterial strains and by the effect of herbicide norfluorazon on plants [16]. Carotenoids are 40 carbon atoms polyisoprenoid compounds, made by the condensation of eight isoprene units. In this class of compounds, we can dis- tinguish between carotenes, linear or cyclic hydrocarbons, and xanthophylls, their oxygenated derivatives. Carotenoids are located in the photosynthetic membranes, within the chloroplast, both free in the lipid bilayer (1–2%) and bound to the subunits of PSI and PSII in specific binding sites whose oc- cupancy is necessary for the folding of these pigment–protein complexes. In higher plants, β-carotene binds to reaction center subunits of both PSI (PsaA and PsaB) and PSII (PsbA,PsbB,PsbC,PsbD). Xanthophylls (lutein, violax- anthin, neoxanthin, and zeaxanthin) are, instead, bound to Lhca and Lhcb proteins forming the outer antenna complexes of PSI and PSII, respectively. In C. reinhardtii, the additional xanthophyll species loroxanthin is present, also bound to Lhc complexes. Location of carotenoid species in both PSI and PSII supramolecular complexes is shown in Fig. 3.2. Together with β-carotene, xanthophylls act both as photoreceptors, ab- sorbing light energy, which is used in photosynthetic electron transport, and as photoprotectants of the photosynthetic apparatus from excess light energy as well as from the reactive oxygen species that are generated during oxygenic photosynthesis. Carotenoids exert their photoprotective role through several mechanisms: (a) Chlorophyll triplet quenching: this action protects thylakoid lipids from peroxidation by quenching triplet chlorophyll species, which prevents pro- duction of reactive oxygen species (ROS) [17–19]; (b) ROS scavenging: in- complete quenching of 3Chl∗ may yield |
into ROS production, which can be scavenged by reaction with carotenoids; (c) Activation of heat dissipation of excess energy (NPQ): specific xanthophylls (lutein, zeaxanthin) are needed for operation of nonphotochemical quenching of excess light energy (NPQ), thus decreasing probability for IC and 3Chl∗ formation. All these mechanisms are cooperatively activated for protection of photosynthetic apparatus when environmental conditions promote photooxidative stress. It is worth nothing that all these mechanisms are rapidly activated: carotenoids detoxify short-living reactive species such as singlet oxygen (1O2), 34 G. Bonente et al. Antenna: xanthophylls Core complex: Antenna: β -carotene xanthophylls Core A complex: β -carotene B Fig. 3.2. Location of carotenoid species in both PSI (a) and PSII (b) supramolecular complexes. (a) PSI image by [58], based on PSI crystal structure by [59]; (b) PSII image by [60] whose lifetime is ∼200 ns, while the activation of heat dissipation of excess en- ergy (NPQ) requires less than a minute. In fact, carotenoids are involved in many of the mechanisms that allows survival of the photosynthetic cell when light intensity and photosynthetic electron transport rate undergo sudden changes, thus providing a more efficient response in conditions of repetitive excess light exposure, such as under sun flecks deriving from the overcast- ing of the leaf canopy; particularly, those mechanisms that are localized in the hydrophobic bilayer of the photosynthetic membrane, hosting reaction centers, and antenna proteins. Additional mechanisms located on the soluble phase of the chloroplast stroma are also of great importance for photoprotec- tion and are based on the activity of superoxide dismutase, ascorbate perox- ydase [20]. Their role has been the object of excellent reviews and will not be discussed here. The study of xanthophyll function in plants and algae has been carried on in the most recent years through an integrative approach, using genetics, phys- iology, biochemistry, and molecular biology [21]. First of all, it can be asked why plants do actually need xanthophylls at all. In fact all the main photopro- tective functions of xanthophylls (namely, triplet quenching and ROS scaveng- ing) are well-known properties of β-carotene [22]. Thus, the extreme conser- vation of carotenoid composition in plant species has little or no explanation on the basis of published chemical properties of these molecules. Nevertheless, the fact that all plant species and to a large extent green algae contain, be- 3 Carotenoids Proteins in Photoprotection 35 OH O O HO violaxanthin OH O O HO neoxanthin OH HO lutein OH HO zeaxanthin Fig. 3.3. The three xanthophylls, namely lutein, violaxanthin, and neoxanthin, contained by all plant species and to a large extent green algae in low light, and the xanthophyll zeaxanthin, synthesized in high light side β-carotene, the same three xanthophylls, namely lutein, violaxanthin, and neoxanthin (Fig. 3.3) in low light; and that violaxanthin is deepoxidized to zeaxanthin when light is in excess through the green lineage [23] is a clear biological demonstration that each of these xanthophylls have a specific func- tion. As mentioned earlier, xanthophylls in the thylakoid membrane are bound to Lhc proteins. Moreover, each Lhc protein has several (2–4) xanthophyll- binding sites. The affinity of each site for xanthophyll species is summarized in Fig. 3.4 for the major LHCII protein, which constitute by far the most abundant xanthophyll–protein binding complex in the chloroplast. Detailed biochemical and spectroscopic analysis has shown that the binding site L1 is occupied by lutein in all Lhc proteins, site L2 can bind lutein or violaxan- thin, site N1 is specific for neoxanthin, and site V1 bind violaxanthin [24–26]. Site L2 can exchange violaxanthin with zeaxanthin, produced in excess light, and undergoes a conformational change, which increases heat dissipation and decrease the lifetime of the 1Chl∗-excited states [5, 6, 27]. As mentioned ear- lier, xanthophylls have a light harvesting function whose importance can be evaluated by spectroscopic methods. Figure 3.5 shows the deconvolution of 36 G. Bonente et al. Fig. 3.4. Occupancy of the xanthophyll-binding sites in the major LHCII antenna complex. The affinity of each site for xanthophyll species is summarized in the figure. From: [45] 1-T spectra and of fluorescence excitation spectra of a typical antenna pro- tein, LHCII. This procedure [28] allows identification of xanthophyll spectral contributions, while the ratio between the amplitude of each component in 1-T vs. fluorescence excitation spectra yields the efficiency of energy transfer to Chl a. On these basis, it can be obtained that xanthophylls contribute to light harvesting by less than 10%, the balance being the effect of Chl a and Chl b absorption. This simple consideration suggests that the major function of xanthophylls is not light harvesting but photoprotection. 3.5 Analysis of Xanthophyll Function In Vivo Genetic has been used as the main strategy for the understanding of the spe- cific function of each carotenoid species in photosynthesis. Single and multiple mutants targeting gene products catalyzing enzymatic steps in the carotenoid biosynthesis pathway have been produced by several laboratories yielding col- lection of mutants with altered xanthophyll composition. Most used mutants have been npq1 (without zeaxanthin), npq2 (without violaxanthin and neox- anthin), lut2 (without lutein), and aba4 (without neoxanthin), which have been useful in elucidating the basic function of the different xanthophyll species. Ideally, the physiologist would like to work on: 3 Carotenoids Proteins in Photoprotection 37 1800000 A abs 1600000 a1 a2 1400000 b1 b2 1200000 neo viola 1000000 lute1 lute2 800000 fitting 600000 400000 200000 0 400 420 440 460 480 500 520 λ (nm) 1800000 B exc 1600000 a1 a2 1400000 b1 b2 1200000 neo viola 1000000 lute1 lute2 800000 fitting 600000 400000 200000 0 400 420 440 460 480 500 520 λ (nm) Fig. 3.5. Deconvolution of 1-T spectra (a) and of fluorescence excitation spectra (b) of a typical antenna protein, LHCII. The ratio of the amplitude of each spectral form in (b) vs. (a) yields the efficiency of excitation energy transfer from each pigment to Chl a, the final sensitizer of photosynthesis. The figure obtained is that light absorption by xanthophylls (vs. Chls, 350–750 nm) = 16.8%; contribution to Chl a fluorescence by xanthophylls (vs. Chls) in trimeric LHCII = 9.9% 1. Genotypes lacking one specific carotenoid 2. Genotypes retaining only one carotenoid species 3. Genotypes lacking one or more carotenoid-binding proteins Although not complete, a considerable collection is now available, whose analysis will gave us a detailed functional survey. Mutant collections were obtained both in higher plants (A. thaliana) and in the green algae model organism C. reinhardtii. Table 3.1 includes several genotypes available, with indications relative to laboratories that contributed to their isolation. These mutant plants were fundamental for the initial work. Major findings are sum- marized in the following paragraphs. fluorescence excitation 1-T 38 G. Bonente et al. Table 3.1. State of the art in genetics of carotenoids biosynthesis and function. Mutants affected in carotenoids or in carotenoids-binding proteins composition are listed 3.6 Nonphotochemical Quenching In strong light, the energy pressure on the photosynthetic apparatus dramat- ically increases thus causing increase of the fluorescence lifetime of Chl and oxidative stress (see earlier). In higher plants and some green algae, a mecha- nism is activated in these conditions for downregulation of chlorophyll-excited states concentration by opening a dissipation channel into heat of the excess energy absorbed over the rate that can be utilized by photochemistry, which is saturated in these conditions. Thus, quenching by reaction centers (photo- chemical quenching), when saturared, is integrated by an additional quenching effect originated outside reaction centers (NPQ, Nonphotochemical quench- ing) (Fig. 3.1). In doing so, the probability of chlorophyll intersystem crossing is decreased and the formation of reactive oxygen species down rated. NPQ can be measured through the decrease of the leaf fluorescence upon high light exposure. It was soon realized that NPQ involves several functional compo- nents: state transitions (see 3) account for a small reduction in leaf fluorescence 3 Carotenoids Proteins in Photoprotection 39 (called qT); the formation of quenching species within PSII core complex also contribute, together with similar effect within antenna system (qI), while the largest contribution is provided by a quenching mechanism located in the an- tenna proteins, which is activated by the transmembrane pH gradient and is, therefore, called “Energy quenching” or qE. 3.7 Feedback Deexcitation of Singlet-Excited Chlorophylls: qE qE is the most relevant NPQ component. It can quench up to 75% of 1Chl∗- excited states with an half time of 1–2 min. qE is triggered by trans-thylakoid ∆pH formation: absorption of excess photons causes the buildup of a high ∆pH, and the resulting decrease in lumen pH is essential for qE. Thus, qE acts as a feedback-regulated mechanism in photosyntheis, as it is modulated by the extent of trans-thylakoid ∆pH, generated by photosynthetic electron transport [29]. The need for stroma/lumen ∆pH and qE is clearly demonstrated by the inhibitory effect on qE of ionophore molecules as nigericin, which is able to abolish pH gradients. Moreover, DCCD (dicyclohexylcarbodiimide), a mole- cule able to covalently bind protonatable residues of proteins, is a powerful inhibitor of qE [30]. Recently, the target site for DCCD has been localized in the PsbS subunit [31], a PSII subunit whose deletion abolishes qE [32]. Xanthophylls have a key role in qE. The lut2 mutant, lacking lutein, shows both a reduced qE amplitude and a slower induction kinetic and an even stronger effect is observed in the npq1 mutant [3]. In this genotype, the en- zyme violaxanthin deepoxidase (VDE) is knocked out. VDE is usually acti- vated by lumen acidification, thus leading to deepoxidation of violaxanthin to zeaxanthin. This event of light-dependent, reversible deepoxidation of the violaxanthin pool is referred as “xanthophyll cycle” [33]. Lack of high light- induced Zea synthesis is thus in npq1 mutant and is thus the reason for the strong reduction in qE. This general figure is supported by the phenotype of the double mutant npq1lut2, where qE is completely abolished leading to photooxidative stress [34], and of the npq2 mutant, where zeaxanthin is con- stitutively accumulated, which exhibits qE similar to WT in amplitude but kinetically faster in onset and slower in recovery [3]. These evidences support a role for zeaxanthin as positive allosteric modulator of qE, expressed upon its reversible binding to Lhc proteins [35]. The mutation npq4 of A. thaliana, the gene encoding the PSII subunit PsbS, is epistatic over carotenoid biosynthesis mutations [32], suggesting the step it catalyzes is upstream with respect to carotenoid function in qE mecha- nism. Conversely, qE amplitude depends on the stoichiometric amount of PsbS polypeptide and on the specific protonation of two acidic lumenal residues, E122 and E226, the sites of DCCD inhibition. Inactivation of one of these sites (A. thaliana point mutants E122Q and E226Q) halves qE, while the 40 G. Bonente et al. double mutant E122Q-E226Q gives equals the deletion mutant npq4 [31, 36]. All together these results suggest that qE mechanism is triggered in excess light by the protonation of PsbS, acting as a sensor of lumen pH, while the quenching event itself is activated downstream and involves the carotenoids lutein and zeaxanthin. Information on the quenching mechanism itself have been obtained by femtosecond transient absorption in vivo [37] suggest that the quenching reaction consists into the transient formation of a Chlorophyll– Zeaxanthin radical cation, which then recombines with heat dissipation of the excitation energy. This radical cation is proposed to trap excitation en- ergy from PSII chlorophylls (Chl bulk) because of a high rate of formation (0,1–1 ps). Npq4 and npq1 mutants did not show any radical cation forma- tion. A tentative model can be proposed based on the recent finding that (a) PsbS it is not a pigment-binding protein, thus leading to the conclusion that quenching is not located in this subunit [38]; (b) by the detection of the radical cations in purified minor chlorophyll-proteins (CP24, CP26, CP29) [61]; and (c) by the finding that zeaxanthin binding induces conformational change and quenching in the very same antenna proteins [27, 39, 40]. Thus, PsbS would act as a pH sensor, activated by protonation of E122 and E226 residues, triggering a conformational change in neighbor PSII antenna subunits (CP24, CP26, CP29), whose ability to undergo formation of the radical cation quench- ing species is modulated by their capacity for exchanging violaxanthin with zeaxanthin [41,42]. 3.8 ∆pH - Independent Energy Thermal Dissipation (qI) When zeaxanthin is synthesized in high light and replaces violaxanthin in site |
L2 of Lhc antenna proteins, a second type of quenching is produced, which is not dependent on the formation of radical cations but rather on energy transfer from Chl a to the short living Zea S1-excited state [43]. This kind of quench- ing, although weaker, is not restricted to the minor proteins CP24, CP26 [27], CP29 [39]; but is also observed in the major LHCII complex [6,43]. A further difference with respect to qE consists in its lack of nigericine sensitivity, once that zeaxanthin synthesis has occurred and is thus constitutively active in npq2 mutant, even in the dark [27]. In WT plants, it is induced upon strong illumination that induces accumulation of zeaxanthin. Plants that have previ- ously accumulated zeaxanthin becomes less sensitive to strong light because of a molecular shift in their antenna proteins, which are set to their short life- time state through a zeaxanthin-induced protein conformational change that can be easily detected through isoelectric point shift and spectroscopy [27,44]. This mechanism, together with qE and qT, contributes to the quenching phenomenon on the whole called NPQ. Although qE is very fast (t1/2 min=1 min) and qT somehow slower (8 min), qI appears to have long relaxation time (20 min–1 h) related to the release of Zea from Lhc proteins. The interplay of these mechanisms ensure photoprotection under changes of light intensity with different time constants. 3 Carotenoids Proteins in Photoprotection 41 3.9 Chlorophyll Triplet Quenching The above-described mechanisms concur to avoid overexcitation of PSII. Nev- ertheless, by increasing light and/or decreasing temperature or metabolic sink activity, overexcitation eventually occurs. As described in section 1, IC (inter- system crossing) converts chlorophyll from its singlet-excited state into triplet, that, ultimately, can react with oxygen yielding ROS. However, carotenoids can largely prevent reaction with oxygen by quenching 3Chl∗. The transition energy level of carotenoid triplet state with nine or more conjugated double bonds is lower than that of chlorophyll. Carotenoid triplet states, thus, can directly receive energy from triplet chlorophyll (triplet chlorophyll quench- ing), thus quenching 3Chl∗. This reaction is located within Lhc proteins and prevents the formation of singlet oxygen. Indeed, xanthophylls bound to Lhc proteins are located in proximity to Chl for efficient quenching of 3Chl∗. As energy of carotenoid triplet state is too low to be transferred to other acceptor molecules, it is directly dissipated as heat: 3Chl∗ +1 Car →3 Car∗ +1 Chl 1) 3Car∗ → (3. 1 Car + heat In LHCII, the major PSII light-harvesting complex, chlorophyll triplet quench- ing is mainly catalyzed by lutein bound in site L1, through transfer of excita- tion energy from nearby chlorophylls [5]. Lutein appears to be the xanthophyll species most efficient in 3Chl∗ quenching as determined by direct measure- ments of the kinetics of carotenoid triplet formation upon excitation of Chl. Transient absorption spectroscopy of lutein vs. violaxanthin containing Lhcb1 has shown lower Car triplet yield and slower kinetics of Chl a to Car triplet transfer in the latter [45]. Consistently, increased photo-damage has been ob- served in vivo [45] because of formation of 1O∗ 2 in chloroplasts and purified complexes. Thus, changes in the xanthophyll occupancy of sites L1 and L2 of Lhcb proteins, particularly LHCII, affect photoprotection. In other Lhc proteins, such as Lhca4, also xanthophylls bound to site L2 are active in triplet quenching [18,19]. Lhca4 is a subunit of PSI characterized by red shifted spectra forms absorbing at >700 nm. Although the function of these spectroscopic features is the object of debate, analysis of recombinant Lhca4 WT vs. mutant missing red-forms showed that xanthophylls efficiency in triplet chlorophyll quenching was improved in the presence of red-forms. It is proposed that “red” chlorophylls, located near to xanthophyll in site L2 [46], act as a funnel for Chl triplet states to xanthophyll molecules bound to site L2, thus allowing 100% efficiency in triplet quenching [18]. 3.10 Scavenging of Reactive Oxygen Species Earlier, we have discussed the key role of carotenoids in mechanisms prevent- ing ROS formation. However, these mechanisms can be saturated and ROS formed in vitro and in vivo [47,48]. Carotenoids are well suited as antioxidants 42 G. Bonente et al. as they are active in scavenging reactive oxygen species generated in the chloroplast. Carotenoids protect from oxidative damage by two general mech- anisms: (1) quenching of singlet oxygen with dissipation of energy as heat; (2) scavenging of radical species thus preventing or terminate radical chain reactions. Carotenoids are hydrophobic antioxidants located in the thylakoid mem- branes. Xanthophylls bound to the Lhc proteins can catalyze 1O∗ 2 scaveng- ing, while β-carotene perform this function in the PS II core complex on 1O∗ 2produced from interaction of 3P680∗ and O2. After reaction with ROS, the carotenoid is excited to a triplet state (3Car∗), and then relaxes into its ground state (1Car) by loosing the extra energy as heat. 1O∗ 2 +1 Car →3 O2 +3 Car∗ 3Car∗ → (3.2) 1 Car + heat Carotenoids can act also as chain-breaking antioxidants in the peroxidation of membrane phospholipids [49] and, therefore, protect unsaturated-rich lipid membranes from rapid degradation. It has been reported that A. thaliana npq1 mutant, unable to synthesize zeaxanthin, has higher lipid peroxidation levels than WT; this phenotype is not related to the lower thermal energy dissipation ability (qE) in npq1 because of the absence of zeaxanthin [47]. Differently on mechanisms like qE or qI, where carotenoids active in the process are bound to Lhc proteins, an important role in ROS scavenging and detoxification is played by carotenoids, which are free to diffuse in the lipid bilayer. In particular, zeaxanthin has been proposed to favor membrane thermostability and protecting from lipid peroxidation [50]. Zeaxanthin has a major role in scavenging: the A. thaliana npq2lut2 double mutant has zeaxanthin as the only available xanthophyll, and is more resistant than WT to photooxidative stress and lipid peroxidation [7]. Nevertheless, zeaxanthin-enriched plants have a decreased growth [27] implying that lutein and neoxanthin play a role in the photoprotection against ROS, during normal growth in the absence of Zea. Lutein has a synergistic effect in photoprotection together with zeaxan- thin. A. thaliana lut2 mutant produces more singlet oxygen than WT under photooxidative conditions; nevertheless, stress symptoms are partially rescued by enhanced zeaxanthin accumulation. Indeed, plants lacking both zeaxanthin and lutein (npq1 lut2 double mutant) show much more photosensitivity and higher lipid peroxidation with respect to each single mutant [45]. The same effect can be observed in green algae: C. reinhardtii single mutants npq1 and lor1 are altered in photoprotective mechanisms both uphill (singlet and triplet chlorophyll quenching) and downhill (scavenging) ROS production, although they are still able to survive in high light. On the contrary, npq1 lor1 double mutant shows a lethal phenotype in high light, because of its lower ability to 3 Carotenoids Proteins in Photoprotection 43 detoxify singlet oxygen and superoxide anion, as it has been demonstrated by the exogenous addition of pro-oxidants chemicals [51]. A similar synergistic effect of zeaxanthin has been shown for neoxanthin. The A. thaliana aba4-1 mutant specifically lacks neoxanthin, and is only slightly more sensitive than WT; however, the double mutants aba4-1 npq1 is prone to photoxidative stress with respect to npq1, suggesting that Zea compensate for lack of Neo, whose specific function appears to be the scavenging of superoxide anion (O2) [52] mainly produced in the Mehler reaction. A role in chloroplast protection from ROS has been recently reported for the small amphiphilic lipid tocopherol. Early in vitro experiments have demonstrated that this compound can efficiently terminate chain reactions of polyunsaturated fatty acid free radicals and quench singlet oxygen [53–55]. It was found that zeaxanthin-supplemented human cells, in the presence of either α-tocopherol or ascorbic acid, were significantly more resistant to photoinduced oxidative stress. The authors postulated that the underlying mechanism responsible for the synergistic action is based on prevention of zeaxanthin depletion, because of its free radical degradation. α-tocopherol would be the final radical scavenger, thus preventing carotenoid consump- tion. This model may be applied also in plants; A. thaliana vte 1 mutant is impaired in tocopherol biosynthesis. When vte1 mutation is coupled with xanthophyll cycle mutation in double mutant npq1 vte1, a higher PSII pho- toinhibition with respect to that of single mutants is reported [56]. These data together with the observation that npq1 plants accumulate higher tocopherol level than WT [47, 57], while vte1 plants accumulate zeaxanthin led to the hypothesis that zeaxanthin and tocopherol have overlapping functions in protecting from photodamage by ROS. 3.11 Conclusions Oxygen, essential for animals, is produced by PSII in photosynthetic organ- isms. These organisms use chlorophylls as sensitizers for light-absorption water photolysis leading to O2 evolution. The chloroplast is thus a biological compartment where the highest O2 concentrations coexists with reducing species produced by light-driven electron transport thus leading to high probability of ROS formation and photodamage. Mechanisms have evolved for preventing formation of photooxidant and detoxify them once they are formed. Photosynthesis heavily depends on their efficiency. First, target of photoprotection mechanisms is maintenance of the balance between light ab- sorption and utilization in the ever-changing natural environment; second, the target is quenching of Chl triplets when balance is lost. Finally, when ROS are eventually formed, the last target consists in their scavenging. 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Diaspro 4.1 Introduction Non-linear interactions between light and matter have been extensively used for spectroscopic analysis of biological, natural and synthetic samples [1–5]. In the 1990s, the development in laser technology allowed to apply these principles to the light microscopy field [6–8]. In this context multi-photon exci- tation (MPE) fluorescence microscopy and second harmonic generation (SHG) imaging are representative of the continuing growth of interest in optical mi- croscopy. Although other modern imaging techniques like scanning near-field microscopy [9], scanning probe microscopy [10] or electron microscopy [11] pro- vide higher spatial resolution, light microscopy techniques have unique char- acteristics for the three-dimensional (3D) investigation of biological structures in hydrated states, including the direct observation of living samples [12–14]. Multi-photon microscopy relies on the property of fluorescent molecules to simultaneously absorb two or more photons [15]. In this context, the advances in fluorescence labelling and the development of new fluorescent/luminescent probes like the so-called quantum dots [16] and the visible fluorescent proteins (VFPs), that can be expressed permanently bound to proteins of interest by genetically modified cells, allow the study of the complex and delicate re- lationships existing between structure and function in the four-dimensions (x -y-z -t) biological systems domain [17, 18]. MPE shares with the confocal microscopy the intrinsical 3D exploration capability and provides some ad- ditional interesting features. First, MPE greatly reduces photo-interactions and permits to image living samples for long time periods. Second, it allows high sensitivity measurements due to the low background signal. Third, since most of the fluorescent molecules show a wide two-photon absorption spec- trum, MPE allows simultaneous excitation of multiple fluorescent molecules with only one excitation wavelength, reducing the effects of chromatic abber- ations of the optical path. Fourth, two-photon microscopy can penetrate into thick and turbid media up to a depth of some 100 µm. Fifth, MPE can induce chemical rearrangement and photochemical reactions within a sub-femtoliter volume in solutions, culture cells and living tissues. 48 D. Mazza et al. Another non-linear process that can be applied to the optical microscopy field is the generation of second harmonic signal. This phenomenon is related to the capability of non-centrosymmetrical matter to scatter light at the dou- ble of the illumination wavelength. Since highly organized biological matters such as myosin or collagen fiber are excellent SHG sources, SHG allows for 3D non-invasive imaging of biological matter in general. It is important to remember here that other kind of non-linear microscopy has been developed in the recent years. One example is Coherent anti-Stokes Raman (CARS) microscopy; since this signal is directly derived from molec- ular vibrations that are characteristic of the chemical composition and mole- cular structure of the sample, this third-order non-linear technique allows for the 3D analysis of both the chemical and the morphological information about the sample, without needing of external labelling. Furthermore, the development of non-linear microscopy favoured pro- gresses of several investigative techniques as fluorescence correlation spec- troscopy [19–22], image correlation spectroscopy [23,24], fluorescence lifetime imaging [25–28], single molecule detection schemes [29–32], photodynamic therapies [33] and two-photon photoactivation and photoswitching of VFPs [34–36]. Finally, an exciting scenario that has opened in recent years is the non- linear optical nanoscopy [37], related to the possibility of breaking the optical resolution limit by combining the light coming from different sources, so that interference patterns, as in 4Pi microscopy [38], or sequential excitation and depletion of fluorescent molecules, as in STED microscopy [38], allow to in- vestigate 3D samples at a nanometric level. 4.2 Chronological Notes on MPE The TPE story starts in 1931, with the theory originally developed by Maria Göppert Mayer in her Ph.D. Thesis [15]. The keystone of TPE theory lies in the prediction that one atom (or molecule) can simultaneously absorb two photons in the same quantum event, within a temporal window of 10−16– 10−15 s. This temporal scale reflects the rarity of a two photon event: in the daylight an efficient fluorescent molecule undergoes a two-photon absorption once every 10 million years, while one photon absorption occurs once a sec- ond [39]. Therefore, to increase the probability of the two-photon event, a high flux of photons is necessary, or in other words a laser source. As for confocal microscopy [40], in fact, the development of laser technology has been a key factor in the experimental evidence of two-photon events and in the spread- ing of the technique. The first observation of a two-photon signal in CaF2 dates back to 1961 [1], just one year later than the introduction of the first Ruby laser. Some years later, the third-order absorption (three-photon effect) was observed in naphthalene crystals [2]. In the same years second harmonic scattered signal was measured by illuminating quartz crystals with a ruby 4 Non-Linear Microscopy 49 laser beam [41]. For many years the application of these non-linear processes was limited to the spectroscopic studies of inorganic samples. The first ob- servation of TPE of organic dyes is dated to 1970 [42], while in 1976 Berns reported a two-photon effect as a result of focusing an intense laser beam onto chromosomes of living cells [43]. The application of these principles to the microscopy field required 20 more years. Even if the original idea of generating 3D microscopy images by means of non-linear effects was first suggested and attempted in the 1970s by Sheppard, Kompfner, Gannaway and Choudhury of the Oxford group [44,45], the practical realization of a ‘two-photon’ microscope is related to the pioneering work of W. Denk in W.W. Webb Laboratories (Cornell University, Ithaca, NY), which was responsible for spreading the technique that revolutionized fluorescence microscopy imaging [6]. 4.3 Principles of Confocal and Two-Photon Fluorescence Microscopy 4.3.1 Fluorescence The term fluorescence is related to the capability of certain molecules to emit light (in a time scale of 10−9 s) when they are illuminated with a proper wavelength. More precisely, the energy required to prime fluorescence is the energy that is necessary to produce a molecular transition to an electronic excited state [46,47]. In other words, if λ is the wavelength of the light delivered on the sample, the energy provided by photons E = hc/λ (where h = 6.6 × 10−34 J s is the Plank’s constant and c = 3 × 108 m s−1 is the speed of light) should be equal to the molecular energy gap ∆Eg between the ground state and one vibrational or rotational level of the electronic excited state: ∆Eg = E = hc/λ. (4.1) Once the molecule has adsorbed the photon, it has several pathways for relaxing back to the ground state, including non-radiative phenomena, phos- phorescence (associated to the forbidden transition to the triplet state) and fluorescence (Fig. 4.1). In fluorescence the internal conversion from the lower vibrational level of the excited state is not associated with light emission while the relaxation to the ground state is achieved by emitting one photon. For this reason, the fluorescence emission is generally shifted towards a longer wave- length than the one used for exciting the molecule. This phenomenon is known as Stokes’ shift and it ranges from 50 to 200 nm depending on the fluorescent molecule in consideration. Conventional imaging techniques use ultraviolet or visible light for the excitation. In multi-photon excitation the jump between the ground state and the excited one is due to the simultaneous absorption of two or more photons [5]. As the sum of the energy of the absorbed photons 50 D. Mazza et al. Fig. 4.1. Perrin–Jablonski diagram representing the possible pathways for one mole- cule to relax back to the ground state once excited must match the molecular energy gap, multi-photon processes allow using longer excitation wavelength. Multi-photon microscopy generally use infrared (IR) light for the excitation, resulting in the increase of penetration depth in turbid media and in the decrease of the photo-toxicity in biological samples. Because of the fact that the molecule is memoryless of the way in which excitation is accomplished, the two-photon induced fluorescence retains the same characteristics of conventional emission process. Therefore, in multi- photon excitation, the fluorescent emission is generally at shorter wavelengths than the one absorbed by the molecule. 4.3.2 Confocal Principles and Laser Scanning Microscopy In conventional wide-field microscopy a large portion of the sample is entirely illuminated with a light source and viewed directly by eye or through any image collection system (charged coupled device (CCD), for instance). With this method the sample undergoes continuous excitation and all the points of the sample, both in-focus and out-of-focus, will contribute to the image. As a consequence, the out-of-focus contribution will appear blurred in the image, resulting in the decrease of the axial resolution and in the hazing of the collected image. In this context, as reported by Minsky in 1961, the maximum performance of a microscopy imaging system should be met if it would be possible to investigate point by point the observed sample in order to collect at each position the light scattered or emitted by that point alone, rejecting all the contributions from the other parts of the sample, especially from those belonging to different focal planes [48]. Even if it is not possible to eliminate every undesired ray because of multiple scattering, it is straightforward to remove all the rays that do not focus on the point of interest, by using a 4 Non-Linear Microscopy 51 point-like source. This can be achieved by using the condenser lens to project a pinhole aperture (a small aperture in an opaque screen) on the |
focal plane or by using a laser source focused on the focal plane. In this way the amount of light delivered on the sample is reduced by order of magnitudes without affecting the focal brightness. However, even in this way, all the points along the focal axis will be able to scatter light (or to produce fluorescence signal) that will contribute to the image. The solution to this problem resides in placing a second pinhole aperture in the image plane that lies beyond the back aperture of the objective lens, so that all the light coming from out of focus scattering sources (or fluorescence sources) will be rejected. As shown in Fig. 4.2, the final result is a symmetrical set-up, made up of two lenses at the two sides of the specimen with two point-like apertures beyond them: in this way the role played by the lens on the excitation side is identical to the one on the detection side and their combined effect results in a relevant improvement of the axial response of the imaging system [49]. The strong symmetry of the system results also in the possibility (adopted by modern raster scanning confocal microscopes) of using the same lens both for the excitation and for the acquisition, in an epi-fluorescence scheme. Because of the point-like nature of the focused beam, in order to acquire microscopic images of an extended portion of the sample, it is necessary to perform a scan. This is usually obtained with one of these methods. The first one is based on raster scanning (point by point in a line and then line by line) of the image field. This operation requires a finite time in order condenser lens objective lens (L1) (L2) specimen detector light source excitation pinhole detection pinhole Fig. 4.2. Simplified view of a confocal microscope. A point-like source (a lamp with an excitation pinhole mask or a laser) is projected on the specimen by a condenser lens L1. All the fluorescent molecules illuminated by the cone of light will emit. This emission is brought to the detector by the objective lens L2. Right before the detector the collection pinhole allow to exclude all the light emitted by out of focus planes focal plane 52 D. Mazza et al. to collect a sufficient signal from each point of the sample. In the first confocal microscopes the scanning was performed by mechanically moving the sample under the investigating beam. However, a faster and more effective way of performing the raster scanning is related to the use of galvanometric mirrors, which are capable to direct the imaging ray on the different points of the sample. With this method the emitted light is usually detected through a photomultiplier tube (PMT) and is displayed by memorizing the detected intensity at each point of the sample. The resulting imaging rate is typically of about one image per second, but modern confocal heads with resonant scanning mirrors can scan images up to 10 times faster. A second possible approach to obtain confocal images consists in using a disk containing multiple sets of pinholes, namely a Nipkow spinning disk, placed in the image plane of the objective lens [50,51]. A large parallel beam is pointed at the disk and the light passing through the pinholes is focused by the objective. The light emitted by the sample crosses again the pinholes and it is brought to a high-efficiency and high time-resolution CCD. When spinning the disk at a high rate it is possible to produce excitation several times per second, reaching an imaging rate comparable to video rate. Despite the scanning architecture, both systems allow for optical section- ing: the sample is placed in a conjugate focal plane, and by moving the ob- jective along the optical axis it is possible to scan different fields of view at different depths in the sample and to collect a series of in-focus optical sections that allow for 3D reconstruction. The quality of the 3D reconstruction will be strictly dependent on the capability of the system to reject out-of-focus light and, therefore, to the pinhole aperture size: the use of smaller pinholes improves the discrimination of in-focus light and an improvement in axial resolution but will also result in a lower signal output. However, axial resolution and optical sectioning do not depend only on the pinhole size but also on the optical properties of the lenses (numerical aperture), on the excitation and emission wavelength and on the properties of the sample (its refractive index) as well as on the overall optical alignment of the instrument. 4.3.3 Point Spread Function of a Confocal Microscope A simple way for schematizing an optical microscope is a linear space in- variant (LSI) system. In this context it is necessary to determine the re- sponse of the system to an impulsive signal (in this specific case the image obtained when observing a point-like object). We will refer at this response as to the point spread function of the microscope (PSF). If the LSI approxi- mation holds, it is possible to idealize any observed object as a collection of point-like sources, and the image can be properly described by the convolu- tion product of the PSF with the object. Let us consider the schematic view of a confocal microscope that we depicted earlier: a monochromatic point-like source is focused onto a sample through a lens L1 and the emitted radiation 4 Non-Linear Microscopy 53 (also supposed to be monochromatic) is collected through the second lens L2 by a point detector. If hex and hem are, respectively, the impulse response of the lens L1 and L2, the radiation distribution delivered on the sample will be Uex = (hex ⊗ δs)(x) = hex, where the point-like excitation source has been modelled with a Dirac delta. The fluorescence emitted by each point x of the sample, Uem(x), will be proportional to the product of the field intensity delivered on the sample and on the distribution of the fluorescent dye D(x), Uem(x) = Uex(x)D(x). This radiation will be then brought by the second lens to the point-like detector, leading to the signal recorded by the detector, Udet(x) = [(hem ⊗ Uem)δd](x), where the detector has been modelled by a delta function. The overall collected signal will be therefore ∫ Itot = Udet(x) dx ∫ ∫ = δd(x) dx hem(x − y)Uem(y) dy ∫ ∫ = δd(x) dx hem(x − y)hex(y)D(y) dy ∫ ∫ = hex(y)D(y) dy δd(x)hem(x − y) dx ∫ = hex(y)hem(−y)D(y) dy. (4.2) If we consider the particular case of a point-like object, (4.2) provides the impulsive response of the system, i.e. the total PSF of the confocal microscope. By modelling the point-like object as a Dirac impulse δ0 (4.2) becomes ∫ Itot = hex(y)hem(−y)δ0(y) dy = hex(0)hem(0). (4.3) If we consider the confocal epi-fluorescence scheme L1 = L2, and if we assume that λex = λem,1 we end up with hex = hem = h. We can generally extend the previous formulas for an x-y-z scanning coupled to the imaging process. We therefore obtain for a general point P (x, y, z), Itot = h2(x, y, z), which is the general expression for the PSF. The mathematical expression for h(x, y, z) can be formulated through the electromagnetic waves scalar theory [52] and through Fraunhofer diffraction, leading to ∣∣ ∣ h(u, v) ∣ ∫ 1 ∣ J − iuρ2 ∣2∝ 0(vρ) e 2 ρdρ∣∣ , (4.4) 0 1 The equivalence of the excitation and the emission wavelength is an approxima- tion for fluorescence where generally λex ≤ λem due to Stokes’ shift. A more precise expression for the fluorescence ca√se is to con√sider a weighted mean of the excitation and emission wavelength λ̄ = 2λ 2 2 emλex/ λem + λex. 54 D. Mazza et al. where J0 is a Bessel function of the 0th order and u and v are dimensionless variables that depend on the lens parameters and wavelengths and are respec- tively proportional to the axial and the radial coordinates in the object space: u ∝ z, v ∝ (x2 + y2)1/2. By making use of the asymptotic expansions of the Bessel function we have therefore expressions for the points along the optical axis and in the focal plane: ( ) ( ) h(0, v) ∝ 2 2 2 J1(v) sin(u/4) h(u, 0) ∝ . (4.5) v u/4 The expressions above can be considered lateral and the axial PSF compo- nents of a conventional microscope, as only one lens is used. We can compare these relation with the confocal PSF: ( ) 2 4 ( ) Itot(0, v) = h(0, v)2 ∝ J1(v) sin(u/4) 4 Itot(u, 0) = h(u, 0)2 ∝ . v u/4 (4.6) In this context the calculation of the full width at half maximum (FWHM) of these expressions represents the system resolution. By limiting our attention to the axial resolution it can be shown that confocal imaging results in an improvement of a factor 1.4 [53,54]. In particular, the expression of the axial resolution for a pinhole aperture diameter smaller than 1 AU2 results rz = √0.64λ (4.7) n − n2 − NA2 where n is the refractive index of the medium and NA is the numerical aperture of the objective. Despite the theoretical formalism for evaluating the ideal PSF of a confocal system shows the improvement in lateral and axial resolution, some considera- tion must be given about the real response of a system when imaging non-ideal samples. First it must be considered that the pinhole aperture is strictly re- lated to the improvement in resolution: by opening the pinhole, the detector cannot be considered anymore as punctual and the resolution gets worse. Therefore, in the observation of dim samples some compromise between the resolution and the intensity of the collected signal must be found. Table 4.1 reports the dependence of axial and lateral resolution on the pinhole size. The experimental results have been obtained by imaging sub-resolved fluorescent beads (Polyscience, diameter (64 ± 9 µm)) with a × 100, 1.3 NA objective under 488 nm excitation. Furthermore, the real PSF will depend on the physical and optical charac- teristics of the observed sample. In particular, the refractive index mismatch 2 AU is the Airy-Unit, the diameter of the Airy disk. 4 Non-Linear Microscopy 55 Table 4.1. FWHM of confocal PSFs for different pinhole sizes Oil (n = 1.5) Lateral (nm) Axial (nm) Pinhole 20 µm Pinhole 50 µm Pinhole 20 µm Pinhole 50 µm Experimental 186 ± 6 215 ± 5 489 ± 6 596 ± 4 Theoretical 180 210 480 560 Table 4.2. FWHM of confocal PSFs for different focusing depths and objective media Air Glycerol Oil Depth Lateral Axial Lateral Axial Lateral Axial (µm) (nm) (nm) (nm) (nm) (nm) (nm) 0 187 ± 8 484 ± 24 183 ± 14 495 ± 29 186 ± 6 489 ± 6 30 244 ± 10 623 ± 9 221 ± 5 545 ± 12 197 ± 10 497 ± 21 60 269 ± 11 798 ± 10 252 ± 7 628 ± 9 186 ± 12 196 ± 19 90 277 ± 5 1063 ± 24 268 ± 8 797 ± 26 191 ± 9 484 ± 12 between the objective immersion medium and the sample solution can play a crucial role as some spherical aberration effects can arise, leading to a loss of axial resolution and deforming the actual shape of the PSF [55]. In par- ticular, the effects of spherical abberation get more important when focusing deep in the sample. Table 4.2 reports the broadening of the lateral and axial resolution when imaging planes at different depths of focus. 4.4 Two-Photon Excitation Let us consider the special case of two-photon excitation. All the consid- erations made here can be easily extended to the more general case of multi-photon excitation. In TPE, two photons are absorbed by a fluorescent molecule in the same quantum event. The interaction may occur only if the global energy delivered by the photons is identical to the energy gap of the molecule between the ground state and some vibrational level of the excited electronic state, or in other words if the sum of the energy of the two photons (that do not have to be necessarily identical) is equal to the energy required for |
prime excitation with a conventional one-photon absorption (ref. Fig. 4.3). This means in terms of the wavelength of the two photons that prime the non-linear excitation, λ1 and λ2: ( ) 1 1 −1 λ1P = + , (4.8) λ1 λ2 56 D. Mazza et al. Fig. 4.3. Perrin–Jablonsky simplified diagram representing the differences between confocal and two-photon excitation microscopy. Fluorescence emission does not de- pend on the way in which excitation is performed where λ1P is the wavelength required to induce one-photon excitation. For practical purposes the wavelength of the two photons is chosen identical, so that 2hc λ2P = λ1 = λ2 = 2λ1P and ∆Eg = . (4.9) λ2P Considering the two-photon process as non-resonant, we can assume the existence of a virtual intermediate state; the electron will reside in the virtual state for a time, τvirt, that can be calculated using the time–energy uncertainty principle: ∆Egτvirt h̄/2 → τvirt 10−15 − 10−16 s. (4.10) The two photons will have to be delayed no more than τvirt to induce the non-linear interaction. It is clear that for a high flux of photons it is therefore required to have the two-photon interaction. In TPE the photons spatial and temporal concentration flux will have a crucial role. The shorter wavelength required for TPE allows using near IR to excite UV and visible electronic transitions. The rigorous development of multi-photon theory re- quires to apply perturbation quantum theory [56]. In particular, by solving the perturbation expansion of time-dependent Shrödinger equation and by us- ing a Hamiltonian containing the dipole interaction of the molecule with the incoming radiation, it is possible to show that the first-order solution corre- sponds to one-photon interaction, while higher order solution are the n-photon ones [57]. Such a derivation allows to show that TPE depends on the square of the intensity delivered on the molecule; however, the same result can be obtained with classical or semi-quantic considerations [58]. 4 Non-Linear Microscopy 57 Therefore, the fluorescence intensity emitted by one molecule can be con- sidered proportional to the two-photon molecular cross section δ2(λ) and to the square of the intensity delivered on the sample: [ ] If(t) ∝ δ2I(t)2 ∝ δ2P (t)2 (NA)2 2 π , (4.11) hcλ where P (t) is the laser power, and the intensity of the incoming radiation is calculated by using the paraxial approximation in an ideal optical system. Two-photon excitation is generally induced with ultra-fast pulsed lasers. The time averaged intensity emitted by one molecule over a time T will be therefore ∫ [ 〈 1 T ] (NA)2 2 ∫ 1 T If(t)〉 = If(t) dt ∝ δ2 π P (t)2 dt. (4.12) T 0 hcλ T 0 We can now consider fp = 1/T as the repetition rate of the pulsed laser and τp as the pulse width. If we consider that the emission of the laser is described by the profile P (t) = Pmax for 0 < t < τp P (t) = 0 for τp < t < T, (4.13) ∫ then the mean square power delivered on the sample P 2 ave = 1 T /T P (t)2 dt 0 becomes P 2 ave = P 2 maxτpfp and (4.12) becomes [ ] 〈 P 2 I ave (NA)2 2 f(t)〉 ∝ δ2 π . (4.14) τpfp hcλ Writing the same relationship for a continuous wave (CW) laser, we obtain [ ] (NA)2 2 〈If(t)〉 ∝ δ2P 2 ave π . (4.15) hcλ Comparing (4.13) and (4.15) it is evident that the excitation of molecules with a pulsed laser is more efficient: in order to obtain the same fluore√scence emission with a CW laser, it is necessary to use an average power 1/ τpfp higher relatively to the pulsed laser case. From (4.13) it is possible to easily evaluate the probability na for a fluo- rophore to absorb two photons simultaneously during a single pulse: [ ] P 2 n ave (NA)2 2 a ∝ δ2 π . (4.16) τpf2 p hcλ The selection of the repetition rate and of the width of the beam pulse is related to the necessity of avoiding saturation of the fluorescence when na approaches unity. In other words, during the beam pulse the molecule must 58 D. Mazza et al. have enough time to relax back to the ground state as this is a prerequi- site for the absorption of another pair of photons. If not, saturation effects arise, leading to a worsening of the axial and radial resolution of the sys- tem [59]. The application of (4.16) allows to choose for proper optical and laser parameters that maximize excitation out of the saturation regime. In particular, the typical values for the ultra-fast lasers used in two-photon mi- croscopy have τp = 80−150 fs and fp = 80−100MHz. The last considerations must be given about the two-photon cross section δ2. Because of the fact that the quantum-mechanical selection rules for two-photon excitation differ from their one-photon counterpart, it is not easy to extend the data for conven- tional absorption to the non-linear case even if a simple “rule of thumb” can be considered. In fact, in general we can expect to have a two-photon excitation peak at a wavelength that is the double of the one-photon excitation maxi- mum. However, the knowledge of the global trend of two-photon cross-section for a particular molecule requires the direct measurement. Figure 4.4 shows the TPE cross section for some of the most common fluorescent molecules. Because of the wide nature of the excitation spectra it can be noted that one single wavelength can be used for the simultaneous excitation of multiple dyes [60, 61]. It has been showed that endogenous fluorescent molecules like flavoproteins, NAD(P)H and tryptophane exhibit TPE fluorescence [61, 62] and also the fluorescent proteins like GFP (green fluorescent protein) are capable of undergoing TPE [63–65]. The two-photon cross-sections are gen- erally expressed in GM (Goppert-Mayer, 1GM = 10−58m4 s). GFP variants 103 Rhodamine B 102 Bodipy FI 10 Bis-MSB Coumarin Dil 100 Dansyl Fluorencein 10−1 DAPI Lucifer Yellow 10−2 Pyrene Cascade Blue 10−3 600 700 800 900 1000 1100 Wavelength (nm) TI: Sapphire SHG of Cr: YAG SHG of Cr: Forsterite Cr: LiSAF Cr: LiSGAF Nd: YLF or Nd:glass Fig. 4.4. Exemplary two-photon cross-sections for some typical fluorophores for biological imaging. The bars represent typical emissions of commonly used laser sources for TPE Two photon Excitation Cross Sections (GM) 4 Non-Linear Microscopy 59 have cross sections between 4 and 60 GM [66]. As comparison we can con- sider NADH that at its excitation maximum, 700 nm, shows a cross section of 0.02 GM [61]. Quantum dots can show cross sections up to 2,000 GM. 4.5 Two-Photon Optical Sectioning In the following we will describe how two-photon (or multi-photon) excitation allows to spatially control excitation along the optical axis, limiting the ex- citation volume to a sub-femtoliter volume, thus allowing optical sectioning. We discussed above that the fluorescence signal emitted under two-photon excitation will depend on the second power of the excitation intensity. We can therefore consider that the intensity delivered point by point on the sample, Iex, is proportional to the lens PSF, which in the paraxial approximation is described by (4.4). Therefore, we can expect that the fluorescent signal emit- ted by the sample Iem(x, y, z) will be proportional to I2 ex and for the axial and radial components of Iem – introducing the dimensionless variables u, v – we get [67] ( ) 2 4 ( ) Iem(0, v)=h(0, v)2 ∝ J1(v) sin( 4 u/4) Iem(u, 0)=h(u, 0)2 ∝ . v u/4 (4.17) These expressions are identical to the axial and radial parts of the total PSF of a confocal system (ref. (4.6)). However, it must be noted that while confocal microscopy achieves this result at the detection side, by means of the pinhole, in two-photon excitation the dependence of the fluorescence intensity on the inverse of the 4th power of the axial coordinate is achieved directly in the excitation step. This means that while in confocal microscopy a thick volume of the sample is excited and the selection of the in-focus contribution is obtained through the pinhole at the detection level, in two-photon excitation, only the molecules in a small volume (order of the femtoliter) around the focal point are properly excited and emit fluorescence [68,69]. This fact also implies that the contributes far-off the focal plane (depending on the NA of the lens) will not be affected by photobleaching [70,71] or phototoxicity [72–74] and do not contribute to the signal detected if a TPE architecture is used. In this way we have no need for a pinhole as all the fluorescent emission is supposed to be originated at the focal plane. This means that TPE is intrinsically three-dimensional as it allows for optical sectioning by collecting images of the sample plane-by-plane and then reconstructing the three-dimensional map of the emitted fluorescence (ref. Fig. 4.5). It must be underlined that since all the fluorescence collected is nec- essarily originated at the focal point, the efficiency of the collection of the signal is much higher than in the confocal case. In TPE, in fact, over 80% of the signal arise from a 700–1,000 nm (depending on the NA of the objective) thick region around the focal plane [54, 75]. This results in a reduction in 60 D. Mazza et al. Fig. 4.5. Exemplary optical sectioning obtained with two-photon excitation at 720 nm. The sample is Colpoda cists, and the signal is produced by autofluores- cence of the membranes background and an increase in the image contrast. This compensates the de- crease in resolution due to the longer wavelength compared to the one used in conventional excitation (4.7). However, the use of infrared wavelength in- stead of UV–visible allows deeper penetration as the long wavelength used in TPE (and in general in MPE) will be scattered less than the UV–visible light [55, 76]. Because of the high intensity delivered in the focal point (com- pared to conventional excitation), it must be noted that the laser power must be finely controlled in order to prevent photo-damage effects [73]. On the other side, the confinement of the excitation processes allows to photochem- ically modify the sample properties with a high 3D spatial control, opening new frontiers in the context on micro- and nano-surgery [77, 78]. Finally, the localization of the photobleaching effect allows to acquire 3D maps of the sample for longer times than in conventional and in confocal architecture, as when observing a specific slice of the sample, the out-of-focus contributes will not be affected by photobleaching [70]. 4.6 Two-Photon Optical Setup The basic setup for multi-photon imaging includes the following elements: a high-intensity ultra-fast infrared laser source (femto- or picosecond pulse width with a 80–100 MHz repetition rate); a laser beam scanning system 4 Non-Linear Microscopy 61 (i.e. a pair of galvanometric mirrors); high numerical aperture objectives (NA 1.0–1.4) in order to deliver high peak intensity in the focal point; and a highly efficient detection system. Laser source plays an essential role in the production of MPE signal. Be- cause of relatively low cross-section of the non-linear processes, high photon flux is required, >1024 photons cm2 s−1 at the focal plane [79]: considering the spectral range of 600–1,100 nm this means a peak intensity in the MW cm−2– GW cm−2. By using an ultra-fast pulsed laser in combination with a high numerical aperture objective, this results in a mean laser power of 50 mW or less. This allows for a sufficient peak intensity to induce TPE, with a mean power levels that are biologically tolerable [72,80]. The most common ultra-fast pulsed lasers are Ti:sapphire femtosecond laser sources [40], which can be tuned in wavelength between 700 and 1,050 nm allowing most of the common fluorescent molecules to be excited in TPE regime. Typical parameters for Ti:sapphire lasers include an average power of 700 mW–1 W, pulse width of 100–150 fs and repetition rate of 80–100 MHz. Other laser sources for MPE include Cr-LiSAF, pulse-compressed Nd-YLF in the femtosecond regime, and mode-locked Nd-YAG. Picosecond pulse width can also be reached by using a pulse stretcher. It must be noted that due to time–energy uncertainty principle, ∆E∆T ≥ h̄/2, a shorter pulse width will return |
in a broader emission in terms of wavelengths: by considering ∆T = τp, ∆λ/λ = ∆E/E and E = hc/λ, we obtain λ2 ∆λ = , (4.18) 2πτpc which for a central laser emission wavelength of 1,000 nm and pulse width τp = 100 fs gives an uncertainty on the wavelength of about 5 nm. Furthermore, it must be considered that, when the laser light crosses the sample, a temporal broadening of the pulse occurs. Direct measurement of the pulse width at the focal plane is not an easy task [81–83]. For practical purposes, it can be assumed that at the focal volume 1.5–2 times broadening is obtained. Some notes also must be given to the scanning system. Generally, a x-y raster scan is performed by means of galvanometric mirrors [84]. Therefore, the image acquisition rate is generally limited by the mechanical properties of the scanner. Fast scanning methods based on resonant scanners can be performed as well; however, it must be considered that fast scanning and sub- sequent high temporal resolution may require some compromises about spatial resolution and sensitivity, since by scanning faster the laser will illuminate for shorter times each point of the sample, resulting in a lower collected signal. Axial scanning can be performed by a variety of solutions, the most common being a single objective piezo nanopositioner or a galvanometric object table. Two-photon architecture usually allows to switch between confocal and TPE imaging retaining the x-y-z focal position [85]. Furthermore, the use of electro- optical (like electro-optic modulators, EOMs) or acusto-optical devices allow 62 D. Mazza et al. Excitation light Scanning system Detector DM Pinhole X-Y Scanner Non-descanned detector Detector DM Objective lens Focal Plane Scattering specimen Fig. 4.6. Laser sources in use at LAMBS Microscobio research center of the University of Genoa for TPE to rapidly modulate the laser intensity: in this way it is possible to define regions to be imaged with different powers during the very same scan. As depicted in Fig. 4.6 it is possible to consider two popular approaches to TPE microscopy, the descanned and non-descanned modes. The first con- figuration uses the same optical pathway as used in confocal microscopy, and the light emitted by the sample is deflected back by the scanning mirrors and brought to the photomultipliers. The pinhole is usually kept open – or removed – as it is not necessary for TPE optical sectioning. In the non-descanned mode the microscope architecture is optimized for collecting the maximum TPE signal: pinholes are removed, the emitted light is collected through a dichroic mirror and it does not cross the scanning system. [61,76,86] Despite the configuration used, the fluorescent light is brought by the detection system by a dichroic mirror. Several types of detectors can be used, Fluorescence 4 Non-Linear Microscopy 63 Fig. 4.7. Optical scheme of two-photon microscope in descanned and non-descanned configuration (Courtesy of M.Cannel and C. Soeller) including PMTs, CCD cameras and avalanche photodiodes. PMTs are the most commonly used for their low cost, good sensitivity in the visible range (the quantum efficiency is about 20%–40% in the blue green range, dropping down to 1% in the red) and for the large photosensitive area that allows a good dynamic range; thus an efficient collection of the signal (Fig. 4.7). Avalanche photodiodes are extremely efficient in terms of sensitivity (quantum efficiency of about 70%–80%). Unfortunately, they are pretty expensive and their small photosensitive area requires the descanned architecture, limiting the global signal collection efficiency. CCD cameras are used for fast imaging, and are particulary useful in the context of tandem or multi-focal imaging. Finally, regarding the collection of the signal, it must be noted that due to the fact that the fluorescent signal is generated only in the focal point, we could think of an ideal TPE microscope with a detector that is able to collect all the light coming from the sample, as the fluorescence signal can be easily recognized by the scattered excitation light by means of the wavelength – i.e. with a low-pass filter. 4.7 Second Harmonic Generation (SHG) Imaging In conventional microscopy, contrast methods consist of phenomena like ab- sorption, reflection, scattering and fluorescence: in this conditions the speci- men response is linearly dependent with the incoming light. This means that a linear relationship exists between the electric field strength Ẽ of the light and the induced polarization of the object P̃ . In particular, if we consider an incoming oscillating field with a non-resonant frequency (i.e. a frequency that is not absorbed by the molecules of the material), the optical response can be approximated to be a first-order response: P̃ (t) = ε0χ (1)Ẽ(t), (4.19) 64 D. Mazza et al. where ε0 is the dielectric constant of the vacuum and χ(1) is the linear susceptibility of the specimen. Moving to the non-linear domain, high power intensity cause a large variety of unusual responses, with a non-linear depen- dency on the applied electric field. Second harmonic generation is one of these nonlinear optical effects in which the incident light is coherently scattered by the specimen at twice the optical frequency and at certain angles [87]. We can generally write the non-linear correspondent of (4.19) as ( ) P̃ (t) = ε χ(1)Ẽ(t) + χ(2)Ẽ2 + χ(3)Ẽ3 0 . , (4.20) where the first left term on the right is the linear scattering, the second is related to SHG, the third to third harmonics, etc. In the above equation, because the fields are vectors, the nonlinear susceptibilities are tensors. As each atom acts as an oscillating dipole that radiates in a dipole radiation pattern, the radiation phase among the enormous number of atoms must be matched to induce constructive interference and thus non-linear generation is allowed under phase-matching conditions (i.e. when the scattered light is in phase). For the same reason, only molecules that exhibit a specific symmetry. In particular, only non-centrosimmetric materials can originate SHG. The SHG signal depends strictly on the relative orientation between the polar- ization of the incoming light and the direction of the symmetry constraints; a polarization analysis of the second harmonic signal can provide useful in- formation about the orientation of molecules, impurities in crystal structures and characteristics of surfaces and optical interfaces. As for linear scattering, second harmonic generation is not associated with absorption and involves only virtual state transitions that are related to the imaginary part of the nonlinear susceptibilities, and so no energy is deposited in the specimen and no damage can be produced. This is one of the major advantages of applying SHG to the microscopy field and in particular to the investigation of biological samples [88, 89]. Furthermore, SHG imaging preserve the intrinsical capabil- ity of 3D investigation of matter, since the high photon flux required for generating this non-linear signal is achieved only within a femtoliter volume around the focal point of the lens. Since both SHG and TPE can be observed simultaneously from the same sample, the correlative analysis of these two signals provide additional insight about the specimen, allowing not only to identify the molecular source of the SHG, but also to probe radial and lateral symmetry within structures of interest. Recognition of the SHG relies on the property that the emitted light has double wavelength of the incoming radiation. Therefore, by changing the color of the illumination laser we expect to observe an analogue shift in the emis- sion wavelength. The emission of SHG signal is not isotropic, as it will be more efficient in the forward direction. However, in dense samples, multiple scattering allow for a detection in the backward direction too. Therefore, SHG can be usually collected both along the transmitted light and along the epi- pathways of the microscope. 4 Non-Linear Microscopy 65 A number of plant structures are capable of efficiently generating second harmonic signals. These structures include stacked membrane structures such as grana, starch granules, secondary cell wall, cellulose, cuticle and cuticular waxes, and silica deposits (bio-opals). Starch granules exhibit high conver- sion efficiency in SHG; in fact, a piece of potato tuber placed in an unfocused, ultra-fast laser beam can efficiently generate a bright SHG beam in the forward direction. For example, the SHG signal from a potato (Solanum tuberosum L.) starch granule is so strong that it is visible to the naked eye even under ambient room light. Cellulose is a linear molecule without branching. Neigh- boring cellulose chains may form hydrogen bonds leading to the formation of micro-fibrils (20–30 nm) with partially crystalline parts called micelles. All the highly organized structure may be responsible for strong SHG signal such as collagen or myosin fibers. SHG allows therefore for three-dimensional imaging and microscopical investigation of tissue organization with typical confocal resolution, without the need for any external labeller [90–92]. The possibility of selecting the wavelength in the IR range permits to perform practically non-invasive in vivo deep tissue imaging, being a promising tool for skin en- doscopy. 4.8 Conclusions The investigation of soft and living matter requires instruments capable to investigate the relationships between the 3D structure of the specimen and its functions and activity. Optical microscopy and especially the advances brought by confocal and non-linear microscopy in the last decades offers a powerful tool for this aim. Confocal microscopy allows to generate 4D (x-y-z-t) views of the specimen in a non-invasive way, preserving the vital condition of the sample, with a strong reduction of the out-of-focus haze, allows the multi- ple observation of different dyes by spectral fingerprinting and recent scanning heads provide a good tool for visualizing at video rate fast in vivo dynam- ics. The advances in confocal microscopy, together with the development of new fluorescent labellers and the spreading of visible fluorescent proteins have favoured the development and the spreading of two-photon and multi-photon imaging. Compared to confocal microscopy, we can mainly recognize two im- portant properties by MPE. 1. The MPE excitation is confined both in the x-y directions and along the optical axis. Therefore, no fluorescence signal arises from elements out of the focal point. Consequently, optical sectioning can be performed without the use of a pinhole or deconvolution algorithms. Furthermore, photo-bleaching and photo-damage are also confined to the focal point, and the signal-to-noise ratio increases as well as the image contrast. The development of proprietary schemes, like the non-descanning acquisition, further improves the efficiency of the signal collection. 66 D. Mazza et al. 2. The use of near-infrared wavelengths allows to minimize biological damage as biological tissues in general poorly absorb infrared light. This combined with the in-focus photobleaching allows for long-term observation of liv- ing samples. Because of the wide two-photon cross section of most of the fluorescent molecules, it is possible to excite different dyes at once with only one laser line, reducing the average power delivered on the sample. Furthermore, the use of IR light permits deeper penetration into the sam- ple (up to 0.5 mm) as both absorption and scattering are reduced with this wavelength. Finally, because of the fact that the separation between excitation and emission (excitation in the IR, emission in the UV–visible) is usually wider than in conventional excitation, it is considerably eas- ier do discriminate between the actual emission and scattered/reflected excitation light. The advances of TPE is strictly connected to the developments in an- other non-linear microscopy technique, second harmonic generation imaging. It offers a practically non-invasive tool for deep tissue imaging as it does not require fluorescent labelling of the samples and it retain the 3D investigation and optical sectioning properties of multi-photon excitation. Also in this case all the advantages using IR light for the illumination are conserved. Since relevant generation of second harmonic signal is associated with the presence of tissue constituents as collagen, SHG may play an important role in skin disease diagnosis and in the medical research field in general, being a promising endoscopy tool. The combination of MPE microscopy and SHG imaging provides both a deep insight into biological matter and offers possibilities in the nano- manipulation of living cells and tissues and in the 3D micro- and nano-surgery field. The range of application of these techniques is rapidly increasing in the biomedical, biotechnological and biophysics sciences and it is now facing the clinical applications. References 1. W. Kaiser, C.G.B. Garrett, Phys. Rev. Lett. 7, 229 (1961) |
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It is a powerful method that allows noninvasive monitoring of specifically labeled targets within living cells, and simultaneous detection of multiple tar- gets using different labels. The spatial resolution in fluorescence microscopy is limited because of the diffraction limit; the resolution in transverse direction is proportional to λ/2NA = λ/2nsinθ (where n is the refractive index in the object space, and θ is the half-angle of the largest cone of rays that can enter or leave the optical system), whereas the longitudinal resolution is given by 2λn/NA2. High-spatial resolution to detect fluorescent molecules below the diffraction limit can be achieved in several ways, such as by increasing the effective numerical aperture (as in 4Pi confocal microscopy) [1], introducing spatial variation in the excitation light creating finer spatial features in the image (as in standing wave microscopy) [2], using multiple-photon fluores- cence absorption or emission mechanisms that lead to nonlinear effects in the light field (as in 2-photon microscopy) [3], and by selectively quenching the fluorescence from a focal spot to obtain a very small fluorescing volume (as in stimulated emission depletion microscopy) [4]. 5.2 Self-Interference Imaging Fluorophores, when immobilized on surfaces, are often quenched because of mechanisms of energy transfer, standing wave nodes in the excitation field, and destructive interference in the emission. Fromherz and coworkers noted that the intensity of the total fluorescence oscillates as a function of the fluo- rophore height above a reflecting substrate [5]. Their technique, fluorescence interference contrast microscopy (FLIC), is based on measuring the intensity of fluorophores located within ∼λ of vertical distance from a reflecting sur- face. On the one hand, this proximity to the surface causes the entire emission 72 M.S. Ünlü et al. Fig. 5.1. The interferometric experimental configurations. (Left): WL reflection spectroscopy is based on spectral variations of reflection from thin transparent films. Interference of light reflected from the top surface and a buried reference surface results in periodic oscillations. (Right): The SSFM technique maps the spectral oscillations emitted by a fluorophore located on a layered reflecting surface into a precise position determination. spectrum to oscillate as light undergoes constructive and destructive interfer- ences. The fluorophore height can be determined from these oscillation curves; however, careful calibration of fluorescence intensity as a function of distance from the surface is required. On the other hand, for larger separations between the fluorophore and the reflecting surface (on the order of 10–20λ), light can go between constructive and destructive interferences multiple times even at the same height. This creates interference fringes in the emission spectrum, and spectral self-interference fluorescence microscopy (SSFM) [6–8] utilizes this principle to reveal height information. Using a combination of a traditional reflection technique and an interfer- ometric fluorescence spectroscopy technique, the average optical thickness of biological layers and the height of fluorescent markers can be measured with subnanometer accuracy. Figure 5.1 summarizes the two techniques schemat- ically. The first technique, white light (WL) reflection spectroscopy, is based on spectral variations of reflection from thin transparent films. Interference of light reflected from the top surface and a buried reference surface results in periodic oscillations in the reflection spectrum. The principle is similar to the interference-based detection technique using color variations due to increased path length as a consequence of surface binding on optically coated silicon [9]. The spectral measurements result in very high accuracy (∼0.2 nm) for measur- ing average optical density, comparable to ellipsometry, and provide a precise relative measure of the additional “optical” mass on the substrate. The second 5 Spectral Self-Interference Microscopy 73 Fig. 5.2. Emission spectra of fluorescein immobilized on a glass slide and on top of a Si-SiO2 chip with two different thicknesses of the oxide layer. technique, SSFM, analyzes the spectral oscillations due to self-interference of direct and reflected emission by a fluorophore located on a layered reflect- ing surface and yields a precise position determination (Fig. 5.1). Figure 5.2 illustrates the effect of self-interference on the emission spectrum of fluores- cein immobilized at different heights on a silicon–silicon oxide wafer. This is compared with the smooth emission envelope of fluorescein immobilized on a glass slide where there is no self-interference. As shown, small height differences (10 nm in the figure) shift the fringes and change the period of oscillation, although the latter is less apparent. It should be noted that |
in contrast to fluorescence interference-contrast mi- croscopy [5], the axial position of the fluorophores in SSFM is determined from the spectral oscillations and not from the total intensity; therefore, variations in fluorophore density, emission intensity, and the excitation field strength do not affect the result. 5.3 Physical Model of SSFM 5.3.1 Classical Dipole Emission Model An emitting fluorophore can be represented as a radiating dipole [7]. The emitter transition dipole, µ, the wave vector, k, and the electric field vector, E, lie in the same plane, which is the plane of polarization of the radiated light. In the far field, if the environmental factors remain constant, the amplitude of the electric field of a fluorescently emitted wave is proportional to the sine of the angle (α) between the dipole µ and the wave vector k. The emission of a classical dipole is, therefore, nonuniform, and has a donut shape (Fig. 5.3). The emission pattern of a dipole near a surface can be described by considering the intensity and polarization of both the direct and the reflected waves. Consider that light emitted from a dipole with polar tilt angle θ placed at 74 M.S. Ünlü et al. Electric field E µ k Direction of Dipole a propagation E a sina Fig. 5.3. Left : Intensity and polarization of electric field emitted by a classical dipole. Right : 3D emission pattern of a classical electric dipole. z q Plane of err q E incidence k Plane of dtr ETE incidence µ Dielectric ain surface ETM krefl k1 qe j Fig. 5.4. Left : Direct and reflected waves emitted by a randomly oriented dipole. Right : Transverse electric and transverse magnetic components of the electric field of incident wave. a distance d from a stack of mirrors is collected by a microscope objective, where the numerical aperture is defined by the collection cone of the objective (Fig. 5.4). Every emission direction within the collection cone can be described by θem, the tilt angle from the surface normal, and ϕ, the azimuthal angle to the dipole. The dipole radiates two coherent waves: one sent directly to the detector, and one incident on the mirror surface, which is then reflected so that it propagates parallel to the direct wave. As the distance between the dipole and the mirror is significantly smaller than the distance between the dipole and the microscope objective, these parallel waves arrive at the same spot on the detector. The ETM component of both the incident and direct waves lies in the plane of incidence, and ETE is perpendicular to it. After reflection, the inci- dent wave is modified by reflection coefficients RTE and RTM of the stack of mirrors. Also, there is an additional phase shift between the direct and the 5 Spectral Self-Interference Microscopy 75 reflected waves because of the distance the incident light travels from the source to the stack of mirrors and back (ei2kd cos θem). Light intensity collected by a microscope objective with maximum collection angle θmax em is integrated over dθem sin θem. To account for fluorophores with different transition dipole orientations, the total collected light intensity is integrated over all possible dipole orientations. Usually, fluorophores in monolayers are randomly distrib- uted in the horizontal plane, so their emission is integrated over ϕ; however, the range of polar tilt angles can sometimes be restricted. For large enough separations between the fluorophores and the mirror, several oscillations of constructive and destructive interference appear within a small span of emitted wavelengths. This creates interference fringes in the emission spectrum, and the phase and contrast of the oscillations will depend on the location and orientation of dipoles above the mirror. The emission of the dipole can be formulated as: I = |ETE dir + ETE refl|2 + |ETM dir + ETM refl|2 , (5.1) where √ ETE dir = ETE inc ∝ cos θ sin ϕ, (5.2) ETM dir/inc ∝ 1 − (sin θem sin θ cos ϕ ± cos θem cos θ)2 − sin2 θ sin2 ϕ, (5.3) ETE refl = ETE incRTEei2φ, (5.4) ETM refl = ETM incRTMei2φ, (5.5) 4πn φ = d cos θem, (5.6) λ and n is the index of refraction of the medium surrounding the dipole. The total emission of a monolayer of random dipoles measured with an objective of maximum collection angle θmax em is: ∫π/2 ∫π/2 θ∫max em Itotal = I (θ, ϕ, θem)dθdϕdθem sin θem. (5.7) θ=0 ϕ=0 θem=0 5.4 Acquisition and Data Processing 5.4.1 Microscope Setup The measurements were performed with a system that combines an upright Leica DM/LM microscope and a Renishaw 100B micro-Raman spectrometer. The sample was positioned and scanned using a motorized submicrometer precision stage. An 1,800 grooves per millimeter grating was used with a spectral resolution of 2 cm−1 at 500 nm. The light dispersed by the grating 76 M.S. Ünlü et al. Optical path 488 nm notch filter Spectrometer Laser 488 nm Confocal aperture Objective θ Fluorophore max monolayer Substrate Fig. 5.5. Microscope setup for SSFM. was imaged onto a thermoelectrically cooled charge-coupled device (CCD). For WL measurements, normal Koehler illumination with a halogen lamp was used, whereas for fluorescence measurements, the light source was an argon ion laser with a 488 nm line. The emission from the fluorophores was collected with a 5× (NA=0.12) objective and transmitted into the spectrometer through a notch filter, blocking the excitation light (Fig. 5.5). 5.4.2 Fitting Algorithm Self-interference spectra are raw data composed of the envelope function rep- resented by the emission profile of the free fluorophore, the oscillatory interfer- ence component, and high-frequency Gaussian noise introduced by the spec- trophotometer. Both WL reflectivity and fluorescence self-interference spectra were fitted using a custom-built MATLAB application that separates the os- cillatory component from the envelope function. This program automatically calculates the parameters of the system such as the thickness of thin films or position of the emitters above the mirror. The variation in the index of refraction of silicon oxide within the used wavelength span was taken into ac- count in the fitting algorithm. The model also takes into account the complex reflectivity of the underlying stack of dielectrics and the orientation of the dipole moments of the emitters. Because the curve-fitting algorithm extracts the oscillatory term to determine the label height, the measurements are im- mune not only to potential quenching of the entire spectrum, but also to any spectral modifications or nonradiative transfer effects. 5 Spectral Self-Interference Microscopy 77 5.5 Experimental Results 5.5.1 Monolayers of Fluorophores on Silicon Oxide Surfaces: Fluorescein, Quantum Dots, Lipid Films SSFM can be used to measure the vertical position of fluorophores separated from a silicon mirror by a transparent layer of silicon oxide. Monolayer surfaces of fluorescein, quantum dots [7], and lipid films [6] are investigated in the experiments below. Fluorescein To demonstrate detection of the axial position of a fluorescein monolayer from the silicon mirror, a sample was prepared with a spacer layer of silicon oxide etched to different heights, and a monolayer of fluorescein was immo- bilized on surface via isothiocyanate-aminosilane chemistry. The surface was then scanned in a grid pattern with fluorescence emission spectra taken every 200 µm, and the spectra were processed to yield the axial position of the fluo- rophores at each spot. Figure 5.6 displays the height data as a 3D gray-scale image, where it is apparent that nanometer axial resolution is obtained. Note that this is not the fluorescence intensity, which is relatively uniform. Quantum Dots Quantum dots (QDs) are a new area of research with potential applications in many fields. The excitation spectrum of most QDs is unusually broad. However, the emission properties of a QD depend on its size: smaller dots emit light with shorter wavelengths. Samples of QDs that are uniform in size have Fig. 5.6. Surface profile reconstructed from self-interference spectra. 78 M.S. Ünlü et al. b) 4.955 chip.wl a) 4.950 QDs.wl Quantum dots Fluorescein QDs.sfm 4.945 4.940 3nm <1nm 4.935 4.930 0 2 4 6 8 10 12 14 16 18 20 Position on the chip, mm Fig. 5.7. (a) Average position of fluorescein and quantum dots above a Si-SiO2 surface. (b) WL and SSFM measurements of a Si-SiO2 chip before and after quantum dot deposition. QDs are sparse enough that their effect on determining the spacer layer thickness is insignificant. SSFM measurements show the source of emission is about 3 nm above the surface. sharp emission peaks; for a broader distribution of sizes, the spectra widen as well. On the one hand, as a fluorescent label for biological research, quantum dots hold a number of advantages over conventionally used organic dyes as they are photostable, can withstand many excitation-emission cycles, and can provide a whole range of nonoverlapping spectra. On the other hand, QDs are less commonly used in staining biological specimens as they cannot be as easily functionalized with reactive groups for selective attachment. Chemical methods of adapting QDs to new environments are being developed by many research groups; in a recent achievement, QDs were used as fluorescent labels inside living cells [10]. To demonstrate visualization of a monolayer of QDs immobilized on a surface, a silicon oxide chip was silanized with aminopropyltriethoxysilane (APTES). CdSe quantum dots capped with ZnS were treated with mercap- toacetic acid to render them hydrophilic and negatively charged at neutral pH. The QDs were then electrostatically attached to the aminated surface [11]. SSFM measurements show that the average position of the emitters is ele- vated above the surface by about 3 nm (Fig. 5.7), which reflects the thickness of the QDs themselves. When compared with a monolayer of fluorescein, the best-fitting curve for the QD spectrum corresponds to a random orientation of the transition dipoles, whereas, in the case of fluorescein, the molecules seem to be more concentrated in the horizontal position. Lipid Films Deposition of lipid bilayers on surfaces has attracted considerable attention because of the possibility of creating biomembrane-based biosensors and for studying the fundamental properties of biomembranes. Artificial lipid films can be used as substitutes for biomembranes in investigating the structural and functional properties of various transmembrane properties in conditions similar to those found in cells. Height (µm) 5 Spectral Self-Interference Microscopy 79 4.938 Top Layer Bottom Layer 4.936 3.5 nm 4.934 5.5 nm (fluorescence) (white light) 4.932 4.930 4.928 0.0 2.0 4.0 6.0 8.0 10.0 Position on sample (mm) Fig. 5.8. Left : Structure of the lipid bilayer film. Right : Axial fluorophore positions measured across the surface. The interaction between the lipid films and various membrane-bound com- ponents can be probed by diagnostic tools such as infrared spectroscopy, neutron reflectivity, surface-plasmon resonance (SPR), and atomic force mi- croscopy (AFM). The finest resolution of lipid surface features can be achieved by AFM, but AFM is unsuitable for probing structures that are too delicate and cannot resolve buried components. SSFM offers a unique opportunity to look inside the lipid membrane and detect the position of a fluorescently labelled component. To determine the position of a fluorescent label attached to the head groups of either the top or the bottom leaflet of a lipid bilayer separated by only about 4 nm, a layer of DPPE containing 2% DHPE-fluorescein was deposited by the Langmuir- Blodgett technique, followed by a layer of DPPE without the dye. The fluores- cence emission spectra as well as WL reflectivity measurements were taken at the same locations on the chip. The chip was cleaned, followed by deposition of a new bilayer of lipids with the fluorescein-containing leaflet on top, and the measurements were repeated. The results are summarized in Fig. 5.8. The WL reflectivity measurements before and after lipid deposition were the same for both experiments (not shown). The fluorescence emission spectra show the average position of the fluorescent dye attached to either the top or the bottom head groups very accurately, fractions of a nanometer, inside the lipid bilayer. 5.5.2 Conformation of Surface-Immobilized DNA DNA array technology, offering highly paralleled detection capability, has be- come a widespread tool in biological research with applications in expression screening, sequencing, and drug discovery. One of the defining characteristics of a DNA array is the availability of the single-stranded probes for hybridiza- tion with the target. The conformation of DNA molecules in an array may significantly affect the efficiency of hybridization; immobilized molecules lo- cated farther away |
from the solid support are closer to the solution state, and Fluorophore Position (µm) 80 M.S. Ünlü et al. are more accessible to dissolved analytes. Recently, advances have been made to characterize surface-bound DNA probes, using optical or contact methods such as ellipsometry, optical reflectivity [12,13], neutron reflectivity [14], X-ray photoelectron spectroscopy [15], FRET [16,17], SPR [18,19], and AFM [20,21]. However, most experimental techniques characterize the DNA layer as a sin- gle entity, parameterizing its thickness or density. The advantage of SSFM is that unlike these other methods, it has the capability of examining the specific positions of internal elements of the DNA chain. To study the conformation of single-stranded DNA (ssDNA) and double- stranded DNA (dsDNA) on glass surfaces with SSFM, 50 and 21-nt oligonu- cleotides were used [8]. In all studies, the first strand of the DNA was covalently bound to the surface at its 5′ end. Experiments were performed with fluorescein markers bound to either the first strand at its distal 3′ end, or the second strand at its 3′ or 5′ end. WL Reflectivity Measurements As schematically shown in Fig. 5.9, oligonucleotides carrying a 5′-amino tag are covalently bound to an aminated surface via a homobifunctional crosslinker. Using WL reflectivity, progressive growth of the surface-bound thin films during DNA immobilization steps is determined. The thickness of the silane layer is 0.8–1.0 nm, which roughly corresponds to a monolayer; phenylene isothiocyanate adds another 0.5–0.6 nm. Immobilization of DNA leads to a further increase in the optical thickness. The average film thickness is determined by WL reflection spectroscopy assuming an index of refraction of 1.46 for DNA as was measured for dense layers [22]. The optical film thickness for ssDNA of 21- and 50-nt is measured as 1.0–1.5 nm and 2.0–2.5 nm, respec- tively. Although the precision of absolute thickness will depend on the index, WL reflectivity provides an accurate relative measure of additional mass on the surface and can thus monitor the efficiency of hybridization. As shown in Fig. 5.9. The WL interference measurements of 50mers immobilized on the surface before (left) and after (right) hybridization. The height of the transparent layers (2 and 3 nm for left and right, respectively) correspond to average optical thickness of the DNA layer. 5 Spectral Self-Interference Microscopy 81 Fig. 5.10. The fluorescence interference measurements for the 50-bp dsDNA labeled at the proximal (left) and distal end (right). Fig. 5.9 (right), adding complementary second strands to 50-mers results in an increase in the film thickness by ∼1.0 nm corresponding to a hybridization efficiency of ∼50%. Determination of the Position of a Fluorescent Label The optical thicknesses of the layers obtained by WL reflectivity measure- ments can be used together with the SSFM measurements to determine the position of the fluorophores relative to the surface. Figure 5.10 illustrates the schematics of the experiment to study the elevation of dsDNA fragments. The maximum elevation of the label is limited by the length of the double helix, which is ∼7 nm for 21-bp and ∼17 nm for 50-bp fragments. As dsDNA has a persistence length of ∼50 nm [23], short fragments in the experiments can be viewed as rigid rods on hinges. A rigid rod hinged to the surface would have an average height of the distal label with an average tilt angle of 60◦ from normal, assuming free rotation of one half of the length. Factors such as steric hindrance or surface interactions may limit this free rotation. Using SSFM, we measured the elevation of the label on top of DNA double helices to be 5.5 nm for 21-bp fragments and 10.5 nm for 50-bp fragments, which represents tilt angles from the normal of 40◦ and 50◦, respectively. These values are a mea- sure of the average distribution of heights within the microscope focal spot. If the label on the second strand is at the 3′ end, its location in the double helix will be at the bottom, close to the surface. Although we did not expect to see a significant variation, the proximal, 3′-end label on 50-bp DNA is elevated by ∼2–3 nm compared with only 0.5–1 nm in the case of the 21-bp fragment. This is because a 50-mer is long enough to have stable partial hybridization that could free the proximal end and yield an elevated label position, and also because of steric hindrance. We also studied the conformation of ssDNA by measuring the height of flu- orescent tags attached to the free end of surfacebound DNA oligonucleotides. Unlike dsDNA, ssDNA is flexible and little is known about the shape or size of ssDNAs on the surface. AFM measurements suggest that ssDNA immobilized on a surface exists in a globular conformation [21]. 82 M.S. Ünlü et al. However, there are reports stating that because of steric hindrance from nearby molecules, ssDNA may change its conformation from a random coil to more extended forms [22,23]. The fluorescent label attached to the distal end of surface-bound single-stranded 21-mer is found to be close to the surface, within 1 nm. In a similar situation, 50-mers show much higher location of the label: 5.5 nm above the surface as illustrated, pointing out to a considerably more extended conformation compared with 21-mers, which may be due to steric effects from closely located grafts in the DNA layer. The surface den- sity of immobilized ssDNA measured using a radiolabel is ∼35 fmol/mm2 for both 21- and 50-mers, which translates into 11 nm distances between adjacent molecules or a 5.5 nm radius of free space around each. As the length of a fully extended 50-mer is 27.5 nm, it is enough to interact with its neighbors, at least intermittently. When a second, unlabeled strand is hybridized, the label at the distal end of the newly formed duplex extends out as well. Unlike the dsDNA in Fig. 5.10, the DNA layer now consists of two species: the dsDNA and the unhybridized strands, both of which are carrying a fluorescent marker but at different heights. The average position of the marker should be somewhere in between depending on the degree of hybridization. The binding efficiency is estimated by comparing the average height of the fluorescent layer above the surface for the two cases, and found to be between 30% and 50%. This rough estimate is close to the result obtained by WL reflectivity: 50% hybridization for both 21 and 50-mers, demonstrating self-consistency. As a further check, we have performed density measurements with radiolabeled DNA and found it consistent with 50% hybridization. Because of intrinsic limitations such as substrate-related quenching of radiative emission, we do not consider the ra- diolabeling for absolute determination of DNA densities, but rather only for relative estimation of DNA densities. We also studied how the conformation of a surface-bound 50-nt labeled oligonucleotide changes when it is annealed with a 21-mer complementary to either its top or bottom part. When a 21-mer complementary to the top section was annealed, the position of the distal end increased from 5.5 to 6.5 nm. However, when an ssDNA–dsDNA construct has the doublestranded part proximal to the surface, the position of the label is lower than that of an unhybridized oligo, decreasing in average height from 5.5 to 3.5 nm. These data suggest that the distal bound 21-mer construct is nearly vertical, and the proximal bound construct has formed a rotation point allowing the flexible distal end to approach closer to the surface. The selected sequence of the oligonucleotides rules out the possibility of intramolecular DNA structures. 5.6 SSFM in 4Pi Configuration SSFM is a powerful tool in determining the position of fluorescent labels and giving insight about the conformation of biological structures bound to surfaces. However, despite its high precision axial position determination 5 Spectral Self-Interference Microscopy 83 capability, its lateral resolution is limited by several micrometers as low NA objectives have to be used to ensure spectral fringe visibility. The large spacer layer thickness results in a focal mismatch between the direct and the reflected image of the sample when high NA objectives are used. Also, with high NA objectives, the rapid change of fields for large angles due to the interference of direct and reflected signals smears out the spectral interference fringes and lowers the signal contrast. One remedy for increasing lateral resolution by using high NA objectives while maintaining the nanometer level axial local- ization capability is to use a second objective instead of a mirror and collect the emission from both sides (Fig. 5.11a). As the spherical wavefronts of the emission are collected by a symmetrical configuration, path length mismatch for higher collection angles for two interference pathways is eliminated unlike using one objective and a reflecting substrate. The path lengths of the two interference arms are adjusted by one of the mirrors mounted on a piezo con- troller, such that they are different by several tens of micrometers to induce modulations in the spectrum of the emitter. This configuration is similar to a) b) 1 0.8 0.6 0.4 0.2 0 18500 18750 19000 19250 19500 wavenumber [1/cm] c) 4 3.5 3 2.5 2 1.5 1 5.1 nm 0.5 28 30 32 34 36 38 axial position [nm] Fig. 5.11. (a) A 4Pi microscope. (b) Normalized spectra of a monolayer of Alexa Fluor 488 measured using 4Pi microscope at two axial positions that are 5 nm apart. (c) Minimization of error in fitting spectral responses of two positions. Error [a.u.] normalized intensity 84 M.S. Ünlü et al. 4Pi confocal fluorescence microscopy introduced by Stefan Hell [1]. However, instead of using the 4Pi microscope for 3D image scanning, the emitted signal is collected by two high NA objectives and coupled to a spectroscopy system to localize monolayers with nanometer accuracy. Scanning of the tight collec- tion spot is performed in lateral dimensions and axial profile is extracted form the spectral fringes similar to SSFM. In Fig. 5.11b, typical spectral response of a 4Pi SSFM system is shown. The two distinct spectra are from the same spot of a monolayer that was moved by 5 nm in axial direction between the data acquisition. The fluorescent emitter is a monolayer of Alexa Fluor 488 dye deposited on a glass cover slip and placed in the common foci of the two high NA oil immersion objectives of the 4Pi microscope. The sample was excited by a 488 nm continuous wave laser using the two objectives. The spectral response clearly shows the shift in spectral fringes because of the change in the axial position of the monolayer of fluorophores. A simple fitting algorithm that utilizes a geometrical model and only includes the position of monolayer with respect to the geometrical focus and the external path length difference can be used to determine the relative difference in the axial positions of monolayers. As seen from Fig. 5.11c, the axial position values that minimize the error in fitting the spectral responses of two positions of the monolayer were actually 5.1 nm apart, which agrees well with the 5 nm physical change in the axial position of the layer. 5.7 Conclusions We have developed a new technique, SSFM, which transforms the variation in emission intensity for different path lengths used in fluorescence interferome- try to a variation in the intensity for different wavelengths in emission, encod- ing the high-resolution information in the emission spectrum. Using SSFM, we have demonstrated analysis of systems that include monolayers of fluores- cein, quantum dots, and lipid films. More importantly, we have demonstrated conformation of surface-immobilized DNA by estimating the shape of coiled ssDNA, the average tilt of dsDNA of different lengths, and the amount of hybridization. We have also shown that localization of axial position of fluo- rescent structures with high precision is possible without sacrificing the lateral resolution using SSFM in 4Pi configuration. These data provide important proofs of concept for the capabilities of novel optical methods for analyzing the molecular disposition of DNA and protein on surfaces. The determination of DNA conformations on surfaces and hybridization behavior provide information required to move DNA interfacial applications forward and thus impact emerging clinical and biotechnological fields. 5 Spectral Self-Interference Microscopy 85 Acknowledgments The authors acknowledge Prof. İrs.adi Aksun of Koc University for his contributions to modeling of fluorophore emission on layered surfaces and Dr. Stephen Ippolito for his help in setting up the microscope, the |
National Science Foundation (Grant DBI0138425), Air Force Office of Scientific Re- search (Grant MURI F-49620-03-1-0379), and National Institutes of Health– National Institute of Biomedical Imaging and BioEngineering (Grant 5R01 EB00 756-03) for their support. The views and conclusions contained in this document are those of the authors and should not be interpreted as representing the US Government. References 1. S. Hell, E.H.K Stelzer, JOSA A 9(12), 2159 (1992) 2. B. Bailey, D.L. Farkas, D.L. Taylor, F. Lanni, Nature 366, 44 (1993) 3. P.T.C. So, C.Y. Dong, B.R. Masters, K.M. Berland, Ann. Rev. Biomed. Eng. 2, 399 (2000) 4. M. Dyba, S.W. Hell, Phys. Rev. Lett. 88, 163901 (2002) 5. A. Lambacher, P. Fromherz, Appl. Phys. A 63, 207 (1996) 6. A.K. Swan, L. Moiseev, C.R. Cantor, B. Davis, S.B. Ippolito, W.C. Karl, B.B. Goldberg, M.S. Unlu, IEEE J. Select. Top. Quantum Electron. 9, 294 (1996) 7. L. Moiseev, C.R. Cantor, I. Aksun, M. Dogan, B.B. Goldberg, A.K. Swan, M.S. Unlu, J. Appl. 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Tarlov, J. Am. Chem. Soc. 119, 3401 (1997) 19. L.K. Wolf, Y. Gao, R.M. Georgiadis, Langmuir 20, 3357 (2004) 20. S.O. Kelley, J.K. Barton, N.M. Jackson, L.D. McPherson, A.B. Potter, E.M. Spain, M.J. Allen, M.G. Hill, Langmuir 14, 6781 (1998) 86 M.S. Ünlü et al. 21. L.S. Shlyakhtenko, A.A. Gall, J.J. Weimer, D.D. Hawn, Y.L. Lyubchenko, Biophys. J. 77, 568 (1999) 22. Q. Du, M. Vologodskaia, H. Kuhn, M. Frank-Kamenetskii, A. Volodogski, Biophys. J. 88, 4137 (2005) 23. S. Elhadj, G. Singh, R.F. Saraf, Langmuir 20, 5539 (2004) 6 Applications of Optical Resonance to Biological Sensing and Imaging: II. Resonant Cavity Biosensors M.S. Ünlü, E. Özkumur, D.A. Bergstein, A. Yalc.in, M.F. Ruane, and B.B. Goldberg 6.1 Multianalyte Sensing Interrogating binding interactions between proteins, segments of DNA or RNA, and biospecific small molecules is critically important for a great num- ber of applications in biological research and medicine. Microarray technology has emerged over the past decade to address applications that seek to mea- sure thousands or even millions of binding interactions at once. A microarray consists of a solid support, or substrate, with multiplicity of spots on its top surface each containing a different type of fixed capturing molecule. A sample solution containing unknown target molecules, or analytes, is introduced to the microarray surface typically via a small fluid chamber or flow cell. The amount of target material bound to any feature after washing the array gives an indication of the affinity between the target and capturing agent at that spot. There are a number of important applications in biological research that benefit from microarray throughput such as gene expression profiling or antigen–antibody interaction monitoring. Aside from research applications, microarrays may play a crucial role in a new era of medical diagnostics. Bio- medical research is continuing to point toward molecular biomarkers that can be used to help doctors diagnose diseases sooner, with greater accuracy, and provide information that helps doctors personalize treatment plans. Present microarray technology requires that the target molecules in the sample solution be labeled or attached with a fluorescent dye molecule for the purpose of determining how much target material has bound to each mi- croarray feature using a fluorescence detector. The need to label the target molecules to visualize the results is a significant shortcoming of present tech- nology and has been described previously [1–4]. In general, fluorescent labeling may suffer from bleaching of the labels, quenching effects from the surface, or low contrast between the label and the autofluorescence of the microarray substrate. In addition, proteins in particular may suffer from altered binding properties once they are labeled. For these reasons, label-free detection for 88 M.S. Ünlü et al. both DNA and protein is preferred, particularly for use in medical diagnostics where variation and cost must be minimized. A vast number of label-free detection techniques have surfaced over the last decade or two to address the problems of label-based detection [5–16]. The important figures of merit for these techniques are sensitivity to bound targets, throughput (number of features evaluated in a single test) and the ability to scale throughput, and cost per test. Sensitivity is often quantified by mass per area (pg mm−2) or average height (pm) where 1 pm corresponds to roughly 1 pg mm−2 for protein or DNA [17–25]. 6.2 Resonant Cavity Imaging Biosensor 6.2.1 Detection Principle Two partially reflecting substrates are positioned such that their reflecting surfaces face each other and form the optical cavity (Fig. 6.1) [26]. The tun- able wavelength laser light is collimated and incident from the back of one of the reflectors. The wavelength of the laser is swept in time and at specific wavelengths the resonant condition of the cavity is met, the light resonates inside the cavity and couples out. Beyond the cavity, the transmitted light is imaged on a camera, so that the resonant response at each location of the cavity is recorded by a corresponding pixel on the camera. The probe biomole- cules are patterned on one of the reflector surfaces. When target biomolecules bind to their specific capturing agents, the local resonant response shifts in Fig. 6.1. Resonant cavity imaging biosensor (RCIB) setup. 6 Resonant Cavity Biosensors 89 wavelength. The presence of biomolecules bound to the surface is fundamen- tally detected by a small perturbation of the electric field inside the cavity. The effect of a slight phase delay is amplified by the resonant behavior of light within the cavity. The cavity enhancement can provide a significant sensitiv- ity improvement over interference techniques that do not benefit from a high finesse optical cavity. The reflectors can be constructed with alternating di- electric layers of silicon and SiO2 and may end with a layer of SiO2 that allows the surface immobilization chemistries that are developed for glass slides to be used. The use of a parallel optical cavity, tunable wavelength laser, and a digital camera allows high throughput. Aside from the alignment and stability of the reflectors, and the internal operation of the tunable laser, there are no moving parts in the setup. Additionally, the reflectors described here may be made very inexpensive and hence disposable. In the present implementation, the cavity is formed with reflectors fabricated from alternating layers of Si and SiO2, constituting a Bragg grating (Fig. 6.2) [27], and the wavelength of light is varied between 1,510 and 1,515 nm. The electric field distribution in the cavity forms a standing wave pattern with a minimum at the Si surface of the reflectors. It is beneficial to place the sensing surface on an approximately quarter wavelength thick layer of similar low-index material, namely SiO2. Adding this extra oxide layer brings the binding surface some distance into the cavity where the field is maximum (Fig. 6.3). When it is placed at the field maximum, the sensing layer interacts with the highest number of photons and slight thickness changes will have a maximum effect on the field distribution, thereby shifting the resonant wavelength a maximum amount. If the sensing surface is positioned at a field minimum, small changes on the surface will have no effect on the resonant cavity and will therefore be undetectable. Adding an extra layer of SiO2 also has the previously mentioned benefit of providing −1µm Si SiO2 Si SiO2 Si Substrate Fig. 6.2. The Bragg structures that are used as the reflectors in RCIB setup with alternating layers of 1/4 wavelength thick Si and 3/4 wavelength thick SiO2. The image is taken with SEM. 90 M.S. Ünlü et al. Fig. 6.3. Electric field pattern in the optical cavity. Fig. 6.4. Simulation of the transmittance of the cavity. The reflectors are 40.0 µm apart from each other with air in between. A final SiO2 layer is included and shift corresponds to 5 nm step on the oxide. a chemically compatible surface for microarray fabrication. Simulation of the cavity transmittance vs. wavelength with changing top surface oxide thickness is plotted in Fig. 6.4. Simulation was done in Matlab using the matrix method for calculating transmittance through layered media [28]. 6.2.2 Experimental Setup, Data Acquisition, and Processing An RCIB setup has been constructed. The illumination source is an IR tunable laser with a fiber-coupled output. Light is coupled into the system with a SMF- 28 fiber and collimated to 10 mm FWHM. All the optics are antireflection coated to avoid interference effects. The laser power is set to 15 µW, and the wavelength is continuously swept from 1,510 to 1,515 nm in 10 s during data collection. The reflectors, which are 15 mm by 15 mm, are placed 40 µm apart 6 Resonant Cavity Biosensors 91 from each other. One reflector is mounted on a tip-tilt stage to bring the reflectors parallel. The second reflector is mounted on a translational stage to adjust the reflector separation. The transmission from the cavity is imaged on an indium–gallium–arsenide (InGaAs) sensor array with 5 × magnification and an aperture setting the NA to 0.1. With these settings, 12 × 12 µm2 areas are imaged on the 60 × 60 µm2 pixels of the sensor array, which has 128 × 128 pixels. The laser wavelength is swept at a rate of 0.5 nm s−1 while the camera captures images at a rate of 30 fps. Images are digitized to 12 bits (4,096 gray-levels) and transferred to a PC. These data are transferred to Matlab and fit using several different meth- ods. There is a trade-off between the accuracy and the speed with which the data can be fit. A more accurate (noise-resilient) curve fitting method is to fit the data to resonant line shape given by (6.1), which models a simple resonant cavity where ∣ ∣ ni ∣ T ∣ e k n ∣ t2 j d ∣∣2= 2 −2j f 1 − , (6.1) r e kd ∣ ni and nf are the refractive indices in and beyond the cavity, t and r are the transmission and reflection coefficients, respectively, of the reflectors, k is the wavenumber inside the cavity, and d is the reflector separation (Fig. 6.5). Further image processing subtracts the curvature inherent to the reflectors. Finally, a surface profile of small height variations on the SiO2 surface can be obtained. 6.2.3 Experimental Results The system sensitivity was tested by imaging etched features on the oxide surface (Fig. 6.6). The reflector that has a SiO2 layer on top is used as the Fig. 6.5. Resonant curve recorded and fitting to this data using (6.1). 92 M.S. Ünlü et al. Fig. 6.6. Left : RCIB scan of the overlapping boxes. Right : Line cut of the image in two consecutive measurements. sensing surface. Standard photolithography and wet (BOE) etching were used to pattern these features on the surface. Two overlapping rectangles, with depths of 3 and 6 nm were etched resulting in four regions with different oxide thickness; not etched, 3 nm deep, 6 nm deep, and 9 nm deep etched. These values are consistent with the AFM measurements of the same sample. System repeatability was tested by making consecutive measurements of the same sample. Averaging boxes measuring 5 pixels by 5 pixels, or an area of 50 × 50 µm2, the RMS deviation in consecutive measurements was 0.01 nm (Fig. 6.6). 6.2.4 Spectral Reflectivity Imaging Biosensor The phase delay added by extra material can also be imaged using a |
sim- pler approach. We propose a surface profilometry technique to be used as a label-free microarray imaging device. Spectral reflectivity imaging biosen- sor (SRIB) uses wavelength dependent reflectivity of a silicon substrate with thick thermally grown SiO2(5–10 µm), to accurately find the film thickness at tens of thousands of different spots, and thus image the surface profile. This technique, having a much lower finesse, is expected to be less sensitive than RCIB, but should be more robust and offer higher throughput. A collimated laser beam that can be tuned from 764 to 784 nm is reflected from a pellicle beam splitter and is incident on the sample surface (Fig. 6.7). At any fixed wavelength, reflection from the sample is imaged on a CCD camera (cam- era pixel size is 13.7 µm and the magnification of the imaging system is 2.3). Then the wavelength is stepped, and another image is taken. Repeating this through the sweeping range of the laser, one can form a spectral reflectivity curve for every 36 µm2 area on the sample in the field of view. The separate 6 Resonant Cavity Biosensors 93 Fig. 6.7. Spectral reflectance imaging biosensor diagram. reflections from oxide and silicon surfaces form an interference pattern and an exact oxide thickness corresponding to each pixel is found by curve fitting the recorded data. Reflection of a single layer can be well estimated by Airy’s formulas [28]. The reflection coefficient for an incoming field is given by: r12 + r r = 23e −2iφ ( 1 + r12r23e− 6.2) 2iφ 2πd φ = nSiO cos θ λ 2 , (6.3) where r12 and r23 are the Fresnel reflection coefficients from the SiO2 surface and SiO2-Si interface, respectively, and φ is the optical phase difference be- tween the two reflections. Here, d is the SiO2 thickness, nSiO2 is the refractive index of SiO2, λ is the wavelength of the incident light, and θ is the incidence angle to the SiO2–Si interface, which will be 0◦ for perpendicular incidence. Curve fitting tools are used to fit this equation to the recorded data at each pixel and to find the d. The setup was tested with etch marks of overlapping rectangles that was mentioned in the previous section. Silicon wafers with 6.1 µm of thermally grown SiO2 were etched by photolithographic techniques. Three different depths of etch steps were created on the sample, which were 4, 12, and 16 nm, and their depths were confirmed with AFM scans. The sam- ple was then imaged by SRIB, and data were processed with curve fitting. A line-cut from the image can be seen in Fig. 6.8. These etch marks were imaged with an accuracy of 0.5 nm. 94 M.S. Ünlü et al. 6134 6132 6130 6128 6126 6124 6122 6120 6118 6116 6114 0 50 100 150 pixels Fig. 6.8. Line-cut from a processed image taken by the SRIB setup. Etch marks at three different heights are seen: 4, 12, and 16 nm. The thickness of the oxide was ∼6,130 nm, initially. Although the present data show that the system can image 250 spots with subnanometer sensitivity, balancing the laser intensity fluctuations and using a better imaging system is expected to improve sensitivity to 0.1 nm with more than 10,000 spots. 6.3 Optical Sensing of Biomolecules Using Microring Resonators 6.3.1 Basics on Microring Resonators Integrated devices featuring resonant microcavities with high quality factors (Q) such as toroids, disks, rings, and spheres have been used as add-drop filters, optical switches, and in laser applications [29–33]. These devices have recently become popular for research in biochemical sensing [34–38] as the demand for highly sensitive and compact devices to detect biomolecules in- creased. Light is confined within the microring cavities by total internal re- flections resulting in high Q resonant modes. When the refractive index of the cladding or the outside medium changes (e.g., due to binding of molecules), a new guiding condition is obtained for the mode, causing a shift in the resonant wavelength. detected oxide thickness (nm) 6 Resonant Cavity Biosensors 95 Microring resonators provide sensitivities comparable to surface plasmon resonance because of their high Q (∼12,000 in this study) [16,39], and they are robust and can be mass-manufactured using well-established silicon-integrated circuit fabrication techniques. However, they lack the very high-throughput potential that is demonstrated with RCIB. The sensitivity of the system presented here is quantified through mea- surement of resonance shifts induced by a change in the refractive index of the medium (a bulk change). Additionally, sensing of biomolecules is demon- strated through observation of the change in resonant condition caused by molecular binding of the well-documented avidin–biotin couple on the surface. 6.3.2 Setup and Data Acquisition The microring resonators used in this experiment are 60 µm in radius, and vertically coupled to waveguides (Fig. 6.9). The effective index of the cavity is measured as neff ≈ 1.5 in deionized water (DI-H2O) ambience, and the penetration depth for the guided mode at 1,550 nm in this environment is calculated as ∼360 nm. A fiber-coupled tunable wavelength IR laser is used as the light source. The cavity can support both TE and TM modes and with the use of a polarizer one of the modes is selected. An optical splitter placed after the polarizer separates the signal into two paths: one arm is chopped in free space at a frequency of 220 Hz and then coupled to the input waveguide of the microring resonator. The output waveguide is coupled to a photodetector as signal input. The second arm acts as the reference input to the photodetector for balanced detection. Common-mode noise cancellation ring resonator lateral offset out in vertical offset Input/output waveguides Fig. 6.9. Microring resonator is vertically coupled to input/output waveguides. Lat- eral and vertical offsets of the resonator allow control over the coupling coefficient. Receptor molecules are immobilized on the resonator surface, and target molecules are released on the surface in a solution during flow. The top view of the resonator is shown in the inset. 96 M.S. Ünlü et al. (mainly laser intensity fluctuations) is achieved through balanced detection, improving the signal-to-noise ratio, thus the overall system sensitivity. A flow- cell is used for controlled solution flow over the microring surface, and real time signal during solution flow is acquired and analyzed with LabVIEW. Direct measurement of a shift in resonance through repeated spectral scans is time consuming. Alternatively, the resonance shift can be measured indirectly by collecting data at a single wavelength and observing the intensity in real time, and the change in intensity can then be mapped to a shift in resonance. To obtain the maximum change in intensity due to a change in effective index, the wavelength at which the measurements are performed is selected to correspond to the point of maximum slope of the resonance curve. The high intensity stability necessary for these measurements is achieved by eliminating the noise components of laser output through balanced detection. 6.3.3 Data Analysis and Discussion The bulk experiments to determine the sensitivity of the system were con- ducted by flow of a solution of known refractive index with respect to the refractive index of DI-H2O that is used as a calibration reference. In Fig. 6.10, the measured shift in resonance with respect to a change in the refractive index of the medium (corresponding to various concentrations of phosphate buffered saline (PBS) solution) is plotted. Notice that the relation is highly lin- ear. Using standard deviation δ (pm) of signal levels during PBS flow and the slope (m) of the plot in Fig. 6.10, the limit of detection (LOD) for a change in refractive index can be approximated as LOD(n)=3δ m−1. We conclude that LOD(n) = 1.8 × 10−5 refractive index units (RIU) for our setup. The experi- ment to demonstrate detection of biomolecules consists of two parts: binding of biotinylated lectins to an avidin covered surface, and breaking the avidin– biotin bonds for surface regeneration. For binding, 3.5 µM biotinylated lectin solution is flowed over the surface. Once the signal level reached by the binding of biotin molecules to avidin covered surface is stabilized, DI-H2O is flowed to wash away the unbound molecules. For surface regeneration, the surface is first washed with a chemical pH-7 buffer that breaks the avidin–biotin bonds, and then with DI-H2O. The real-time intensity recording of binding/regeneration processes is shown in Fig. 6.11. The actual amount of change in the signal is measured between levels of DI-H2O flow before and after the introduction of biotinylated lectin solution. At these levels, the outside medium is identical, so the change in intensity is caused only due to binding of molecules. The signal level at the end of the regeneration phase is close to the initial level indicating that partial recovery of surface is achieved. 6 Resonant Cavity Biosensors 97 140 120 100 y = 141332x-188397 R2=0.99458 80 60 40 20 0 1.3330 1.3332 1.3334 1.3336 1.3338 Refractive Index Fig. 6.10. Shift of resonance is linearly related to change in refractive index. The slope of the plotted curve is used to determine the limit of detection for refractive index changes. 0.12 Binding Regeneration BL 0.10 0.08 Intensity DI-H change 0.06 2O 0.04 pH 0.02 Break due to data acq. 0.00 DI-H2O 0 2000 4000 6000 8000 10000 Time (s) Fig. 6.11. The detected intensity in a real time binding experiment is plotted. The plot illustrates both binding and regeneration phases (BL: biotinylated lectin solu- tion, DI-H2O: deionized water, pH: pH-7 buffer). Binding efficacy is only measured between the signal levels when DI-H2O is flowed across the sensor. Intensity (V(rms)) Resonance Shift (pm) 98 M.S. Ünlü et al. 6.4 Conclusions We have presented three optical techniques aimed at molecular sensing that employ optical resonance. A planar optical resonant cavity is used in the case of RCIB, where the high finesse of the optical cavity can provide high sensitivity, while the planar configuration tied with a camera and tunable laser can provide for simultaneous detection from many parallel locations. The technique was demonstrated using nanometer scale etches in a SiO2 surface that model microarray features. We have also presented a second technique that observes the wavelength-dependent reflectivity from a silicon substrate with a thermally grown oxide layer to detect molecules binding to the top oxide surface. In this case, the Fresnel reflection measured can be considered to be the result of a low finesse optical cavity formed by the oxide layer together with the bound molecules. The benefit of this technique compared with the high finesse RCIB technique is that the monolithic structure avoids the difficulty maintaining the reflector alignment necessary in the latter technique. In the final technique presented, microring resonators employ a very strong resonant coupling to achieve very high sensitivity at the cost of added complexity and a larger minimum feature size. Acknowledgments The authors acknowledge Boston University Office of Technology Develop- ment for their support, the National Institute of General and Medical Sci- ences (NIH R21 GM 074872-02), and the Army Research Laboratory (ARL) for their support under the ARL Cooperative Agreement Number DAAD17- 99-2-0070 and under the AMCAC-RTP Cooperative Agreement DAAD19-00- 2-0004. The authors also thank Dr. Brent Little and the Little Optics Division of Nomadics Inc. for providing the microring resonator devices. The views and conclusions contained in this document are those of the authors and should not be interpreted as representing the Laboratory or the US Government. References 1. S. Niu, R.F. Saraf, Smart Mater. Struct. 11, 778 (2002) 2. R. Nadon, J. Shoemaker, Trends Genet.18, 265 (2002) 3. 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Ouyang 7.1 Photonic Crystals: A Short Introduction 7.1.1 Electromagnetic Theory Lord Rayleigh was the first to study electromagnetic wave propagation in one- dimensional (1D) periodic media and to identify the angle-dependent, narrow band in which light propagation is prohibited. However, it was not until a full century later, when Yablonovitch [1] and John [2] in 1987 combined Maxwell’s equations with solid-state physics theorems to introduce the concept of pho- tonic bandgaps in two and three dimensions. Many subsequent developments in fabrication, theory, and applications (e.g., fiber optics, integrated optics, and negative refraction materials) have since followed. As shown in Fig. 7.1, photonic crystals (PhCs) are periodically structured media [3] in one, two, or three dimensions. PhCs can be designed to produce photonic bandgaps. Light with photon energies or frequencies that fall inside this bandgap cannot propagate through the PhC. The periodicity in length scale is proportional to the wavelength of light inside the bandgap. PhCs are the electromagnetic analog of crystalline atomic lattices, in which interference in the electron wavefunction produces the forbidden bands. Hence, the study of PhCs is also governed by the Bloch-Floquet theorem. To study the propagation of light, Maxwell’s equations are solved as an eigenproblem. We assume that light propagates in a mixed dielectric medium with no free charges or currents. Maxwell’s equations are then given by: ∇ 1 H(r, t) = 0 ∇× ∂H(r, t) E(r, t) + = 0 c ∂t (7.1) ∇ε(r)E(r, t) = 0 ∇× ∂E(r, t) H(r, t) − ε(r) = 0 , c ∂t where ε(r) = εr+ai is the dielectric function, ai is the primitive lattice vector in all three dimensions, and c is the speed of light in vacuum. By combining 102 P.M. Fauchet et al. Fig. 7.1. 1D, 2D, and 3D photonic crystals. The periodicity is comparable to the wavelength of light. After Joannopoulos, [3] the source-free Faraday’s and Ampere’s laws at a fixed frequency ω, one can obtain a harmonic mode solution: H(r, t) = H(r)eiωt (7.2) E(r, t) = E(r)eiωt. The two curl equations can then be written as: ∇× iω E(r) + H(r) = 0 c (7.3) ∇× iω H(r) − ε(r)E(r) = 0 , c leading to the following (equation: ) 1 ( ) ∇× ∇× ω 2 H(r) = H(r). (7.4) ε(r) c By casting (7.4) as a periodic eigenproblem in analogy with Schrodinger’s equation, the solution can be obtained from the Bloch-Floquet theorem: H(r) = Hn,k(r)eik·x, (7.5) with eigenvalues ωn(k) that satisfy: ( ) ( ) (∇ + ik) × 1 2 (∇ ω + n ik) × (k) H k(r) = Hn,k(r). (7.6) ε(r) n, c Equation (7.6) yields a different Hermitian eigenproblem over the primi- tive cell at each Bloch wavevector k. These eigenvalues ωn(k) are continuous functions of k and can be plotted in a usual dispersion diagram. As the eigen- solutions are periodic functions of k, a numerical computational approach, such as the plane-wave expansion method, is often used to calculate the PhC bandgap diagram in reciprocal space. 7 Biodetection Using Silicon Photonic Crystal Microcavities 103 7.1.2 One-Dimensional and Two-Dimensional PhC Because Maxwell’s equations are scale-invariant, it is convenient to use nor- malized frequencies ω or wavelengths (a/λ), where a is the lattice constant and λ is the wavelength of light in vacuum. Thus, the same solutions can be applied to any wavelength simply by choosing the appropriate a. Figure 7.2 shows the dispersion curve in a 1D PhC if light propagation is restricted along the optical axis z. If the incident wave travels off-axis, the symmetry in the xy plane is destroyed, and the degeneracy of the bands is lifted. This angle of incidence dependent bandgap imposes practical constraints. Indeed, the take advantage of the 1D bandgap, the light beam must be well collimated and aligned along the z-direction. Let us now consider 2D PhC structures. In these structures, the electro- magnetic field can be decomposed into two polarizations. In the transverse electric (TE) mode, the E field is in plane, and the H field is perpendicu- lar to the plane. For the transverse magnetic (TM) mode, the situation is reversed. There are two common topologies for making 2D PhCs (Fig. 7.3): square lattice with high index rods surrounded by a low index medium and hexagonal lattice with low-index holes embedded in high index medium. The rod structure is better suited for TM light, and the holes are better suited for TE light. It is also desirable to have bandgaps that overlap in frequency in each symmetry direction of the crystal. Thus, geometries with a periodicity Fig. 7.2. Dispersion curve for a 1D PhC, for light propagating on the z-axis only (left) and for light propagating off axis (right). On the right, the thick curves labeled n = 1 correspond to the x-polarized band and the thin curves labeled n = 2 represent the yz-polarized band. When light propagates on axis, the bands are degenerate. After Joannopoulos, Ref. 3 104 P.M. Fauchet et al. a X M Γ a M K Γ Fig. 7.3. From left to right : Schematic of 2D PhC topography, Brillouin zone and 3D view of a PhC slab. The top row describes a square lattice of dielectric rods in air; the bottom row corresponds to a triangular lattice of air holes in a dielectric substrate Fig. 7.4. Projected band diagram for a finite-thickness slab of air holes in a dielectric slab. The shaded region indicates light cone; only modes below the light cone can propagate and the rest of the modes suffer from severe radiation loss. The “bandgap” below the light cone exists only for the even (TE-like) modes that is nearly the same in different directions are preferred, and in the case of 2D PhCs, this is an hexagonal lattice. It is impossible to fabricate a 2D PhC with an infinite slab thickness. An alternative solution is to use thin PhC slabs. The major feature that distinguishes the two systems is the light cone, which is a continuum of states indicated by a shaded region in Fig. 7.4. The modes in the light cone suffer from severe radiation loss, while states that lie beneath the light cone are guided modes. These guided modes are confined to the slab because of the high refractive index contrast between the slab material (e.g., Si) and the substrates (e.g., air or SiO2). The bandgap is then determined by the range 7 Biodetection Using Silicon Photonic Crystal Microcavities 105 Fig. 7.5. Gap size (as a fraction of the mid-gap frequency) versus Si slab (n = 3.45) thickness for PhC slabs with air hole radius to periodicity ratio r/a of 0.3 and 0.45, respectively. The optimum slab thickness is different for these two cases because of the difference in slab effective refractive index of frequencies for which no guided modes exist. For a slab surrounded by identical top and bottom layers, two categories of slab modes [even (TE-like) and odd (TM-like)] are then defined according to the reflection symmetry with respect to the horizontal plane of midthickness of the slab. The slab thickness plays an important role in determining whether a PhC slab has a bandgap. For example, if the slab is too thin (i.e., less than half a wavelength), the mode cannot be confined within the slab. As shown in Fig. 7.5, the photonic bandgap width depends on the slab thickness, and the optimal slab thickness varies with different r/a – or different effective slab refractive index. However, the bandgap width is not be the only consideration in selecting the slab thickness. With the microcavities to be discussed later, longer photon decay times or higher quality factors Q can be achieved by using slightly thicker slabs. 7.1.3 Microcavities: Breaking the Periodicity By intentionally introducing a defect in the PhC, localized electromagnetic states can arise inside the photonic bandgap. These localized modes are the optical analog of the donor or acceptor states produced inside the bandgap of semiconductor crystals. Figure 7.6 illustrates the electric field distribution of a fundamental mode in a PhC microcavity. Instead of staying inside the high refractive index Si, the electric field concentrates in lower refractive index air holes. This property makes it possible for the E-field to interact with the analyte infiltrated inside the air holes. 106 P.M. Fauchet et al. Fig. 7.6. Field confinement at the symmetry-breaking defect in a 2D PhC micro- cavity. The electric field is mostly confined in defect region 7.1.4 Computational Algorithms There are two standard computational approaches for studying PhCs: frequency-domain and time-domain methods. The plane-wave expansion al- gorithm belong to the frequency-domain group. It does a direct computation of the eigenstates and eigenvalues of Maxwell’s equations, and is thus better suited for calculating band diagrams. By solving for the eigenvalues, one can precisely predict eigenstates without missing very closely spaced modes, and each field is computed at a definite frequency. One of the disadvantages using this method is that all of the lowest eigenstates have to be computed before reaching the states of interest near the bandgap. It is especially problematic in calculating defect states, in which the lower bands are “folded” many times (depending on the array size) in the Brillouin zone. Moreover, frequency- domain approaches require a supercell with periodic boundaries. Hence, a large array size has to be chosen when computing a single-point-defect PhC microcavity because the interactions between repeated supercells must be minimized. In contrast, the FDTD (finite-domain-time-difference) method, which is a time-domain approach, computes the fields at each computational cycle at a definite time. |
This approach is well-suited to compute field distributions and resonance decay-times (quality factor Q). By finding transmission peaks in the Fourier transform of the system response to the input, resonant modes can be identified. One advantage of the FDTD methods is that by sending a pulse into the structure, the response can be obtained at all the frequencies at the same time. Ideally, FDTD methods can be used to calculate structures with arbitrary geometries.As the frequency resolution of the Fourier spectrum 7 Biodetection Using Silicon Photonic Crystal Microcavities 107 is inversely related to the simulation time, a long runtime is required to obtain an acceptable resolution, and the disadvantage in terms of computation time can be severe. 7.2 One-Dimensional PhC Biosensors 7.2.1 Preparation and Selected Properties of Porous Silicon One-dimensional PhC biosensors have been made using porous silicon (PSi). PSi is obtained by the electrochemical etching of a crystalline silicon wafer in a hydrofluoric acid (HF) solution [4]. The properties of PSi, including the thickness of the porous layer, its porosity, the average pore diameter, and the pore nanomorphology, can be controlled precisely. As a result, the optical properties of PSi can be tuned widely, which makes it a very flexible material for optical biosensors. The internal surface of PSi is very large, ranging from a few to hundreds of square meters per gram, which makes PSi very suitable for capturing biological targets. The dissolution of silicon requires the presence of fluorine ions (F−) and holes (h+). The pore initiation and growth mechanisms are qualitatively un- derstood. Pore growth can be explained by several models [5–7]. If the sili- con/electrolyte interface becomes rough shortly after etching starts, the sur- face fluctuations of the Si/electrolyte interface either grow (PSi formation) or disappear (electropolishing). In forward bias for p-type substrates, the holes can still reach the Si/electrolyte interface as the electric field lines are focused at the tip of the pores. Thus, holes preferentially reach the Si/electrolyte in- terface deep in the pores, where etching can proceed rapidly (Fig. 7.7). In contrast, no holes reach the end of the Si rods, effectively stopping the etch- ing there. In addition to this electrostatic effect, the random walk of the holes toward the Si/electrolyte interface makes it more likely that they reach it at or near the pore’s tip, also resulting in preferential etching at the pore’s tip. When an n-type substrate is used, porous silicon formation takes place in reverse bias. Another important mechanism becomes predominant if the Si rods are narrow enough (typically much less than 10 nm). In this size regime, the electronic states start to be affected by quantum confinement. When the motion of carriers is restricted in one or more dimensions, the hole states in the valence band are pushed to lower energy by quantum confinement, which produces a potential barrier to hole transport from the wafer to the Si rods. The holes can no longer drift or diffuse into the Si rods and further etching stops except at the pore’s tip. When the current density decreases, the number of holes at the pore tips drops, which produces smaller pores. Thus, the porosity (defined as the per- centage of void space in the material) can be precisely controlled by the etching current density. Figure 7.8 shows the dependence of the porosity on current 108 P.M. Fauchet et al. Fig. 7.7. The dissolution of silicon when holes are injected from the substrate toward the Si/electrolyte interface. On the left, porous silicon formation takes place when the hole flux is relatively small. On the right, electropolishing takes place when the hole flux is large Fig. 7.8. Dependence of the porosity of the current density for highly-doped n-type silicon 7 Biodetection Using Silicon Photonic Crystal Microcavities 109 Fig. 7.9. Top view and cross-sectional SEM images of mesoporous silicon with an average pore diameter of approximately 20 nm, formed in a highly doped p-type silicon substrate (0.01 ohm-cm), using an electrolyte with 15% HF in ethanol. (c, d): SEM images of 60 nm macropores formed in a very highly doped n-type silicon substrate (0.001 ohm-cm) using an electrolyte with 6% HF. (e, f): SEM images of 120 nm macropores formed in highly doped n-type silicon substrate (0.01 ohm- cm), using an electrolyte with 6% HF. (g, h): SEM images of 1.5 µm macropores etched from low doped p-type silicon (20 ohm-cm), using an HF/dimethylformamide electrolyte density for highly doped n-type (0.01 ohm-cm) silicon [8]. The pore morphol- ogy is also affected by the choice of doping type and concentration, as illus- trated in Fig. 7.9 [8]. The top view SEM images show PSi samples with differ- ent pore diameters ranging from mesopores (pore size between 10 and 50 nm) to macropores (pore size >50 nm). The bottom-row figures are cross-sectional SEM images of the same samples. The mesopores formed in p+ silicon sub- strates have very branchy pore walls (Fig. 7.9a, b). The macropores formed in n-type wafers (Fig. 7.9c–h) have much smoother pore walls and larger pore sizes. Note that most mesoporous silicon samples and certainly all microp- orous silicon samples (pore sizes <10 nm) exhibit strong luminescence in the visible to near infrared region [9]. This results from quantum confinement of electrons and holes in nanometer-sized quantum structures, which increases the bandgap [4,10,11] and enhances the radiative recombination rate [12]. 7.2.2 Sensing Principle PSi is a good host material for label-free optical biosensing applications be- cause its optical properties (photoluminescence and reflectance) are highly sensitive in the presence of chemical and biological species inside the pores [13]. PSi optical biosensors with a variety of configurations such as single layer Fabry-Perot cavities [14], Bragg mirrors [15], rugate filters [16], and micro- cavities [17–19] have been experimentally demonstrated for the detection of toxins [20], DNA [17], bacteria [21], and proteins [8, 14, 22]. The capture of 110 P.M. Fauchet et al. 1 a. 1 b. c. 0.8 0.8 0.6 0.6 0.4 0.4 0.2 0.2 0 0 Si Substrate 0.6 0.7 0.8 0.9 1 0.7 0.8 0.9 PSi Microcavity Wavelength (µm) Wavelength (µm) Fig. 7.10. (a) A 1D microcavity with a symmetry-breaking defect layer sandwiched between two Bragg reflectors; (b) The reflectance spectrum of such a PSi microcavity displays a sharp dip near the middle of the high-reflectance bandgap; (c) the pho- toluminescence of bulk PSi consists of a broad band ranging from <700 to 900 m, whereas the PL spectrum of the microcavity consists of a narrow peak centered on the reflectance dip biological or chemical molecules inside the pores increases the effective refrac- tive index of the PSi structures, thus resulting in a red shift in the photolu- minescence or reflectance spectra of 1D PSi PhCs. A PSi microcavity is a 1D photonic band gap structure that contains a defect (symmetry breaking) layer sandwiched between two Bragg mirrors [23]. Each Bragg mirror is a periodic stack of layers with two different porosities and quarter wavelength optical thickness. Figure 7.10 shows a PSi microcavity and its typical reflectance and photoluminescence (PL) spectra. Depending on the thickness of the defect layer, the reflectance spectrum contains one or several sharp resonance dips in the bandgap. The reflectance spectrum modulates the measured PL spectrum and produces one or several very narrow PL peaks. The position of the reflectance dip or photoluminescence peak is determined by the optical thickness (refractive index times the physical thickness) of each layer in the structure. A slight change of the refractive index inside the pores causes a shift of the spectrum. The effective dielectric constant of PSi is related to its porosity by the Bruggeman effective medium model [24]: (1 − εsi − εPSi ε P ) + void − εPSi P = 0, (7.7) εsi + 2εPSi εvoid + 2εPSi where P is the porosity, εPSi the effective dielectric constant of porous sili- con, εsithe dielectric constant of silicon, and εvoid the dielectric constant of the medium inside the pores. Equation (7.7) shows that the effective refrac- tive index of PSi (n2 eff = εPSi) increases as the porosity decreases and as εvoid increases. In Fig. 7.11, neff is plotted as a function of porosity [10]. In sensing applications, εvoid increases due to the binding of targets to the inter- nal surface of the pores. Thus, the overall effective dielectric constant of the porous structure εPSi increases, which causes a red shift in the reflectance dips. Reflectance PL (a.u.) 7 Biodetection Using Silicon Photonic Crystal Microcavities 111 4 n-void=1 3.5 n-void=1.3 3 2.5 2 1.5 1 0 20 40 60 80 100 Porosity (%) Fig. 7.11. Effective refractive index of porous silicon as a function of porosity for two values of the index of refraction of the pores Figure 7.11 shows that, for a given increase of εvoid, the effective refractive index change is larger for higher porosity layers. To selectively detect the targets of interest, the internal surface of PSi needs to be functionalized. Highly selective elements, such as DNA segments and antibodies, can be immobilized on the internal surface of the pores as the bioreceptors or probe molecules. When the sensors are exposed to the target, the probe molecules selectively capture the target molecules. The molecular recognition events are then converted into optical signals via the increase of the refractive index. 7.2.3 One-Dimensional Biosensor Design and Performance The figure of merit describing the sensitivity of affinity sensors is ∆λ/∆n, where ∆λ is the wavelength shift and ∆n is the change of the ambient re- fractive index. For a PSi microcavity sensor with an average porosity of 75%, ∆λ/∆nis ∼ 550 nm [25], which is much larger than for sensing platforms that rely on the interaction between the evanescent tail of the field and the analyte [26]. For a system able to detect a wavelength shift of 0.1 nm, the minimum detectable refractive index change of the pores is 2 × 10−4. The sensitivity of PSi microcavity sensors can be improved by increasing its quality factor Q (Q = λ/∆λ, where λ is the resonance center wavelength and ∆λ is the full width half maximum of the resonance dip). For higher Q-values, the spectral features are sharper and smaller shifts can be detected. The Q-factor can be increased by increasing the contrast in the porosity of the different layers. However, a higher contrast in porosity is usually produced by a higher contrast in pore opening, which may not be favorable for biosensing neff 112 P.M. Fauchet et al. applications when easy infiltration of the target throughout the entire mul- tilayer structure is required. For a given porosity contrast, the Q-factor can also be increased by increasing the number of periods of the Bragg mirror. In practice, the number of periods cannot be increased arbitrarily for two reasons. First, uniform infiltration of the molecules becomes more difficult in thicker devices. Second, maintaining a constant HF concentration at the tip of very deep pores is difficult [27], which may lead to an undesired porosity or refractive index gradient. PSi-based PhC sensors are not perturbed by large, unwanted bioparticles. When the PSi sensor is exposed to a complex biological mixture, only the molecules that are smaller than the pores can be infiltrated into the sensor. Furthermore, an increase of refractive index on the top of the microcavity due to the presence of large, unwanted objects only causes changes to the side lobes in the reflectivity spectrum, not to the resonance dip [28]. Thus, PSi microcavities are more reliable than planar sensing platforms, where the nonspecific binding of large size objects present in a “dirty” environment may produce a false-positive signal. The pore size also affects the sensitivity of PSi biosensors because the targets do not completely fill the pores but instead are attached to the pore walls. For a PSi layer of a given porosity, the internal surface area decreases as the pore size increases. The effective refractive index change of a layer with larger pores is thus smaller as the percentage of the pore volume occupied by the biological species is smaller. The spectra for microcavities with fixed porosities (e.g., 80% for the high porosity layer and 70% for the low porosity layer) but different pore diameters (ranging from 20 to 180 nm) have been calculated. For a given coating thickness (with nlayer = 1.42, a typical value for |
biomolecules), the resonance red shift decreases as the pore size increases, as shown in Fig. 7.12 [8]. For a 0.3 nm thick coating layer, a microcavity with 40 nm pores produces a red shift of 10 nm, while a microcavity with 100 nm pores produces a red shift of only 3 nm. Thus, to optimize the sensitivity, the pore size should be as small as possible while still allowing for easy infiltration of the biological material. Note that the amount of red shift can be used to precisely measure the amount of material captured inside the pores [29]. 7.2.4 Fabrication of One-Dimensional PhC Biosensors The porosity can be controlled by the etching current density. Once a layer has been etched, further etching using a different current density does not affect it, as explained in Sect. 2.1. Stacks of layers with different refractive indices can thus be formed by switching the current density during etch- ing [30]. Figure 7.13 shows a cross-sectional SEM image of a multilayer structure etched, using a periodic current density pulse train. The current den- sity determines the porosity and the pulse duration determines the thickness. Figure 7.14 shows top view and cross-sectional SEM images, and reflectance spectra for two different types of microcavities with different pore sizes. The 7 Biodetection Using Silicon Photonic Crystal Microcavities 113 Fig. 7.12. Calculated red shift of the reflectance dip vs. pore diameter for layers of fixed porosities (70 and 80%) and various thicknesses L of the coating on the pore walls Fig. 7.13. Three low-porosity layers and two high-porosity layers etched in silicon using a periodic current density. Note the continuity of the pores and the sharpness of the interfaces between high and low porosity layers. The period is approximately 1 µm mesoporous silicon microcavity with pore diameters from 10 to 50 nm was fabricated on a p+ wafer (0.01 ohm-cm) with 15% HF in ethanol. The macro- porous silicon microcavity with pore diameters from 80 to 150 nm was etched in an n+ wafer (0.01 ohm-cm) with 5.5% HF in DI water [10]. 114 P.M. Fauchet et al. a b c 200 nm 2 µm Wavelength (nm) d e f 200 nm 2 µm Wavelength (nm) Fig. 7.14. Top view and cross-sectional SEM, and reflectance spectra of PSi mi- crocavities made of mesoporous Si (top) and macroporous silicon (bottom) 7.3 Selected Biosensing Results 7.3.1 DNA Detection Mesoporous silicon microcavities were used for the detection of small DNA segements (22 base pairs) [17, 31]. Multiple photoluminescence peaks as nar- row as 3 nm of full width half maximum (FWHM) were measured from a microcavity with a thick defect layer. The first step in the sensor prepara- tion involved the silanization of the thermally oxidized porous silicon sample with 3-glycidoxypropyltrimethoxy silane, which was hydrolyzed to a reactive silanol by using DI water (pH∼4). The thermal oxidation treatment not only produced a silica-like surface, but also improved the stability of the lumines- cence. After silanization, the probe DNA, which has an amine group attached to the 3′ end of the sequence, was immobilized onto the pore surface via dif- fusion. The amine group attacks the epoxy ring of the silane, thereby opening it up and forming a bond. Finally the DNA attached porous silicon sensor was exposed to the complementary strand of DNA (cDNA). By comparing the photoluminescence spectrum of the sensor before and after the DNA hy- bridization, the DNA/cDNA binding was detected, as shown in Fig. 7.15 [31]. The detection limit of the sensor was determined to be in the picomolar range. 7.3.2 Bacteria Detection Gram(-) bacteria are responsible for a large numbers of deaths resulting from infection. To detect Gram(-) bacteria, we selected a target molecule present in this bacterial subclass and not in Gram(+) bacteria. Lipopolysacharide (LPS) is a primary constituent of the outer cellular membrane of Gram(-) bacteria [32]. LPS is composed of three parts: a variable polysaccharide chain, Reflectance (%) Reflectance (%) 7 Biodetection Using Silicon Photonic Crystal Microcavities 115 (a) PSi / DNA (b) PSi / DNA/ cDNA (c) Differential Signal 600 650 700 750 800 850 900 Wavelength (nm) Fig. 7.15. Photoluminescence spectra of a mesoporous silicon microcavity with a thick defect layer supporting six clearly resolved reflectance dips (PL peaks). (a) After functionalization of the pore surface with probe DNA segments; (b) after capture of complementary DNA segments; (c) differential spectrum a core sugar, and lipid A. As lipid A is common to all LPS subtypes, it is a natural target. An organic receptor, tetratryptophan ter-cyclo pentane (TWTCP) was designed and synthesized as the probe molecule. TWTCP specifically binds to diphosphoryl lipid A in water with a dissociation constant of 592 nM [59]. After the microcavity sensor was exposed to 3-glycidoxypropyltrimethoxy silane, a mixture of TWTCP and glycine methyl ester was applied to the sen- sor. Glycine methyl ester was used as a blocker molecule to avoid the reaction of the four amino groups of the tetratryptophan receptor with the epoxide- terminated PSi surface. As shown in Fig. 7.16, upon exposure of lysed Gram(-) cells (Escherichia coli) to the immobilized TWTCP sensor, a 4 nm red shift of the photoluminescence spectrum of the microcavity was detected [21]. How- ever, when the sensor was exposed to a solution of lysed Gram(+) cells (Bacil- lus subtilis), no shift of the spectrum was observed. These results were con- firmed with all Gram(-) and Gram(+) bacteria tested. 7.3.3 Protein Detection A protein biosensor based on macroporous silicon microcavity was demon- strated with the streptavidin-biotin couple [8]. Biotin is a small molecule, while streptavidin is relatively large (67 kDa), making its infiltration difficult into Normalized PL Intensity (a.u.) 116 P.M. Fauchet et al. 1 1 0.9 0.9 0.8 0.8 0.7 0.7 0.6 0.6 0.5 0.5 0.4 0.4 0.3 0.3 0.2 0.2 0.1 0.1 0 0 675 775 875 675 775 875 Wavelength (nm) Wavelength (nm) 0.6 0.6 0.4 0.4 0.2 0.2 0 0 −0.2 −0.2 −0.4 −0.4 −0.6 −0.6 675 775 875 675 775 875 Wavelength (nm) Wavelength (nm) Fig. 7.16. Photoluminescence spectra of a mesoporous silicon biosensor similar to the one shown in Fig. 7.15 before and after exposure to bacterial cell lysates (top row) and differential spectra (bottom row). The spectra on the left have been obtained for bacteria that are Gram(+) and the spectra on the right for bacteria that are Gram(-) mesopores but easy into macropores. Furthermore, each streptavidin tetramer has four equivalent sites for biotin (two on each side of the complex), which makes it a useful molecular linker. To create a biotin-functionalized sensor for the capture of streptavidin, microcavities were first thermally oxidized, then silanized with aminopropy- ltriethoxysilane to create amino groups on the internal surface. The probe molecules, Sulfo-NHS-LC-LC-biotin, were then immobilized inside the pores. As shown in Fig. 7.17a, the red shift of the resonance increased with increas- ing biotin concentration. A 10 nm red shift corresponds to a nearly complete (95%) biotin surface coverage. Intensity Intensity Intensity Intensity 7 Biodetection Using Silicon Photonic Crystal Microcavities 117 14 100 20 (a) (b) (c) 12 80 16 10 8 60 12 6 40 8 4 20 4 2 0 0 0 0 0.4 0.8 1.2 1.6 2 600 650 700 750 800 850 900 0 20 40 60 80 100 Biotin concentration (mg/ml) Wavelength (nm) Biotin surface coverage (%) Fig. 7.17. (a) Increase of the reflectance dip red shift when more probe molecules (biotin) are added; the shift eventually saturates when all the pore walls are covered by biotin. (b) After exposure to target molecules (streptavidin), the microcavity spectrum shifts to the red. (c) The measured red shift upon capture of the targets depends on the coverage of the pore walls by the probe molecules To study how the biotin surface coverage affects the binding to strepta- vidin, sensors derivatized with different biotin concentrations were exposed to the same amount of the target: 50 ml of streptavidin with a concentration of 1 mg ml−1. After exposure to streptavidin, a red shift was detected, which is at- tributed to the specific binding of streptavidin to the biotin-derivatized macro- porous microcavity (Fig. 7.17b). For the control samples that were silanized but did not contain biotin, no shift was detected after exposure to strepta- vidin, which indicates that nonspecific absorption of streptavidin inside the microcavity is nonexistent or negligible. The red shift caused by streptavidin binding to biotin is a function of the biotin surface coverage. Figure 7.17c shows that there is an optimum biotin surface coverage that maximizes the red shift, and therefore the capture of streptavidin. This behavior is in good agreement with observations by other researchers using different sensing techniques [34]. The spacing between each neighboring biotin needs to be large enough so that biotin can reach the pocket-like binding site of streptavidin. The roll-off of the red shift showing in Fig. 7.17c is due to the fact that when the biotin surface density becomes very high and each biotin is closely surrounded by its neighbors, biotin cannot protrude deep enough into the binding site of streptavidin, thus decreasing the probability of capturing streptavidin. Other proteins have also been detected using 1D PhC microcavities made of PSi, including intimin, a protein secreted by pathogenic E. coli [22]. 7.3.4 IgG Detection Immunoglobulin (IgG) is the most common type of antibody synthesized in response to a foreign substance (antigen). Antibodies have a specific molecular structure capable of recognizing a complementary molecular structure on the antigen, such as proteins, polysaccharides, and nucleic acids. From the X-ray crystal structure, the longest dimension of IgG is approximately 17 nm [35]. ∆λ (nm) Reflectivity (%) ∆λ (nm) 118 P.M. Fauchet et al. Fig. 7.18. A PSi microcavity biosensor functionalized for the capture of one type of IgG can discriminate against another type of IgG with a contrast ratio 10× The detection of rabbit IgG (150 kDa) was investigated through multiple lay- ers of biomolecular interactions in a macroporous silicon microcavity sensor. The silanized sensor was first derivatized with biotin, which can selectively capture streptavidin. Exposure of biotinylated goat anti-rabbit IgG to the sensor results in its attachment to the surface through the binding between biotin and streptavidin. The sensor used goat anti-rabbit IgG as the probe molecule to selectively capture rabbit IgG. A red shift of the spectrum can be detected when each layer of molecules is added to the sensor. As shown in Fig. 7.18 when the sensor was exposed to 50 ml of a solution containing rabbit IgG at a 1 mg ml−1 concentration, a 6 nm red shift was detected [25]. When the sensor was exposed to 50 ml goat IgG (1 mg ml−1), which does not bind to the goat anti-rabbit IgG, the red shift was negligible (<0.5 nm). 7.4 Two-Dimensional PhC Biosensors 7.4.1 Sample Preparation and Measurement Silicon-on-insulator (SOI) wafers were used to fabricate 2D PhC microcavity biosensors [36]. The Si slab thickness was approximately 400 nm. Rigorous 3D simulations using the FDTD method were performed prior to the fabrication. The incident light was coupled along the Γ -M direction because the in-plane leakage of the resonance mode is mainly in the Γ -M direction and hence the coupling efficiency is higher along the Γ -K direction. The ridge waveguide was tapered down from 2 to 0.6 µm with an external surface cross-section of 2 µm× 400 nm, which predominantly select fundamental TE mode as the propagation mode. 7 Biodetection Using Silicon Photonic Crystal Microcavities 119 Fig. 7.19. Fabrication flow starting from a silicon on insulator (SOI) wafer and ending with the 2D PhC microcavity inserted in a silicon wire waveguide Fig. 7.20. Experimental setup for measuring the transmission of a 2D PhC micro- cavity. A microscope objective is used to observe the alignment of all the components The fabrication procedure is depicted in Fig. 7.19. After the SOI wafer was cleaned and an oxide mask grown, negative (PMMA) and positive (FOX) resists were used for pattern transformation. After electron-beam lithography and resist development, reactive ion etching was performed to transfer the pattern onto the Si slab. The ridge waveguide facets were then polished to improve the coupling efficiency. Figure 7.20 illustrates the measurement setup. An HP8168F laser, tunable from 1,440 to 1,590 nm, was used as the light source. The TE polarized laser beam was focused onto the input |
tapered ridge waveguide (from 2 µm to 0.6 µm) using a tapered lensed fiber (with a spot size of 2 µm at focus), and the transmitted signal was measured by a photodetector. To optimize the intensity of the TE polarized-light, a polarization controller was inserted between the laser and the input fiber. 7.4.2 Sensing Principle The sensing principle is very similar to the one used in the 1D PhC biosen- sors. In contrast to chemical sensors [37], in biological sensors, the biomolecule 120 P.M. Fauchet et al. Fig. 7.21. After functionalization of the air hole surfaces with probe molecules (left), targets are captured by the biosensor (middle), which produces a coating on the air hole walls (right) recognition strongly depends on the surface chemistry, thus, instead of filling up the pores, the molecules prefer to form a monolayer coating at the pore (air hole) wall As shown in Fig. 7.21, highly selective probes (e.g., DNA, an- tibody) can be immobilized on the internal surface of the air holes and form a monolayer that can capture specific target molecules (e.g., matching DNA, proteins). Hence, the coating causes a refractive index change only in the vicinity of the pore wall. 7.4.3 Selected Biosensing Results Protein Detection To test the device performance, glutaraldehyde-bovine serum albumin (BSA) coupling was used as the model system. The air hole is ∼30 times larger than the BSA hydrodynamic diameter [38], so that the infiltration proceeds easily, and both glutaraldehyde and BSA can form a uniform monolayer coated on the pore walls. To prepare the surface for capturing biomolecules, the device was first ther- mally oxidized at 800◦C to form a silica-like interface for binding of amino groups. To functionalize the sensor and capture glutaraldehyde, the microcav- ity internal surface was silanized. Subsequently, the target, BSA, was immo- bilized covalently on the sidewall. The experiment protocol is as follows: (1) clean the sensor surface with DI water and dry it under nitrogen flow. Store the sensor in moist ambient; (2) drop 5 µl of 2% aminopropyltrimethoxysilane on the sensor for 20 min; (3) rinse the sensor with DI water and dry with nitrogen flow, and then bake at 100◦C for 10 min; (4) drop 5 µl of 2.5% glu- taraldehyde in Hepes buffer on the sensor for 30 min; soak in buffer for 10 min to dilute the unreacted agents and dry it under nitrogen flow; (5) drop 5 µl of 1% BSA on the sensor for 30 min; then soak in buffer for 20 min and dry it with nitrogen. 7 Biodetection Using Silicon Photonic Crystal Microcavities 121 Fig. 7.22. Transmission near the 2D PhC microcavity resonance for the function- alized sensor (a), after exposure to the probe, glutaraldehyde (b), and subsequent exposure to the target, bovine serum albumin Figure 7.22 illustrates the normalized transmission spectra measured at three different binding stages. The PhC microcavity consisted of a triangular array of cylindrical air pores in a 400 nm-thick silicon (Si) slab. The lattice constant a was 465 nm, and the pore diameter r was 0.3a. A defect was intro- duced by reducing the center pore diameter to 0.18a, leading to a transmis- sion resonance in the bandgap close to 1.58 µm. Curve (a) shows the initial transmission after the oxidation and silanization. Curve (b) was measured af- ter exposure to glutaraldehyde. A resonance red shift of 1.1 nm is observed. Curve (c) shows a red shift of 1.7 nm after BSA binding corresponding to a total shift of 2.8 nm with respect to the initial spectrum. To verify the exper- imental results, a plane-wave expansion calculation with 32 grid points per supercell was performed. The simulation of the resonance red shift assumes that the refractive index of the dehydrated proteins is 1.45. This value agrees with an ellipsometry measurement [29] and with the literature values [39]. Figure 7.23 plots the predicted resonance red shift vs. coating thickness. Using ellipsometry, the thickness of the dehydrated glutaraldehyde monolayer was measured as 7± 1 Å. We then calculated that glutaraldehyde should intro- duce a resonance red shift of 0.98± 0.2 nm, which is in good agreement with the experimental data. The thickness of a dehydrated BSA layer measured with the same method is approximately 15± 5 Å, which should introduce an additional red shift of 2.7± 1 nm or a total red shift of 3.8± 1 nm. The experimental data are again in good agreement with the model. 122 P.M. Fauchet et al. 8 6 4 2 0 0 10 20 30 40 50 Coating thickness t (Å) Fig. 7.23. Calculated red shift vs. coating thickness of the air hole walls for the device structure tested in Fig. 7.22 The biomolecules can also attach to the sidewall, the bottom, or the top of the device. 3D simulations (Fig. 7.24) using FDTD methods were carried to simulate the molecule-position dependence. Within the molecule size range of interests (i.e. <50 nm), the coating on the pore wall contributes the most to the spectral shift, whereas the presence of biomolecules elsewhere produces a much smaller shift. This is not surprising, as the mode is strongly confined in the Si slab and on the air hole sidewalls, the interaction of the mode and the analyte inside the device is much stronger than on top of the device. Toward Single Molecule Detection As the electric field is strongly localized in the defect region, what would happen if the biomolecules were present only in the defect region? [40] The sensitivity ∆λ/∆t decreases by a factor of 4, but the total amount of ana- lyte that is detectable drops by nearly two orders of magnitude, from 2.5 pg to 0.05 pg. To demonstrate the potential for single bioparticle detection, a microcavity with a large defect (diameter ∼900 nm) was fabricated. Latex spheres (density of 1.05 g cm−3, refractive index n = 1.59) with a diameter of approximately 370 nm were dropped on the PhC. As shown in Fig. 7.25, Redshift ∆λ (nm) 7 Biodetection Using Silicon Photonic Crystal Microcavities 123 100 Si 80 Capable of detecting amount of analyte ~ 2.5 fg 60 40 20 0 0 10 20 30 40 50 60 70 Coating thickness ∆t (nm) Fig. 7.24. Calculated resonance red shift vs coating thickness, for a biomolecule layer on the side walls of the air holes, at the bottom of the air holes, or on top of the device Fig. 7.25. Close up of near the central defect of a 2D PhC microcavity in which one latex microsphere has been captured one latex microsphere was inside the defect region and the rest of the spheres remained on top of the device. The normalized transmission spectra of the PhC microcavity shown in Fig. 7.26 demonstrate that the device can detect a single bioparticle. For a device with a quality factor Q of ∼2,000, a spectral shift of 1 nm can be measured very easily. Simulations show that biomolecules smaller than 50 nm can be detected. Resonance shift ∆λ (nm) 124 P.M. Fauchet et al. Fig. 7.26. Transmission near the resonance of the 2D PhC microcavity when the defect air hole contains one latex microsphere (curve b) or no microsphere (curve a). The microsphere’s diameter was 370 nm 7.5 Conclusions PhC microcavities made of silicon are a platform of choice for biosensing. After proper functionalization, both 1D and 2D devices have excellent per- formance in terms of sensitivity and selectivity. It is important to note that a complete biosensor system requires other components (such as prefiltration and concentration modules [41]) that have not been described here but can also be made in silicon and integrated with the PhC microcavity. Acknowledgments The work described in this chapter was supported by the US National Sci- ence Foundation through grant BES 04279191. Fabrication of the 2D PhC microcavities was performed in part at the Cornell NanoScale Facility, which is supported by NSF through grant ECS 03-35765. The technical contribu- tions of Dr. Selena Chan, Dr. Marc Christophersen, and Dr. Chris Striemer are gratefully acknowledged. References 1. E. Yablonovitch, Phys. Rev. Lett., 58, 2059 (1987) 7 Biodetection Using Silicon Photonic Crystal Microcavities 125 2. S. John, Phys. Rev. Lett., 58, 2486 (1987) 3. J.D. Joannopoulos, R.D. Meade, J.N. Winn, in Photonic Crystals: Molding the Flow of Light, (Princeton Press, 1995) 4. P. M. Fauchet, in Encyclopedia of Applied Physics, Update 2, (Wiley-VCH Verlag, 1999), pp. 249–272 5. R.L. Smith, S.D. Collins, J. Appl. Phys. 71, R1 (1992) 6. A.G. Cullis, L.T. Canham, P.D.J. Calcott, J. Appl. Phys. 82, 909 (1997) 7. V. 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Striemer, T.R. Gaborksi, J.L. McGrath, P.M. Fauchet, Nature 445, 749 (2007) 8 Optical Coherence Tomography with Applications in Cancer Imaging S.A. Boppart 8.1 Introduction Optical coherence tomography (OCT) is a rapidly emerging optical imag- ing technique for a wide range of biological, medical, and material investiga- tions [1,2]. OCT was initially developed in the early 1990s, and has provided researchers with a novel means by which biological specimens and nonbiolog- ical samples can be visualized. A primary advantage of OCT is the ability to image tissue microstructure in situ at micron-scale image resolution, without the need for excision of a specimen for tissue processing. The optical ranging in OCT is analogous to ultrasound B-mode imaging, except that OCT |
uses low-coherence light rather than high-frequency sound. Cross-sectional OCT images can be generated, as is commonly done in ultrasound, or en face OCT sections can be acquired, as in confocal and multiphoton microscopy. The OCT imaging principle involves optical ranging, where the optical reflection of light from a low-coherence optical source is spatially localized using in- terferometry. The OCT image that is assembled is a gray-scale or false-color multidimensional spatial representation of backscattered light intensity. The signal intensity represented within an OCT image represents the differential backscattering contrast between different tissue types on a micron scale. OCT performs imaging using light; therefore, it has a one to two order-of-magnitude higher spatial resolution than ultrasound. Because the optical imaging beam can be transmitted readily through air, OCT beam-delivery systems do not require contact with the specimen or sample, as do ultrasound probes. Spectro- scopic characterization of tissue and cellular structures is also possible within the optical spectrum of the light source. Ophthalmology was the first medical application area for OCT, where high-resolution tomographic imaging of the anterior eye and retina is possi- ble [3]. The transparency of the eye at visible and near-infrared wavelengths, and its accessibility using optical instruments and techniques, has enabled extensive research and clinical applications for diagnostic OCT imaging. The 128 S.A. Boppart diagnosis of many retinal diseases is possible because OCT can provide images of retinal pathology with micron-scale resolution. OCT has also been applied in a wide range of nontransparent tissues [4,5]. In nontransparent tissues such as skin, muscle, and other soft tissues, the imaging depth is limited by optical attenuation due to scattering and ab- sorption. Optical scattering decreases with increasing wavelength. Therefore, while ophthalmic OCT imaging has primarily been performed at 800 nm wavelengths, OCT imaging in nontransparent tissues has been typically per- formed with wavelengths of 1.0–1.3 µm. Imaging depths up to 2–3 mm can be achieved using a system detection sensitivity of 100–110 dB. In early ex- ploration imaging studies, OCT has been performed in virtually every organ system to investigate applications in cardiology [6–8], gastroenterology [9,10], urology [11, 12], neurosurgery [13], and dentistry [14], to name a few. Us- ing short coherence length, short-pulsed light sources, high-resolution OCT has been demonstrated with axial resolutions of less than 5 µm [15–17]. High- speed real-time image acquisition rates have also been achieved, which enables volumetric imaging [18, 19]. New imaging modes in OCT have been demon- strated, such as Doppler OCT imaging of blood flow [20–22] and birefringence imaging to investigate laser intervention [23–25]. OCT beam delivery systems including transverse imaging catheter/endoscopes and forward imaging de- vices have enabled OCT imaging of internal structures [26–28], and most recently, catheter-based OCT has been used to perform in vivo imaging in animal models and human patients [29–33]. 8.2 Principles of Operation OCT performs optical ranging in tissue, using high spatial resolution and high dynamic range detection of backscattered light as a function of optical delay. While the speed of ultrasound waves in tissue is relatively slow and detectable using electronics, the velocity of light is extremely high. There- fore, the time delays of the reflected light cannot be measured directly and interferometric detection techniques must be employed. Low-coherence inter- ferometry or optical coherence domain reflectometry are techniques that are used in OCT. First developed in the telecommunications field for measuring optical reflections from faults or splices in optical fibers [34], low-coherence in- terferometry can also be used for localizing the optical reflections in biological tissue. Subsequently, the first applications in biological samples included one- dimensional optical ranging in the eye to determine the location of different ocular structures [35,36]. Time delays of reflected light-off of tissue boundaries are typically mea- sured using a Michelson-type interferometer (Fig. 8.1). Other interferometer designs, such as a Mach–Zehnder interferometer, have been implemented to optimize the delivery of the OCT beam and the collection of the reflected signals [37,38]. Light reflected from the specimen or sample is interfered with 8 OCT with Applications in Cancer Imaging 129 Fig. 8.1. Schematic illustrating the concept of low coherence interferometry. Using a short coherence length light source and a Michelson-type interferometer, interference fringes are observed only when the path lengths of the two interferometer arms are matched to within the coherence length of the light source in a time-domain OCT system. The full-width half-maximum of the envelope of the autocorrelation function is equal to the coherence length (∆lc) and axial resolution in OCT. This envelope also represents the axial point-spread-function of an OCT system light reflected from a reference path of known path length, which spatially determines the location of the reflection in depth. Interference of the light between the two arms of the interferometer can occur only when the optical path lengths of the two arms match within the coherence length (axial resolu- tion) of the optical source. If the reference arm optical path length is scanned, as in a time-domain OCT system, different delays of backscattered light from within the sample are measured, and a single column of depth-dependent data is collected. The interference signal is detected at the output port of the in- terferometer, digitized, and stored on a computer. Following a depth (z) scan, the incident beam is scanned in the transverse direction (x) and multiple ax- ial measurements are performed. A two-dimensional data array is generated, which represents the optical backscattering through a cross-sectional plane in the specimen (Fig. 8.2). Similarly, the OCT beam can be translated in the third (y) dimension, and a series of two-dimensional cross-sectional images can be collected to form a three-dimensional volume. The logarithm of the backscatter intensity is then mapped to a false-color or gray-scale and dis- played as an OCT image. Typically, the interferometer in an OCT instrument can be implemented using fiber optic couplers, and beam-scanning can be 130 S.A. Boppart Fig. 8.2. An OCT image is based on the spatial localization of variations in op- tical backscatter from within a specimen. For depth-priority scanning, images are acquired by performing axial measurements of optical backscatter at different trans- verse positions on the specimen and displaying the resulting two-dimensional data set as a gray-scale or false-color image. OCT images can also be acquired with transverse-priority scanning to collect an en face image similar to optical section- ing in confocal or multiphoton microscopy. Multiple planes in either mode can be assembled for three-dimensional OCT performed with small mechanical galvanometers or another beam-delivery in- strument to yield a compact and robust system (Fig. 8.3). The axial resolution in OCT images is determined by the coherence length of the light source, in contrast to the tight focus and confocal region in confocal microscopy. The axial point spread function of the OCT measurement is defined by the sig- nal detected at the output of the interferometer (detector arm), and is the electric-field autocorrelation of the source. The coherence length of the light is the spatial width of the field autocorrelation, and the envelope of the field autocorrelation is equivalent to the inverse Fourier transform of the optical source power spectrum. The width of the autocorrelation function (axial res- olution), therefore, is inversely proportional to the width of the optical power spectrum. If the optical source has a Gaussian spectral distribution, then the free-space axial resolution ∆z is given by 2 ln 2 λ2 ∆z = , (8.1) π ∆λ where ∆z and ∆λ are the full widths at half maximum of the autocorrelation function and power spectrum, respectively, and λ is the central wavelength of 8 OCT with Applications in Cancer Imaging 131 Fig. 8.3. Schematic representation of an OCT system implemented using fiber op- tics. The Michelson interferometer is implemented using a fiber-optic coupler. Light from the low-coherence source is split and sent to a sample arm with a beam deliv- ery instrument and a reference arm with an optical path-length scanner. Reflections from the arms are combined and the output of the interferometer is detected with either a photodiode or a linear CCD array in a spectrometer. Components for both a time-domain and a spectral-domain OCT system are shown the source. The dependence of the axial resolution on the bandwidth of the optical source is plotted in Fig. 8.4. To achieve high axial resolution (approach- ing 1 µm), therefore, requires extremely broad bandwidth optical sources. The curves plotted in Fig. 8.4 are for three commonly used wavelengths, 800, 1,300, and 1,500 nm. From (8.1), higher resolutions can be achieved with shorter wavelengths. However, shorter wavelengths are more highly scattered in bio- logical tissue, and frequently result in less imaging penetration. Evident from this discussion, the axial and transverse resolutions in OCT are not directly related, as in conventional microscopy. However, the transverse resolution in an OCT imaging system is determined by the focused spot size, according to the principles of Gaussian optics, and is given by 4λ f ∆x = , (8.2) π d where d is the beam diameter on the objective lens and f is the focal length of the objective lens. Large numerical aperture optics can be used to fo- cus the beam to a small spot size and provide high transverse resolution. 132 S.A. Boppart 30 25 20 15 10 5 1500 nm 1300 nm 800 nm 0 0 50 100 150 200 250 300 350 Source Spectral Bandwidth (nm) Fig. 8.4. Dependence of coherence length (axial resolution) on optical source band- width. Curves are plotted for 800, 1,300, and 1,500 nm, three common wavelengths used in OCT. High axial imaging resolution is achieved with broad spectral band- widths and shorter wavelengths. Shorter wavelengths, however, are more highly ab- sorbed in biological tissue, decreasing imaging penetration depth The transverse resolution is also related to the depth of focus or the confocal parameter 2zR (two times the Raleigh range). π∆x2 2zR = . (8.3) 2λ Increasing the transverse resolution (decreasing the spot size at the focus) subsequently results in a reduced depth of field. For OCT imaging, the con- focal parameter or depth of focus is typically chosen to match the desired depth of imaging. High transverse resolutions are often required and may be utilized in OCT. However, the short depth of field requires additional optical or mechanical techniques to spatially track the focus in depth along with the axial OCT scanning. Relatively small incident powers of 1–10 mW are commonly required for OCT imaging. Typically, the real-time (∼30 frames per second) acquisition can be achieved at a signal-to-noise ratio of ∼100 dB with 5–10 mW of incident optical power. Recent advances in spectral-domain OCT (SD-OCT) [39, 40] and swept-source OCT (SS-OCT) [41, 42] have enabled extremely fast axial scanning and real-time volumetric imaging at micron-scale resolution, while maintaining sufficiently high SNR for imaging larger volumes of tissue. Coherence Length (mm) 8 OCT with Applications in Cancer Imaging 133 8.3 Optical Sources for Optical Coherence Tomography OCT imaging systems have primarily used superluminescent diodes (SLDs) as low coherence light sources [43]; however, new sources are becoming widely available. SLDs are attractive because they are compact, have high efficiency, low noise, and are commercially available at a range of wavelengths including 800 nm, 1.3 µm, and 1.5 µm. Output powers, however, are typically less than a few milliwatts, which frequently limits the use of these sources to slow acquisi- tion rates in order to preserve signal-to-noise ratio. The available bandwidths are relatively narrow, permitting imaging with 10–15 µm resolution. Because of the need for broader bandwidth sources, manufacturers have been devel- oping SLD sources that have bandwidths of 50–100 nm (3–10 µm resolution, depending on wavelength). Advances in short-pulse, solid-state, laser technology make these sources attractive for OCT imaging in research applications. Femtosecond solid-state lasers can generate tunable, low-coherence light at powers sufficient to per- mit high-speed OCT imaging [15–17]. The titanium:sapphire (Ti:Al2O3) laser from 0.7 to 1.1 µm has been a commonly used source, not only for OCT, but most often for multiphoton microscopy. Imaging resolutions of less than 1 µm have been demonstrated at 800 nm using a Ti:Al2O3 laser source [17]. To demonstrate the improvement in resolution afforded by a titanium:sapphire laser, a comparison between the power spectra and autocorrelation func- tions of a 800 nm center wavelength SLD and a short pulse (≈5.5 fs) tita- nium:sapphire laser is shown in Fig. 8.5. An order-of-magnitude |
improvement in axial resolution is noted for the short pulse titanium:sapphire laser source [16]. In an effort to develop more compact and convenient sources compared to a titanium:sapphire laser, superluminescent fiber sources have been inves- tigated [44, 45] as well as pumped optical fibers that can produce extremely broad supercontinuum [46–48]. Since the titanium:sapphire (Ti:Al2O3) laser technology is routinely used in multiphoton microscopy applications for its high peak intensities to enable multiphoton absorption and subsequent emis- sion of fluorescence from exogenous fluorescent contrast agents [49], the si- multaneous generation of OCT and multiphoton microscopy images has been demonstrated, providing complementary image data using a single optical source [50]. 8.4 Fourier-Domain Optical Coherence Tomography OCT has most-commonly been performed by rapidly scanning the reference arm path-length to acquire a single scan in depth. This mode has been termed time-domain OCT (TD-OCT). In this mode, faster image acquisition rates were therefore dependent on the rate at which this optical path-length could be varied, and rapid-scanning optical delay lines were previously developed for 134 S.A. Boppart Fig. 8.5. Comparison of optical output spectra (top) with interference signals (lower left) and envelopes (lower right) for a Kerr-lens modelocked titanium:sapphire (Ti:Al2O3) laser vs. a superluminescent diode (SLD). The broad optical bandwidth of the titanium:sapphire laser (260 nm) permits a free-space axial resolution of 1.5 µm. In comparison, the superluminescent diode with a 32 nm spectral bandwidth permits an axial resolution of 11.5 µm. Figure reprinted with permission [16] this purpose. In the mid 1990s, it was realized, and in recent years, demon- strated that alternatives to this time-domain mode of scanning were tech- niques that simultaneously collected light from all depths into the tissue as the beam was positioned at each transverse position. The light sources for these techniques are either a broadband source in which the collected light is detected by a linear CCD array in a spectrometer (spectral-domain OCT, SD- OCT) [39,40], or a narrow-band source which is rapidly swept over a range of frequencies and the collected light is detected by a photodiode (swept-source OCT, SS-OCT) [41, 42]. For both of these techniques, depth-dependent scat- tering information is obtained by taking the inverse Fourier transform of this acquired spectral data. Recently, these techniques have become preferred for the improvement in acquisition rate and sensitivity that they provide over the time-domain OCT systems. Recent use of the SS-OCT has demonstrated real-time acquisition rates. The primary advantages to using a swept-source is that all the optical power is contained within a narrow wavelength range at any one point in time, and 8 OCT with Applications in Cancer Imaging 135 then is swept to a different wavelength. Wide tuning ranges make high imaging resolution possible and at the rate of hundreds of thousands of axial scans per second, fast volumetric imaging can be performed. The development of fast swept sources has utilized innovative methods, including a rotating polygonal mirror array [41] or Fourier-domain mode-locking [42]. High-speed SD-OCT has been demonstrated in the living human eye and in other real-time applications. Because the acquisition rate is no longer de- pendent on a mechanically scanned reference arm, significantly faster rates are achievable, up to typical rates of 30,000 axial scans per second, depending on the read-out rates of the linear CCD arrays in the spectrometer. The computa- tional complexity has been increased for both SD-OCT and SS-OCT because the inverse Fourier transform of the acquired data is required for each axial scan line. Because dedicated digital-signal-processing (DSP) chips are readily available, it appears that the computational power necessary for this appli- cation is feasible and available to the research investigator. Spectral-domain OCT also has the advantages of improved phase stability, because no mechan- ical scanning is performed, and higher signal-to-noise ratio, because individual wavelengths are now detected by separate elements in a linear detector array. Linear detector arrays, especially indium–gallium–arsenide arrays used for de- tecting the commonly used 1,300 nm OCT wavelength, are expensive, shifting the costs of rapid scanning optical delay lines for time-domain systems to the detector array for SD-OCT. 8.5 Beam Delivery Instruments for Optical Coherence Tomography The OCT imaging technology is modular in design. This is most evident in the optical instruments through which the OCT beam can be delivered to the tissue. Because OCT is fiber-optic based, single optical fibers can be used to deliver the OCT beam and collect the reflected light, thereby mak- ing the beam delivery system potentially very small, on the order of the size of an optical fiber (125 µm diameter) itself. The OCT technology can also be readily integrated into existing optical instruments such as research and surgical microscopes [51, 52], ophthalmic slit-lamp biomicroscopes [3], catheters [26], endoscopes [29], laparoscopes [27], needles [28], and hand-held imaging probes [27] (Fig. 8.6). Imaging penetration is determined by the opti- cal absorption and scattering properties of the tissue or specimen. The imaging penetration for OCT ranges from tens of millimeters for transparent tissues such as the eye to less than 3 mm in highly scattering tissues such as skin. To image highly scattering tissues deep within the body, novel beam-delivery instruments have been developed to relay the OCT beam to the site of the tissue to be imaged. An OCT catheter has been developed for insertion into biological lumens such as the gastrointestinal tract [29]. Used in conjunction 136 S.A. Boppart Fig. 8.6. Beam delivery instruments. The OCT beam can be delivered through a number of new or modified optical instruments including (a) surgical and research microscopes, (b) hand-held probes and laparoscopes, (c) fiber-optic catheters, and (d) optical needle-probes with endoscopy, the 1 mm diameter catheter can be inserted through the work- ing channel of the endoscope for simultaneous OCT and video imaging [31]. Similar catheters have been used to image plaques within the living human coronary arteries [33]. Minimally invasive surgical procedures utilize laparo- scopes, which are long, thin, rigid optical instruments to permit video-based imaging within the abdominal cavity. Laparoscopic OCT imaging has been demonstrated by passing the OCT beam through the optical elements of a laparoscope [27,32]. Deep solid-tissue imaging is possible with the use of fiber- needle probes [28]. Small (400 µm diameter) needles housing a single optical fiber and micro-optic elements have been inserted into solid tissues and ro- tated to acquire OCT images. Recently, microfabricated micro-electro-optical- mechanical systems (MEOMS) technology has been used to miniaturize the OCT beam scan mechanism [53]. 8.6 Spectroscopic Optical Coherence Tomography Spatially distributed spectroscopic information can be extracted from the tis- sue specimen using spectroscopic OCT (SOCT) algorithms and techniques [54–57] (Fig. 8.7). In structural OCT imaging, the amplitude of the envelope of the field autocorrelation is acquired and used to construct an image based on the magnitude of the optical backscatter at each position. Spectral information 8 OCT with Applications in Cancer Imaging 137 Fig. 8.7. Spectroscopic OCT imaging. Spatially resolved spectroscopic information can be extracted from the detected OCT signal. By digitizing the interferogram fringes and transforming the data using a Fourier-transform, the spectrum of the returned light can be determined. When compared to the original laser spectrum, spectral differences can be identified, which are related to the absorption and scat- tering of the incident light. Spectroscopic OCT images of control and experimental botanical specimens where a highly absorbing and fluorescent chemical dye has accumulated in the vascular system. Corresponding fluorescence and bright-field microscopy images are shown. Figure modified with permission from [58] can be obtained by digitizing the full interference signal and applying digital signal processing algorithms to transform the data from the time (spatial) do- main to the frequency (spectral) domain. Transformation algorithms include the Morlet wavelet and the short-time Fourier transform with attention to reduction of windowing artifacts. While spectroscopic data can be extracted from each point within the specimen, there is an inherent trade-off between high spatial resolution and high spectral resolution and novel algorithms are being investigated to optimize this data. Once the spectral data is obtained at a point in the tissue, the spectral center of mass can be calculated and compared with the original spectrum from the laser source. Spectral shifts to longer or shorter wavelengths, from the original center of mass, are displayed on a 2D image using a multidimensional hue-saturation-luminance (HSL) color space. At localized regions within the tissue, a color is assigned with a hue that varies according to the direction of the spectral shift (longer or shorter wavelength) and a saturation that corresponds to the magnitude of that shift. The luminance is held constant. Longer wavelengths of light are scattered less in turbid media. In a homo- geneously scattering sample, one can observe using spectroscopic OCT that shorter wavelengths are scattered near the surface and a smooth color-shift occurs with increasing depth, as longer wavelengths are scattered. In more heterogeneous samples, such as tissue, scattering objects such as cells and sub-cellular organelles produce variations in the spectroscopic OCT data. 138 S.A. Boppart Spectroscopic OCT images can indicate changes in the spectroscopic prop- erties of the tissue; however, further investigation is needed to determine how the spectroscopic variations relate to the biological structures and how this information can be used for diagnostic purposes. 8.7 Applications to Cancer Imaging The noninvasive, noncontact, high-resolution, real-time imaging capabilities of OCT and its many modes of operation have enabled a wide range of new applications in biology, medicine, surgery, and materials investigations. In this section, applications in tumor cell biology and cancer imaging are presented. OCT has successfully made a transition from being a laboratory-based exper- imental technology to one that is useful clinically [59]. In the coming years, results from long-term controlled clinical trials will further demonstrate the usefulness of this technology. 8.7.1 Cellular Imaging for Tumor Cell Biology Although previous studies have demonstrated in vivo OCT imaging of tissue morphology, most have imaged tissue at ∼10–15 µm resolutions, which does not allow differentiation of cellular structure. The ability of OCT to identify the mitotic activity, the nuclear-to-cytoplasmic ratio, and the migration of cells has the potential to not only impact the fields of cell, tumor, and de- velopmental biology, but also impact medical and surgical disciplines for the early diagnostics of disease such as cancer. High-resolution in vivo cellular and subcellular imaging has been demon- strated in the Xenopus laevis (African frog) tadpole (Fig. 8.8) [16, 60]. Many of the cells in this common developmental biology animal model are rapidly dividing and migrating during the early growth stages of the tadpole, provid- ing an opportunity to image dynamic cellular processes. Cell dynamics can also be tracked in three-dimensional volumes of high-resolution OCT data as they migrate through an engineered tissue scaffold along a chemoattractant- induced gradient (Fig. 8.9) [61]. From this 3D data set, time-dependent cell position was color-coded. The ability of OCT to characterize cellular dynam- ics such as mitosis and migration is relevant for cancer diagnostics and for the investigation of tumor metastasis in humans. Combining the coherence gating of OCT with high numerical aperture mi- croscope objectives enables high axial- and transverse-resolution imaging deep within highly scattering specimens. This optical configuration has been called optical coherence microscopy (OCM), and can improve the optical sectioning capability of confocal microscopy. For several years, investigators have recog- nized that OCT and MPM can utilize a single laser source for multimodal- ity imaging. Recently, a microscope that integrates OCM and multiphoton (MPM) fluorescence imaging has been used to image cells in 3D engineered 8 OCT with Applications in Cancer Imaging 139 Fig. 8.8. In vivo morphological and cellular imaging in a developmental biology animal model. Photograph and cross-sectional OCT images of a Xenopus laevis (African frog) tadpole specimen showing developing tissue morphology, as well as individual cells and corresponding histology Fig. 8.9. Tracking cell migration. A population of macrophage cells is tracked in three dimensions over the course of 3 h using OCT. Macrophage cell migration was induced with a chemoattractant at one end of the 3D scaffold, separated by a semi- permeable membrane. The time-dependent positions of the cells are color-coded white (time = 0h) and grey (time = 3h). Modified figure used with permission from [61] tissue scaffolds [62, 63]. OCT and MPM provide complementary image data. OCT can image deep through transparent and highly scattering structures to reveal the 3D structural information. OCT, however, |
cannot detect the presence of a fluorescing particle. In a complementary manner, MPM can lo- calize fluorescent probes in three-dimensional space and provide insight into cell function. MPM can detect the fluorescence, but not the microstructure 140 S.A. Boppart Fig. 8.10. Integrated multimodality microscopy using optical coherence microscopy (OCM) and multiphoton microscopy (MPM). Coregistered image data provides insight into structure (OCM) and function (MPM) of cells in 2D and 3D culture con- ditions, under the influence of external mechanical stimuli, and following pharmaco- logical interventions. Fibroblasts with green-fluorescent-protein (GFP) transfected to be expressed with vinculin, a cell surface adhesion protein, are shown. A nuclear fluorescent dye has also been used to identify the location of the nuclei. OCM im- ages based on scattering show the spatial distribution of the cell and extracellular components. Figure modified and used with permission from [63] nor the location of the fluorescence relative to the microstructure. Hence, the development of an integrated microscope capable of OCT and MPM uniquely enables the simultaneous acquisition of microstructural data and the localiza- tion of fluorescent probes for precisely coregistered structural and functional imaging (Fig. 8.10). 8.7.2 Translational Breast Cancer Imaging OCT has been used to differentiate between the morphological structure of normal and neoplastic tissue for a wide-range of tumors [13, 64–67]. The use of OCT to identify tumors and tumor margins in situ will represent a signif- icant advancement for medical or image-guided surgical applications. Beam delivery systems such as a compact and portable hand-held surgical probe or a modified surgical microscope permits OCT imaging within the surgical 8 OCT with Applications in Cancer Imaging 141 field, while the main OCT instrument can be remotely located in the surgi- cal suite. The use of OCT in image-guided surgical procedures represents a paradigm shift for the surgical oncology community. While the surgical on- cologist typically resects macroscopic solid tumors, consideration must still be given to the microscopic, cellular extent of the disease. To address this, surgeons typically resect with large margins in an effort to remove any occult cells or nests of tumor. Resected tissue is sent to the surgical pathology lab where margins are examined following histological processing to ensure that they are clean. During this time, a fully staffed operating room and anes- thetized patient must often wait for the decision on these margins. Therefore, moving the high-resolution microscopic imaging of tumor margins from the surgical pathology lab into the operating room would represent a substantial reduction in costs, including time, costs, and patient health. The use of OCT for examining tumor margins has many advantages, as well as introducing new challenges. Large surface areas (and three-dimensional volumes) of tissue must be examined in real-time at micron resolutions. Improvements in data management, acquisition rates, automated tissue identification, and zooming capabilities all must be addressed. An example of the use of OCT for identifying tumors at varying stages is shown in Fig. 8.11. Using a well-characterized carcinogen-induced rat mam- mary tumor model that mimics the progression and histological findings of human ductal carcinoma of the breast, OCT images were acquired at varying time-points and compared to corresponding histology [67]. In good agreement, the OCT images identify not only late-stage morphological changes and the clear tumor margin, but also early ductal changes and evidence of abnormal cells located away from the primary tumor. A portable OCT system has been constructed for intraoperative imaging of surgical margins following lumpectomy procedures for the surgical treatment of breast cancer (Fig. 8.12). Different OCT biopsy needles have also been con- structed for imaging into solid tumors or for guiding needle-biopsy procedures in breast cancer [28] (Fig. 8.12). Ongoing intraoperative and image-guided procedure studies are underway to determine the sensitivity and specificity of OCT compared with the gold-standard histopathological analysis. 8.8 Optical Coherence Tomography Contrast Agents When imaging biological tissues, it is often desirable to enhance the signals measured from specific structures. Contrast agents that produce a specific image signature have been utilized in virtually every imaging modalities, in- cluding ultrasound, computed tomography, magnetic resonance imaging, and optical microscopy, among many others. There are multiple optical properties that are amenable for generating contrast in optical images, including OCT images (Fig. 8.13). Recently, new engineered contrast agents and molecular contrast techniques specifically designed for OCT have been developed and 142 S.A. Boppart Fig. 8.11. Cancer imaging using OCT. OCT can identify neoplastic changes at varying stages of tumor growth. Top image set: Morphological OCT and histological imaging of (a,b) normal and (c,d) late-stage carcinogen-induced mammary tumor in a rat model. The tumor (t) mass is evident compared to the low-backscattering adi- pose (a) cells. Bottom image set: Early-stage ductal changes detected in this model using OCT and confirmed with histology. Ducts are imaged in cross-section (a,b) and through a longitudinal section (c,d). Figure modified and used with permission from [67] characterized [68, 69]. In contrast to fluorescent probes, which are commonly used as contrast agents in fluorescence, confocal, and multiphoton microscopy, new classes of optical contrast agents suitable for OCT must be based on mech- anisms other than the detection of incoherently emitted fluorescence because OCT detects only coherent light. Agents that alter the local scattering or absorption properties are used (Fig. 8.14), such as oil-filled microspheres that have scattering nanoparticles of melanin, gold, carbon, or iron-oxide either embedded in their protein shell or encapsulated in their liquid-filled core [70]. Fluorescence-based microscopy techniques have the advantage of very low background signal in the absence of autofluorescence. This advantage can also be obtained in OCT by using novel dynamic contrast agents that are physically modulated in space using an external magnetic or electric field [71,72]. While 8 OCT with Applications in Cancer Imaging 143 Fig. 8.12. Portable OCT system for real-time imaging during surgical operations and procedures. The compact system utilizes a superluminescent diode with a center wavelength at 1,300 nm wavelength and spectral-domain detection. (a) Forward- directed and (b) side-directed fiber-optic OCT needle probes can be used to collect data from within solid tissue masses or during needle-biopsy procedures the resolutions of OCT may be insufficient to resolve individual nanometer or micron-sized scattering agents, the use of modulating particles enables sub- resolution detection of particles moving in and out of the OCT beam, or by modulating not only the agent itself, but also the local tissue environment, producing both changes in the amplitude of the reflected light (for structural OCT) and the phase of the reflected light (for phase-resolved spectroscopic or Doppler OCT). Because OCT detects changes in optical scattering, the sen- sitivity of detecting small relative changes in scattering is less than detecting relatively large changes in local absorption, particularly if absorption is due to an exogenously administered contrast agent. There is an increasing interest in near-infrared contrast agents in fluorescence-based microscopy techniques because longer excitation and emission wavelengths can propagate deeper into and further out of scattering tissue. Spectroscopic OCT techniques, which can detect spatially resolved spectral changes, can be used to identify the presence of a near-infrared absorbing dye if the absorption spectra of the dye overlaps with the optical source spectrum [58]. 144 S.A. Boppart Fig. 8.13. Metaphorical diagram of an optical contrast agent illustrating the mul- tiple optical substrates, properties, and applications that can be utilized for optical probes and contrast agents. There are many properties and principles to exploit, in addition to the more common fluorescence and bioluminescence properties. Figure used with permission from [68] Fig. 8.14. Contrast agents for OCT. As with every other imaging modality, con- trast agents enhance the diagnostic ability of the modality and permit site-specific detection of features. A wide array of novel contrast agents have recently been de- veloped including (top left) microspheres with scattering nanoparticles embedded in the core or shell, (top right) magnetically-modulated agents of free or encapsulated iron-oxide, (bottom left) absorbing near-infrared dyes detected with spectroscopic OCT, and (bottom right) plasmon-resonant nanorods as scattering and absorbing agents 8 OCT with Applications in Cancer Imaging 145 Finally, plasmon-resonant gold nanoparticles in the form of spheres, shells, cages, and rods have been utilized as strong as wavelength-specific absorbers and scatterers [73–75]. By changing the size and structure of these nanopar- ticles, the wavelength-dependent absorption and scattering can be tuned throughout the near-infrared and visible wavelengths. These nanoparticles have been used as contrast agents for OCT, and also proposed as multifunc- tional therapeutic agents because their strong absorption properties can be used to induce local hyperthermia in cells and tissues. All of these agents are expected to be highly biocompatible and composed of materials that have been previously found to be suitable for in vivo use. These agents, with protein, iron-oxide, gold, or biocompatible molecular sur- faces, may be functionalized with antibodies or molecules to target them to specific molecules, cells, or tissue types and thus provide additional selectivity that can enhance the utility of OCT as an emerging diagnostic technique. 8.9 Molecular Imaging using Optical Coherence Tomography The advantages of OCT, compared to other imaging techniques, are numer- ous. In particular, OCT can provide imaging resolutions that approach those of conventional histopathology and imaging can be performed in situ. Despite its advantages, a serious drawback to OCT is that the linear scattering prop- erties of pathological tissue probed by OCT are often morphologically and/or optically similar to the scattering properties of normal tissue. For example, although morphological differences between normal and neoplastic tissue may be obvious at later tumor stages, it is frequently difficult to optically detect early-stage tumors. To improve the ability of discriminating tissue types in this scenario, molecular imaging techniques are being developed. Molecular imaging involves the generation of images or maps of tissue that contain molecular-specific information. The use of exogenous contrast agents in OCT (described above) is one example of molecular imaging, when such agents are functionalized to target and label specific molecular structures on cells or in tissue, such as cell-surface receptors that are over-expressed in tumors. In many instances, it may be more desirable to detect the presence of endogenous molecules without the addition exogenous agents that could alter the biology or the viability of the tissue. Pump and probe techniques have been used to detect the presence of molecules that have transient absorption states in the tissue that are induced by an external pump beam [76]. Spectroscopic OCT has been used to probe for variations in the oxygenation state of tissue, recognizing the spectroscopic differences between oxy- and deoxy-hemoglobin [77]. Both of these methods, however, rely on a limited set of molecules or molecular features that can be detected. A new method called Nonlinear Interferometric Vibrational Imaging (NIVI) has been developed to achieve molecular contrast in OCT imaging by 146 S.A. Boppart exploiting optical nonlinearities [78, 79]. The nonlinear processes, in particu- lar, are coherent anti-Stokes Raman scattering (CARS) and second harmonic generation (SHG), but the general idea could easily be extended to other non- linear processes such as third harmonic generation (THG), coherent Stokes Raman scattering (CSRS). For these, the nonlinear process generates an op- tical signal that remains coherent with the incident light. Therefore in NIVI, the origin of this coherent signal can be spatially resolved in three-dimensions using OCT-like interferometers. The heterodyne detection scheme common in OCT also imparts significant advantages in detecting these nonlinear signals, including increased sensitivity, full reconstruction of the magnitude and phase information of the sample Raman susceptibility [80], background rejection of noncoherent four-wave-mixing processes arising from molecules such as water [81], and in the case where reference molecules are placed in the reference arm of the interferometer, molecular specificity can be obtained. Figure 8.15 shows a schematic of the energy-level diagram involved in CARS, along with how a reference molecular species would be incorporated into Reference Mirror Dm Molecular/Chemical n np s nAS Species np |1> | Tunable 0> Sample Sources np = pump laser frequency BS ns = Stokes laser frequency nAS = anti-Stokes output frequency Detector Delay (µm) −20 0 20 40 60 80 2.0 Lc = 32 µm 1.5 λas = 647 µm 1.0 2 0.5 0.0 0 0 647 Delay (nm) Fig. 8.15. Molecular OCT imaging. Nonlinear optical signatures in tissue are used to identify specific molecular bonds in a technique called Nonlinear Interferometric Vibrational Imaging (NIVI). An energy level diagram is shown (top left) for coherent anti-Stoke Raman |
scattering (CARS). By placing a reference molecular species in the reference arm of the OCT interferometer (top right), coherent CARS signals are gen- erated in the reference molecular species, and from the sample to produce spatially resolved images of molecular composition. An interferogram from two independently generated CARS beams is shown (bottom) for the benzene molecule, demonstrating the familiar-looking OCT interferogram, but with molecular specificity ICARS (a.u.) I (a.u.) 8 OCT with Applications in Cancer Imaging 147 0 500 Meniscus 1000 (a) 1500 2000 3000 4000 5000 6000 7000 8000 9000 10000 11000 12000 Micrometers Nonlinear Interferometric Transmission Vibrational Imaging Light Microscopy 0 200 400 600 800 1000 1200 1400 (b) (c) 1600 1000 µm Fig. 8.16. Multidimensional Nonlinear Interferometric Vibrational Imaging (NIVI). NIVI, tuned to the C–H vibrational resonance, is performed on a cuvette containing acetone. Signal is generated only from the acetone and not from the cuvette or the particulates in the acetone. NIVI images are shown for the meniscus and bottom of the cuvette, and compared with a transmission light microscopy image of the cuvette bottom. Modified figures used with permission from [82] the OCT interferometer. A representative interferogram generated from two independent samples of benzene is shown, demonstrating that a molecularly specific interferogram can be generated, and used to enhance molecular con- trast and perform molecular imaging using NIVI [78]. This has been extended to multidimensional imaging (Fig. 8.16), and on-going research is investigat- ing NIVI for 3D molecular imaging in tissues. While this method is still in development, the ability to image and map the three-dimensional spatial distribution of individual endogenous molecules such as DNA, RNA, pro- teins, or other diagnostic biomolecules [79] is likely to extend the diagnostic capabilities and clinical utility of OCT to the molecular level. 8.10 Conclusions The capabilities of OCT offer a unique and informative means of imaging bi- ological specimens and nonbiological samples. The noncontact nature of OCT and the use of low-power near-infrared radiation for imaging allow this tech- nique to be noninvasive and safe. Conventional structural OCT imaging does not require the addition of fluorophores, dyes, or stains to provide contrast Micrometers Micrometers 148 S.A. Boppart in images under most conditions. Instead, OCT relies on the inherent optical contrast generated from variations in optical scattering and index of refrac- tion. These factors permit the repeated use of OCT for extended imaging. OCT permits the cross-sectional imaging or en face sectioning of tissue and samples, enabling in vivo structure to be visualized in opaque specimens, or in specimens too large for high-resolution confocal or light microscopy. Imaging at cellular and subcellular resolutions with OCT is an important area of ongoing research. While many developmental biology animal models have been commonly used for their scientific value, small size, ease of care and handling, and readily visualized cellular features, cellular imaging in humans, particularly in situ, is a challenge because of the smaller cell sizes (10–20 µm) compared to larger undifferentiated cells in developing organisms. To our ad- vantage, poorly differentiated cells present in many neoplastic tissues tend to be larger, increasing the likelihood for detection using OCT at current imaging resolutions. Clinical applications of OCT are likely to continue to increase, particularly as more commercial OCT systems become available. With successful integra- tion into clinical ophthalmic imaging, OCT applications in gastroenterology, cardiology, and oncology are likely to become more commonplace. Finally, novel means to perform molecular imaging using OCT will be pursued, using targeted OCT-specific contrast agents or advanced nonlinear optical methods for identifying and mapping the molecular and ultrastructural composition of biological tissue. OCT represents a multifunctional investigative tool, which not only complements many of the existing imaging technologies available today, but overtime, is also likely to become established as a major optical imaging modality. Acknowledgments I thank my students, post-doctoral fellows, and research personnel in the Biophotonics Imaging Laboratory at the Beckman Institute for their hard work and dedication in advancing the OCT technology. I appreciate the insight and collaborative contributions of Profs. Kenneth Suslick, Martin Gruebele, and Keith Singletary from the University of Illinois at Urbana-Champaign and Alexander Wei from Purdue University for using OCT to explore new applica- tions. I also thank my colleagues in Biomedical Optics for their contributions to this work, and my clinical collaborators and colleagues at Carle Founda- tion Hospital and Carle Clinic Association, Urbana, Illinois, USA. Additional information can be obtained at http://biophotonics.uiuc.edu. Prof. Stephen Boppart’s email address is boppart@uiuc.edu. 8 OCT with Applications in Cancer Imaging 149 References 1. D. Huang, E.A. Swanson, C.P. Lin et al., Science 254, 1178 (1991) 2. B.E. Bouma, G.J. Tearney (eds.), Handbook of Optical Coherence Tomography (Marcel Dekker, New York, 2001) 3. J.S. Schuman, C.A. Puliafito, J.G. Fujimoto, Optical Coherence Tomography of Ocular Diseases, 2nd edn. (Slack, Thorofare, NJ, 2004) 4. J.M. Schmitt, A. Knuttel, M. Yadlowsky et al., Phys. Med. Biol. 39, 1705 (1994) 5. J.G. 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News 12, 23 (2005) 9 Coherent Laser Measurement Techniques for Medical Diagnostics B. Kemper and G. von Bally 9.1 Introduction Holography and speckle interferometry are well established tools for industrial nondestructive testing and quality control, which are in general performed by the detection of object displacements while applying static stress, tempera- ture changes, shock waves, or by vibration monitoring [1–3]. Therefore, up to the present, various holography and speckle interferometry systems for macroscopic as well as microscopic applications have been developed. Also for biomedical applications holographic and speckle interferometric metrology opens up new perspectives for the visualization and the detection of displace- ments and movements. Here, for the early recognition of malignant cancers, for example, it is of interest to distinguish between different tissue elastic- ities [4–7]. Also, in the fields of Life Sciences and Biophotonics, there is a requirement for an optical instrumentation of timely and spatially high res- olution analysis, measurement, and documentation in supracellular, cellular, and |
subcellular range. For such applications, the special requirements of tis- sues and cellular probes have to be taken into account and a lateral resolution in microscopic dimensions as well as very compact and flexible speckle inter- ferometric arrangements are required. Additionally, for in vivo applications, a high repetition rate of the results for online monitoring is necessary. This can be obtained by the application of micro-optics, optical fiber technology, miniaturized (color) CCD sensors, and fast digital image processing systems. Furthermore, spatial phase shifting methods and suitable techniques for non- diffractive digital holographic reconstruction, which require in contrast to tem- poral techniques considerably less stability of the experimental setup and the investigated specimens, allow for a quantitative determination of the object wave phase. From this information the underlying displacement data as well as the object shape can be obtained under suitable geometric conditions [1,8]. In this contribution, in an overview, experimental results obtained with sev- eral holographic and (speckle) interferometric arrangements based on endo- scope and microscope optics are presented to demonstrate applications and 152 B. Kemper and G. von Bally possibilities of holographic interferometric metrology on biological specimens and cellular samples. The results of these investigations show that it is possi- ble to detect minimal-invasive elasticity differences of biological tissues, which may be used for an early tumor and cancer recognition. Furthermore, data obtained from long-term digital holographic investigations on toxin-induced reactions of adherent cancer cells demonstrate application prospects of digital holographic phase contrast microscopy in marker-free life cell imaging [9,10]. 9.2 Electronic Speckle Pattern Interferometry (ESPI) 9.2.1 Double Exposure Subtraction ESPI Figure 9.1 shows schematically a mainly out-of-plane sensitive ESPI for the investigation of rough surfaces. Coherent laser light is divided into an object illumination wave O and a reference wave R. The image of the object surface that is covered by a speckle pattern due to the speckle effect [11] is imaged on an image recording device (e.g., a charge coupled device (CCD) sensor). The superimposition of O and R is performed by a second beam splitter BS. In double exposure subtraction ESPI, two speckle patterns are recorded sub- sequently on a CCD sensor superimposed by a reference wave. The intensities I and I ′ of the two object states are [1] √ I(x, y) = IO(x, y) + IR(x, y) + 2 IO(x, y)IR(x, y) cos(ψ(x, y)) I ′(x, y) = IO(√x, y) + IR(x, y) (9.1) + 2 IO(x, y)IR(x, y) cos(∆ϕ(x, y) + ∆φ(x, y)). Fig. 9.1. ESPI setup. BS beam splitter; L1, L2, L3 lenses; CCD CCD sensor 9 Coherent Laser Measurement Techniques for Medical Diagnostics 153 In (9.1) IO represents the intensity of the object wave, which can be as- sumed equal before and after the object deformation or movement between two recordings and IR is the intensity of the reference wave. The term ∆ψ(x, y) = φO(x, y)−φR(x, y) represents the stochastic phase difference while ∆φ(x, y) is the phase difference caused by the object deformation d and the sensitivity vector S = kQ + kB, which takes into account the illumination direction kQ, the observation direction kB, and the light wavelength λ [1]: 2π ∆φ(x, y) = S · d. (9.2) λ Subtraction of the two intensities (9.1) yields √ [ ] ∆φ(x, y) ∆φ(x, y) (I − I ′)(x, y) = 4 IO(x, y)IR(x, y) sin ψ(x, y) + sin . 2 2 (9.3) To display the result in video repetition rate on a monitor the modulus |I−I ′| is calculated. By observation of |I−I ′| qualitative information about the object deformation is obtained. 9.2.2 Spatial Phase Shifting (SPS) ESPI Investigations on biological specimen require a method for quantitative deter- mination of the phase difference ∆φ that works even under instable conditions. For this reason, spatial phase shifting (SPS) methods [12, 13] are applied for sign correct phase difference determination that require only a single inter- ferogram to determine the phase distribution of one object state. Another advantage of this technique in comparison with temporal phase shifting pro- cedures [14] is that no movable parts (e.g., Piezo translators) in the optical path and no additional electronic synchronization are necessary. For the realization of SPS, the speckle image of the investigated object sur- face is superimposed in the image plane with spatial carrier fringes, e.g., with vertical or horizontal orientation. This is achieved by an adequately chosen, almost constant spatial phase gradient β(x, y) = (βx, βy) that is generated by a tilt between object wave and reference wave. The intensity of the interference pattern formed on the CCD sensor can be described as [14] I(xm, yn) = [I0(xm, yn) ( ) ] Φ 1 + γ(xm, yn) sinc cos(φ(x 2 m, yn) + mβx + nβy + C) . (9.4) 154 B. Kemper and G. von Bally In (9.4) m = 1, .,M and n = 1, ., N are the indices of the CCD pixel co-ordinates xm, yn. I0(xm, yn) = I0 O(xm, yn) + I0 R(xm, yn) is the sum of the in√tensities of object wave and reference wave. The parameter γ(xm, yn) = 2 IO(xm, yn)IR(xm, yn)/I0(xm, yn) denotes the modulation of the intensity patterns. The term sinc(Φ/2) = sin (Φ/2)/(Φ/2) describes the intensity inte- gration on a single CCD pixel in the angle range Φ. φ(xm, yn) is the distrib- ution of the object wave phase and C an additional constant phase off-set. For the calculation of the object wave phase from each recorded intensity pattern, in addition to Fourier transformation methods [15–17], a variable three-step algorithm [12] can be applied by taking into account three intensity values Ik−1, Ik, Ik+1 (in horizontal or vertical orientation) of neighboring CCD pixels: ( ) 1 − cos β I φk + kβ + C = arctan k−1 − Ik+1 m sin β 2Ik − Ik−1 − odulo 2π. Ik+1 (9.5) The algorithm requires for a correct phase evaluation that the mean speckle size is at least the size of three pixels of the digitized interferograms. For the parameter β, either βx or βy may be chosen. The adjustment of the phase gradients can be performed by analysis of the 2D frequency spectrum of the carrier fringe pattern in the interferograms by 2D digital fast Fourier transform (FFT) [13,16]. The phase difference ∆φk modulo 2π between two phase states φk, φ′ k of the object wave front is calculated: ∆φk = (φ′ k + kβ + C) − (φk + kβ + C) = φ′ k − φk. (9.6) Figure 9.2 illustrates the evaluation process of spatial phase-shifted inter- ferograms for displacement detection. Figure 9.2a,b shows the spatial phase- shifted interferograms I, I ′ obtained from two different displacement states of a tilted metal plate. In Fig. 9.2c,d the corresponding phase distributions φ, φ′ (mod 2π), calculated by (9.5) from Fig. 9.2a,b, are depicted. The correspond- ing correlation fringe pattern calculated by (9.3) is depicted in Fig. 9.2e. The resulting phase difference distribution ∆φ mod 2π obtained by subtraction of Fig. 9.2c,d modulo 2π as well as the according filtered phase difference distri- bution is shown in Fig. 9.2f,g. Figure 9.2h,i finally represents the unwrapped phase difference after removal of the 2π ambiguity and the related pseudo 3D representation of the data. From the data in Fig. 9.2h the underlying dis- placement of the plate is calculated by taking into consideration recording and imaging geometry of the experimental setup by (9.2). 9 Coherent Laser Measurement Techniques for Medical Diagnostics 155 Fig. 9.2. Evaluation of spatial phase-shifted interferograms. (a),(b) Spatial phase- shifted interferograms I, I ′ of two displacement states of a tilted metal plate; (c),(d) Phase distributions φ, φ′ (mod 2π) calculated by (9.5) from (a) and (b); (e) Cor- relation fringe pattern calculated by (9.3); (f),(g) Raw and filtered phase difference distribution mod 2π obtained by subtraction of (c) and (d) modulo 2π; (h),(i) Un- wrapped phase difference and corresponding pseudo 3D representation of ∆φ 156 B. Kemper and G. von Bally 9.3 Endoscopic Electronic Speckle Pattern Interferometry (ESPI) Endoscopy is a wide spread intracavity observation technique routinely used in minimal invasive diagnostics and industrial intracavity inspection. The com- bination of endoscopic imaging with speckle interferometric metrology allows the development of tools for a nondestructive quantitative detection of defects within body cavities, including the analysis of shape, structure, displacements, and vibrations of the object. In this way Electronic-Speckle-Pattern Interfer- ometry (ESPI) opens up new perspectives for biomedical applications, espe- cially in minimally invasive diagnostics in the medical field [18]. Contrary to earlier attempts in holographic endoscopy where single interferograms with Q-switched lasers were recorded [19], an on-line process analysis can be per- formed with a rate near video repetition frequency. This can be achieved by endoscopic ESPI systems with an external (proximal) interferometric arrange- ment using standard endoscope optics [4,20] or by an arrangement where the ESPI system is positioned in the endoscope tip [21] (distal arrangement). Such endoscope ESPI systems open up the possibility to replace the opera- tor’s tactile sense, which is lost in endoscopic surgery, by visual information (“endoscopic taction”) [4, 5, 22]. 9.3.1 Proximal Endoscopic ESPI The setup for proximal endoscopic ESPI with the speckle interferometer po- sitioned outside the cavity is illustrated in Fig. 9.3. The advantage of such Fig. 9.3. Arrangement for proximal endoscopic ESPI. CCD CCD sensor, AOM acousto-optic modulator, DG double pulse generator, BV/PC digital image process- ing system 9 Coherent Laser Measurement Techniques for Medical Diagnostics 157 an arrangement is that standard CCD cameras can be used. Additionally, it is possible to form modular concepts of the ESPI system with flexible as well as rigid standard endoscopes for imaging. The light source is a cw laser (e.g., an argon-ion laser, λ = 514.5 nm or a frequency doubled Nd:YAG laser, λ = 532 nm). The regulation of the light intensity is performed by an acousto- optic modulator (AOM) using the first diffraction order. Behind the AOM, the laser beam is divided into the object illumination wave and the reference wave, with both coupled into single-mode optical fibers. Spatial phase shift- ing (SPS) (see Sect. 9.2.2) is applied to obtain quantitative information about displacements and motions. Therefore, an interference pattern between object wave and reference wave is generated by positioning the end of the reference wave fiber with an off-set to the optical axis. The interference patterns of the superimposed object wave and reference wave are recorded by a progressive- scan (full frame) camera with a CCD sensor. A fast digital image processing system (BV/PC) allows an image acquisition of the intensity patterns up to 25 Hz. Simultaneously, the corresponding correlation fringe patterns obtained by image subtraction are displayed online with a rate of 12.5 Hz on an ex- ternal monitor. The exposure time and the delay between two exposures are variable in order to be suitable for the measurement conditions of biological specimen (for further details see [4,13,23]). Figure 9.4 shows on the left panel a mobile endoscope ESPI system based on an analog CCD camera and an image processing system with frame grabber interface. On the right panel of Fig. 9.4 a modular endoscope ESPI system with IEEE1394 (“FireWire”) technology consisting of a laser unit, a speckle interferometric module, and a notebook-computer for image processing is depicted. One of the current limitations of endoscopic imaging in the gastrointesti- nal tract is the absence of tactile perception, making it impossible to assess gastrointestinal wall elasticity during routine endoscopic examinations. For this reason it is of interest to distinguish between tissue elasticity for the Fig. 9.4. (Left) Mobile endoscope ESPI system based on an analoge CCD camera and an image processing system with frame grabber interface. (Right) Modular endoscope ESPI system with IEEE1394 (“FireWire”) technology consisting of a laser unit, a speckle interferometric module, and a notebook-computer for image processing 158 B. Kemper and G. von Bally Fig. 9.5. In vitro investigations on a human intestinal specimen by stimulation with an endoscopic ultra sound tube. (a),(d) White light images of a tissue part without pathological findings and of the tumorous tissue; (b),(c) Phase difference distribu- tions (mod 2π); (c),(f) Pseudo 3D plots of the displacement along the marked cross sections in (b) and (e) early recognition of malignant cancers, for example. In vitro investigations on intestinal specimens with carcinoma tissue have been carried out. For the experiment, the |
tissue is stimulated by an endoscopic ultrasonic tube (single pulse, amplitude: ≈200 µm). Afterwards, during the relaxation process of the tissue, series of stroboscopic phase difference distributions are captured. Figure 9.5 depicts characteristic results. Figure 9.5a,d shows the white light images of an investigated area of the specimen without pathological findings in comparison to a tumorous part of the tissue. In Fig. 9.5b,e exemplary re- sults of phase difference distributions mod 2π obtained during the relaxation process of the tissue are depicted. The corresponding calculated surface de- formation along the cross sections in Fig. 9.5b,e is plotted in Fig. 9.5c,f. The phase difference shows concentric fringes for the tissue part without patho- logical findings (Fig. 9.5b) and a parallel fringe distribution for the hardened tumorous tissue (Fig. 9.5e). Thus, the tissue part without pathological findings can be distinguished from the tumorous tissue of diminished elasticity. 9.3.2 Distal Endoscopic ESPI To avoid the phase instabilities of image fiber bundles and the aberrations of standard rod lens endoscope imaging systems in endoscopic ESPI, a compact realization of the ESPI camera system in the endoscope tip (distal arrange- ment) is of particular advantage [21]. In addition, a smaller speckle size can be 9 Coherent Laser Measurement Techniques for Medical Diagnostics 159 Fig. 9.6. Concept of a distal endoscope ESPI sensor. CCD CCD sensor (1/4 or 1/6 in.) obtained by short optical path length distances in a compact interferometric alignment, thus achieving a higher lateral resolution up to microscopic dimen- sions. The concept of an out-of-plane sensitive distal ESPI sensor is illustrated in Fig. 9.6. The coherent light source is an amplitude modulated cw laser. The object illumination wave (O) as well as the reference wave (R) are guided by single-mode optical fibers. For the object illumination O as well as for the reference wave R additional microlens systems are applied that are fixed at the end of each optical fiber. To achieve an arrangement suitable for endoscopic applications, the ref- erence wave is redirected by 180◦ with a prism and a beam splitter. The interference patterns of the superimposed waves R and O are recorded by a one-chip color CCD sensor (e.g., 1/4 inch sensor or 1/6 inch sensor; pixel resolution, 752 × 582 pixels; maximum diameter, 8 or 4 mm). For the quan- titative displacement detection, an aperture is used to regulate the speckle size to three CCD pixels for the application of spatial phase shifting tech- niques [9, 23]. Therefore, a spatial carrier fringe pattern between O and R is generated by positioning the end of the reference wave fiber with a lateral off-set out of the optical interferometer axis. Figure 9.7 depicts a photo of a distal endoscope ESPI sensor based on a 1/6 inch CCD sensor. Figure 9.8 shows results of investigations with a distal endoscopic speckle interferometer based on 1/4 inch color CCD sensor. In Fig. 9.8a the white 160 B. Kemper and G. von Bally Fig. 9.7. Distal endoscope ESPI sensor based on a 1/6 in. CCD sensor Fig. 9.8. Results obtained with a distal endoscopic speckle interferometer based on a 1/4 in. CCD sensor. (a) White light image of an USAF 1951 test chart; (b) Corre- sponding speckle image (λ = 514.5 nm), a lateral resolution of 8.7 µm is obtained; (c) Correlation fringe pattern of displacement measurements on a tilted white painted metal plate; (d) Corresponding filtered phase difference distributions 9 Coherent Laser Measurement Techniques for Medical Diagnostics 161 Fig. 9.9. Results of in vitro investigations on a human stomach: Filtered phase difference distributions of a “healthy” area (a) and a region containing an adeno carcinoma (b) light image of a USAF 1951 test chart positioned in a distance of 5 mm in front of the sensor is depicted. Figure 9.8b shows the corresponding speckle image affected by illumination with coherent light (λ = 514.5 nm). Within the speckle image a lateral resolution of 8.7 µm is obtained. Figure 9.8c,d presents results from a recorded series of displacement measurements with a white painted metal plate, which was tilted between two recordings to demonstrate the fringe resolution and quality. Shown are the correlation fringe pattern (Fig. 9.8c) and the respective filtered wrapped phase difference distributions modulo 2π (Fig. 9.8d) converted to 256 gray levels. The maximum displace- ment resolution of the system in this configuration can be estimated from the noise of the phase difference map to λ/7. To demonstrate the performance of the distal ESPI sensor on biological specimens analogous to the experiments described in Sect. 9.3.1, investigations on tumorous human stomach gastric wall (in vitro) have been carried out. In Fig. 9.9 the filtered wrapped phase difference distribution of a “healthy” part of the specimen is depicted (imaged area about 1 cm2), which has been stimulated manually with a test needle. The results are concentric fringe pat- terns around the tip of the needle. In contrast, an area containing an adeno carcinoma shows a parallel fringe pattern (Fig. 9.9b), indicating that the tissue elasticity in this region is diminished, which is in agreement with the results from Sect. 9.3.1. 9.4 Microscopic (Speckle) Interferometry In microscope ESPI, the motivation is to integrate the interferometric metrol- ogy into microscope systems to achieve an enhancement of these techniques by an additional high resolution detection and visualization of movements, displacements [24, 25], and refractive index changes. Figure 9.10 shows the 162 B. Kemper and G. von Bally Fig. 9.10. Setup for a microscope (speckle) interferometer. Light source: frequency doubled cw Nd:YAG laser (λ = 532 nm); SMF: single-mode fibers for object illumi- nation and reference wave; the illumination of the sample can be performed optional with incident light or in transmission; AP: aperture for regulation of the speckle size and suppression of scattered light schematic setup for a microscopic (speckle) interferometer. The object is im- aged by a microscope lens on a CCD sensor. The coherent object illumination is performed either in transmission or with incident light by single mode opti- cal fibers. The speckle size (or in the case of smooth waves disturbing scattered light due to transparent specimens) is regulated by an additional aperture AP that is positioned behind the microscope lens. The detection of optical path length changes affected by micro changes of the sample can be performed by a spatial phase shifting method as described in Sect. 9.2.2. Therefore, the ref- erence wave is generated by an optical single mode fiber placed in the plane of AP that is positioned with an off-set from the optical axis of the interfer- ometer to produce a nearly constant phase gradient between object wave and reference wave. Figure 9.11 shows results obtained by a microscope (speckle) interfer- ometer setup in combination with a ×10 magnification microscope lens. In Fig. 9.11a the white light image of an USAF 1951 test chart is depicted. Figure 9.11b shows the corresponding speckle image effected by illumina- tion with coherent laser light (λ = 532 nm) with a lateral resolution of 7.8 µm. In Fig. 9.11c–f results from displacement measurements on a dyed probe of an intestine carcinoma which was tilted in the experiment are de- picted. Figure 9.11c shows the white light image of the investigated area. The correlation fringe pattern, the corresponding phase difference distribu- tion, and the filtered phase difference fringes are presented in Fig. 9.11d–f. 9 Coherent Laser Measurement Techniques for Medical Diagnostics 163 Fig. 9.11. Results of measurements obtained with a ×10 magnification microscope lens. (a) White light image of an USAF 1951 test chart; (b) Speckle image of (a) by illumination with coherent laser light (λ = 532 nm); (c) White light image of a dyed part of intestine carcinoma fixed on a glass slide; (d) Correlation fringe pattern of the tilted specimen in (a); (e) Corresponding phase difference distribution modulo 2π; (f) Filtered phase difference distribution 164 B. Kemper and G. von Bally Fig. 9.12. (a) Phase difference distribution of optical path length changes due to micro-changes and micro-movements of human liver tumor cells (HepG2) after t = 75 min (×40 magnification immersion microscope lens). (b) Correlation fringe pattern obtained with a ×10 magnification microscope lens during investigations of a living water flea. For both measurements the illumination of the specimen was performed in transmission, λ = 532 nm The results demonstrate that displacements can be detected while the lat- eral resolution of the speckle image is in microscopic dimensions. Figure 9.12a shows results obtained by application of the interferometric arrangement in Fig. 9.10 in combination with a ×40 magnification immersion microscope lens for monitoring microchanges of human liver tumor cells (HepG2) in cell culture medium. The depicted phase difference distribution (illumination in transmis- sion, λ = 532 nm) due to optical path length changes corresponds to micro- movements and microchanges of the specimens after t = 75 min. Figure 9.12b shows a correlation fringe pattern obtained from the investigation of a living water flea utilizing a ×10 magnification microscope lens, which is an exem- plary result of a recorded series of 50 images and demonstrates the feature of online monitoring. 9.5 Digital Holographic Microscopy 9.5.1 Principle and Measurement Setup In Life Sciences and Biophotonics, a quantitative minimal invasive analysis of dynamic life processes with resolution in cellular and subcellular scale is of particular interest. In connection with microscopy, digital holography provides contact-less, marker-free, quantitative phase contrast imaging. Digital holography is based on the classic holographic principle, with the difference that the hologram recording is performed by a digital sensor, e.g., a CCD or CMOS camera [26,27]. The subsequent reconstruction of the holo- graphic image that includes the information about the object wave is carried 9 Coherent Laser Measurement Techniques for Medical Diagnostics 165 Fig. 9.13. Schematics for digital holographic microscopy using incident light (left) and inverse transmission arrangements (right) [28] out numerically with a computer. Digital holographic microscopy (DHM) per- mits nondestructive, marker-free, and quantitative high resolution full-field phase contrast imaging of transparent samples such as living cells [9, 10]. Figure 9.13 depicts the schematics of two “off-axis” setups for digital holo- graphic microscopy, which are particularly suitable for the integration into commercial microscopy systems. The coherent light of a laser (e.g., a fre- quency doubled Nd:YAG laser, λ = 532 nm) is divided into object illumina- tion and reference wave, using singlemode optical fibers. The left panel of Fig. 9.13 shows an incident light illumination arrangement for the investiga- tion of reflective objects. The set-up on the right panel of Fig. 9.13 is designed to investigate transparent objects such as living cell cultures. In both cases the coherent laser light for the illumination of the sample is coupled into the optical path of the microscope’s condenser by a beam splitter. The reference wave is superimposed onto the light reflected or transmitted by the object by a second beam splitter with a slight tilt against the object wave front. Thus, “off-axis” holograms are generated and recorded by a CCD camera. After hologram acquisition, the data is transmitted by an IEEE1394 (“FireWire”) interface to a digital image processing system; thus bypassing the need for cost intensive frame grabber cards with hardware-specific software. This ap- proach provides the advantage that commercial standard microscope optics with high numerical aperture (e.g., water immersion and oil immersion) can be used in combination with an optimized (Koehler-like) illumination of the sample. Furthermore, such a modular integration of the additional optical components for digital holography does not restrict the conventional function of the microscopy systems [28]. 166 B. Kemper and G. von Bally Fig. 9.14. (Left) Inverse microscope with P.A.L.M. MicroBeam and attached dig- ital holographic microscopy module. The illumination of the sample is performed in transmission mode (built in cooperation with P.A.L.M. Microlaser Technologies GmbH, Bernried, Germany). An incubator allows temperature stabilized long-term investigations of livings cells. (Right) Upright fluorescence microscope with digital holographic microscopy module for illumination of the sample in transmission and reflection (built in cooperation with Carl Zeiss Jena GmbH, Germany) [28] Figure 9.14 shows an inverse (left) and an upright (right) microscope with attached digital holographic microscopy modules. The typical hologram cap- turing time depends on the applied imaging device (here CCD cameras) and amounts within the range of 1 ms. The reconstruction rate of the digital holo- grams depends mainly on the capability of the applied image processing sys- tem and the size |
of the digitized holograms. With current computer systems (e.g., a Pentium IV 2.8 GHz) and the reconstruction algorithm described in Sect. 9.5.2 at a frame size of 512× 512 pixels reconstruction rates up to ∼4 Hz can be achieved. 9.5.2 Nondiffractive Reconstruction The reconstruction of the digitally recorded holograms is performed numeri- cally with a computer. In general, Fresnel-transformation based digital holo- graphic reconstruction methods generate not only the information contained in the object wave but in addition the intensity of the reference wave (“zero order”) and a “twin image” [29]. Furthermore, the size of the reconstructed holographic image depends on the reconstruction distance to the hologram plane. Thus, in digital holographic microscopy, the reconstruction of the digitally captured holograms is performed by the application of a non- diffractive reconstruction method (NDRM) [30–34]. The intensity distribu- tion IHP(x, y, z0) in the hologram plane HP, located at z = z0, is formed by the interference of the object wave O(x, y, z = z0) and the reference wave R(x, y, z = z0): 9 Coherent Laser Measurement Techniques for Medical Diagnostics 167 IHP(x, y, z0) = O(x, y, z0)O∗(x, y, z0) + R(x, y, z0)R∗(x, y, z0) +O(x, y, z0)R∗(x, y, z0) + R(x, y, z0)O∗(x, y, z0) = IO(√x, y, z0) + IR(x, y, z0) + 2 IO(x, y, z0)IR(x, y, z0) cos ∆ϕHP(x, y, z0), (9.7) with IO = OO∗ = |O|2 and IO = RR∗ = |R|2 (* denotes the conjugate com- plex term). The parameter ∆ϕHP (x, y, z0) = φR(x, y, z0)− φO(x, y, z0) is the phase difference between O and R at z = z0. In the presence of a sample in the optical path of O, the phase distribution represents the sum φO (x, y, z0) = φO0(x, y, z0)+∆ϕS(x, y, z0), where φO0(x, y, z0) denotes the pure object wave phase and ∆ϕS(x, y, z0) represents the optical path length change that is ef- fected by the sample. For areas without a sample, ∆ϕHP (x, y, z0) is estimated by a mathematical model [31,35]: ∆ϕHP (x, y, z0) = φR((x, y, z0) − φO0(x, y, z0) ) = 2π Kxx2 + Kyy2 + Lxx + Lyy . (9.8) The parameters Kx,Ky in (9.8) describe the divergence of the object wave and the properties of the applied microscopy lens. The constants Lx, Ly denote the linear phase difference between O and R due to the off-axis geometry of the experimental setup. For quantitative phase measurement from IHP(x, y, z0) in a first step, the complex object wave O(x, y, z = z0) in the hologram plane is determined pixel wise by solving a set of equations that is obtained from insertion of (9.8) in (9.7). For that purpose, neighboring intensity val- ues within a square area of 5 × 5 pixels around a given hologram pixel are considered by application of a spatial phase shifting algorithm (for details see [9] and [23]). The utilized algorithm is based on the assumption that only ∆ϕHP (x, y, z0) = φR(x, y, z0) − φO0(x, y, z0) between the object wave O(x, y, z0) and the reference wave R(x, y, z0) varies rapidly spatially in the hologram plane. In addition, because of the spatial phase shifting algorithm, the object wave’s intensity has to be assumed constant within an area of about 5 × 5 pixels around a given point of interest of the hologram. These require- ments can be fulfilled by an adequate relation between the magnification of the microscope lens and the image recording device. Therefore, the magnifi- cation of the microscope lens is chosen in such a way that the smallest imaged structures of the sample that are restricted by the resolution of the optical imaging system due to the Abbe criterion are over sampled by the CCD sen- sor. In this way the lateral resolution of the reconstructed holographic phase contrast images is not decreased by the spatial phase shifting algorithm [31]. The parameters Kx,Ky, Lx, Ly in (9.8) cannot be obtained directly from the geometry of the experimental setup with an adequate accuracy and for this reason are adapted once before the measurements by an iterative fitting process in an area of the hologram without sample [31,34]. The evaluation of digital holographic phase contrast images requires, in correspondence to microscopy with white light illumination, a sharply focused 168 B. Kemper and G. von Bally image of the sample. For the case that the object is not imaged sharply in the hologram plane HP during the hologram recording process, e.g., due to mechanical instability of the experimental setup or thermal effects, in a second evaluation step a further propagation of the object wave to the image plane can be carried out for subsequent focus correction. The propagation of O(x, y, z0) to the image plane zIP that is located at zIP = z0 + ∆z in the distance ∆z to HP can be carried out by a Fresnel transformation [26, 31, 35] or by a convolution algorithm [34,36,37]: { O (x, y, zIP = z0 + ∆z) = F− ( F { ( 1 O (x, y, z0)} ))} exp iπλ∆z ν2 + µ2 . (9.9) In (9.9) λ is the applied laser light wave length, ν, µ are the coordinates in frequency domain and F denotes a Fourier transformation. During the propa- gation process, the parameter ∆z is chosen in such a way that the holographic amplitude image appears sharply, in correspondence to a microscopic image under white light illumination. A further criterion for a sharp image of the sample is that diffraction effects due to the coherent illumination appear min- imized in the reconstructed data. As a consequence of the applied algorithms and the parameter model for the phase difference model ∆ϕHP in (9.8), the re- sulting reconstructed holographic images do not contain the disturbing terms “twin image” and “zero order.” In addition, the method allows in compari- son to propagation by Fresnel transformation, as e.g., in [31, 35], a sharply focused image of the sample in the hologram plane. The propagation of O by (9.9) enables in this way the evaluation of image plane holograms containing a sharply focused image of the sample and effects no change of the image scale during subsequent refocusing. In the special case that the image of the sam- ple is sharply focused in the hologram plane with ∆z = 0 and thus zIP = z0, the reconstruction process can be accelerated because no propagation of O by (9.9) is required. From O(x, y, zIP), in addition to the absolute amplitude |O(x, y, zIP)| that represents the image of the sample, the phase information ∆ϕS(x, y, z0) of the sample is reconstructed simultaneously: ∆ϕS(x, y, z0) = φO (x, y, z0) − φO0(x, y, z0) {O (x, y, z = arctan IP)} {O (x, y, zIP)} (mod2π). (9.10) After removal of the 2π ambiguity by a phase unwrapping process [1], the data obtained by (9.10) can be applied for quantitative phase contrast mi- croscopy, which is the main topic of interest for the described experiments. For an incident light geometry as depicted in the left panel of Fig. 9.13, the topography zs can be calculated on the phase distribution ∆ϕS(x, y, z0): λ∆ϕS(x, y, z zs(x, y, z0) = 0) λ = ∆ϕ (x y z ) ( . 2.2 4 s , , 0 . 9 11) π π 9 Coherent Laser Measurement Techniques for Medical Diagnostics 169 For illumination in transmission the thickness of cells dcell in cell culture medium with a homogenous refractive index nmedium can be determined by measuring the optical path length change ∆ϕcell of the cells to the surrounding medium: λ∆ϕcell(x, y, z0) 1 dcell(x, y, z0) = , 2π ncell − (9.12) nmedium with the integral refractive index ncell and the wave length λ of the applied laser light. For fully adherently grown cells, the parameter dcell is estimated in first order to describe the shape of single cells [34,38]. Figure 9.15 illustrates the evaluation process of digital recorded holo- grams. Figure 9.15a,b shows a digital hologram obtained from a living human pancreas carcinoma cell (Patu8988T) with an inverse microscope arrange- ment in transmission mode (×40 microscope lens, NA = 0.65) and the re- constructed holographic amplitude image that corresponds to a microscopic bright field image at coherent laser light illumination. Figure 9.15c depicts the simultaneously reconstructed quantitative phase contrast image modulo 2π. The unwrapped data without 2π ambiguity, representing the optical path length changes that are affected by the sample in comparison to the sur- rounding medium due to the thickness and the integral refractive index, are shown in Fig. 9.15d. Figure 9.15e depicts a pseudo 3D plot of the data in Fig. 9.15d. Figure 9.15f shows the cell thickness along the marked dashed line in Fig. 9.15d, which is determined by application of (9.12) with ncell = 1.38 and nmedium = 1.337. Fig. 9.15. Example for evaluation of digital holograms. (a) Digital hologram of a human pancreas carcinoma cell (Patu8988T); (b) Reconstructed holographic am- plitude image; (c) Quantitative phase contrast image (mod 2π); (d) Unwrapped phase distribution; (e) Pseudo 3D plot of the unwrapped phase image in gray level representation; (f) Calculated cell thickness along the dashed white line in (d) 170 B. Kemper and G. von Bally 9.5.3 Resolution and Numerical Focus The resolution of digital holographic microscopy can be characterized by in- vestigating calibration test charts. The left panel of Fig. 9.16 shows the recon- structed amplitude (holographic image) of a negative USAF 1951 resolution test chart (illumination in transmission), recorded using a ×40 microscope lens (NA = 0.6). The magnified image section of group 9.5 (resolution limit of the test chart) represents a line width of 620 nm and is resolved clearly. The comparison with the Abbe criterion shows that the lateral resolution is diffraction limited (in correspondence to bright field microscopy) and can be increased by using microscope optics with higher numerical aperture [31]. The right panel of Fig. 9.16 demonstrates the axial resolution for incident light illumination of a reflective metal surface (nano-structured chromium on chromium surface) recorded with a ×5 microscope lens (NA = 0.1). The de- picted elements represent a height of 30 nm and are resolved clearly in the reconstructed phase distribution. Because of the phase noise the axial resolu- tion is determined to be ≈5 nm. The digital reconstruction of different object planes from a single hologram enables a variable (subsequent) numerical fo- cus of digital holographic images (“digital holographic multi focus”) without additional mechanical or optical components. Figure 9.17 demonstrates the digital holographic refocus for the example of a semitransparent USAF1951 test chart (upper panel) and for the holographic image of a living human pancreas tumor cell of the type Patu8988S (lower panel). Fig. 9.16. (Left) Reconstructed holographic amplitude of a negative USAF 1951 test chart (digital hologram recorded in transmission mode), the elements of group 9.6 correspond to a lateral resolution of 550 nm; (b) Reconstructed phase contrast image of a reflective phase object (chromium on chromium sample) with steps of 30 nm in axial direction (digital hologram recorded in reflection mode) [28] 9 Coherent Laser Measurement Techniques for Medical Diagnostics 171 Fig. 9.17. Amplitudes reconstructed from single recorded holograms in different focus planes (left : unfocussed reconstruction in the hologram plane; right : numer- ically focused). (a),(b) Semitransparent USAF 1951 test chart (×40 microscope optic, NA = 0.6) and (c),(d) Living human pancreas tumor cell Patu8988S (×100 oil immersion optic, NA = 1.3) [28] 9.5.4 Digital Holographic Phase Contrast Microscopy of Living Cells Investigations on living pancreas tumor cells [39, 40] were carried out to demonstrate the potential of digital holographic microscopy for the visual- ization of toxin-induced morphology changes. Therefore, pancreatic tumor cells were exposed at 37◦C to taxol. Digital holograms of selected cells were recorded continuously over 4 h. Figure 9.18 shows the results. In the upper row of Fig. 9.18, the obtained unwrapped phase distributions at t = 0, t = 78, 172 B. Kemper and G. von Bally Fig. 9.18. Monitoring of a living PaTu8988S cell after adding a toxin (taxol) to the cell culture medium. Morphological changes such as cell rounding and finally the cell collapse are induced. Upper row : gray level coded unwrapped phase |
distribution at t = 0, t = 78, and t = 262 min after taxol addition. Lower row : Corresponding pseudo 3D representations of the phase data (Cooperation: Dr. Jürgen Schneken- burger, Department of Medicine B, University of Münster, Germany) [28] Fig. 9.19. Cross sections through the measured optical path length changes cor- responding to the dashed white lines in the phase distributions of Fig. 9.18 (up- per row) [28] and t = 262 min after taxol addition are depicted. The lower row of Fig. 9.18 shows the pseudo 3D plot of the phase images. Figure 9.19 depicts cross sec- tions through the measured optical path length changes corresponding to the dashed lines in the upper row of Fig. 9.18. From Figs. 9.18 and 9.19 it is clearly visible that the toxin induces morphological changes as cell rounding and finally the cell collapses. 9 Coherent Laser Measurement Techniques for Medical Diagnostics 173 9.6 Discussion and Conclusions This contribution gives an overview of proximal and distal endoscopic ESPI systems as well as microscope (speckle) interferometric and digital holographic microscopy systems with regard to biomedical applications. The exemplarily shown results obtained from the application of the proximal and distal en- doscopic ESPI systems on biological specimens demonstrate the ability of ESPI to detect displacements and differences in elasticity of biological tis- sues even underneath the visible surface. This could enhance conventional endoscopic investigations by the substitution of the missing tactile sense by a visual information (“endoscopic taction”). In addition, for distal endoscopic ESPI it has been shown that it is possible to develop compact speckle inter- ferometric arrangements with a lateral resolution in microscopic range. The presented (speckle) interferometric microscopy setup provides a simple inter- ferometer adjustment and a fast detection of microchanges even on living organisms. In connection with microscopy, digital holography allows a non- contact, fast, quantitative, minimal invasive, full field detection with high res- olution (<5 nm in axial direction) by quantitative holographic phase contrast imaging of reflective and transparent samples, with the possibility to trans- form the obtained phase data into an illustrative pseudo 3D representation. In this way, digital holographic microscopy enables topography measurements on nanostructured surfaces, quantitative on-line detection of drug effected cel- lular thickness/shape variations. 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Weiss, E. Albrecht, V.E. Samoilova, W. Domschke, M.M. Lerch, Gut 54, 1445 (2005) 40. H.P. Elsässer, U. Lehr, B. Agricola, H.F. Kern, Virchows Arch. B. Cell Pathol. Incl. Mol. Pathol. 61, 295 (1992) 10 Biomarkers and Luminescent Probes in Quantitative Biology M. Zamai, G. Malengo, and V.R. Caiolfa The genome sequence of many organisms is now complete. “However, genome sequences alone lack spatial and temporal information and are therefore as dynamic and informative as census lists or telephone directories” [1]. We know the words of the genetic code, yet we need to associate each of them to a specific function. The challenge of this century is to figure out how proteins work together to make living cells and organisms. Proteins are essential for most biological processes, but they are not simply objects with chemically reactive surfaces, and it is not easy to understand their function. Proteins localize to specific environments in the cells: membranes, cytosol, organelles, or nucleoplasm, undergo diffusive or directed movement, and often are coupled to chemical events. The exceptional capability of the proteins to regulate virtually all dynamic processes in living cells depends on the fine regulation of their topology, movement, and chemistry. In fact, cells respond to stimuli through signaling pathways that orchestrate the recruitment and assembly of proteins into biomolecular machines. Roadmap1 focuses on ways to solve the structures of protein machines. To achieve these goals, it is critical to understand how the cell architecture influences the formation of these protein complexes. Research is, therefore, turning to the study of protein function in their most natural context [2]. 10.1 Fluorophores and Genetic Dyes 10.1.1 Small Organic Dyes and Quantum Dots Small organic fluorophores (<1 kDa) for covalent labeling of macromolecules have been optimized for wavelength range, brightness (extinction coefficient for absorbance, fluorescence quantum yield) photostability, and reduction in self-quenching. Hundreds of such dyes are commercially available [3]. However, 1 (http://www.nihroadmap.nih.gov) 178 M. Zamai et al. core C shell polymer coating biomolecule 10 nm 30 nm A B Fig. 10.1. Biomarkers gallery. (a) Confocal maximal projection of fixed HEK293 cells immunostained for actin (red) and stained for the nuclei with Hoechst 3342 (blue). Only one cell expresses EGFP-labeled uPAR (green). (b) Confocal maximal projection of fixed HEK293 stably transfected with EGFP-labeled uPAR (green), and stained with Cy5-uPA ligand (red). (c) (Top) Scheme of a quantum dot and (bottom) an example of application in cell imaging representing the distribution of GM1 gangliosides found in the plasma membrane in live HeLa cells; nucleus was stained with Hoechst 3342 (reproduced from [4]) these dyes lack specificity for any particular protein; most applications use antibodies in fixed and permeabilized cells or specifically labeled ligands (Fig. 10.1a,b). Quantum dots (QDs) are inorganic nanocrystals that fluoresce at sharp and discrete wavelengths depending on their size [4–6]. Their extinction coefficients is 10–100 times higher than small fluorophores and fluorescent proteins (FPs). They also have good quantum yields and exceptional photo- stability that allow repeated imaging of single molecules [4]. Their absorbance extends from short wavelengths up to just below the emission wavelength, so that a single excitation wavelength readily excites QDs of multiple emis- sion maxima. QDs typically contain a core of CdSe or CdTe and ZnS shell (Fig. 10.1c). For biological applications, a coating that makes QDs water sol- uble is necessary to prevent quenching by water, and allow conjugation to protein targeting molecules such as antibodies and streptavidin. The large size of QDs conjugated to biomolecules (10–30 nm) prevents efficient traver- sal of intact membranes, which restricts their use to permeabilized cells or extracellular or endocytosed proteins. 10.1.2 Fluorescent Proteins Rapid advances in live-cell imaging technologies, combined with the use of genetically encoded fluorescent proteins (FPs), has resulted in a revolution in cell biology, as it is now possible to track the assembly of protein complexes within the organized microenvironment of the living cell. In the next sections, we discuss some of these advances, focusing on fluorescence imaging and spec- troscopic techniques that are front-edge for the analysis of protein movement and interactions in living cells. 10 Biomarkers and Luminescent Probes in Quantitative Biology 179 Since the discovery of the GFP in 1961 by Osamu Shimomura and colleagues [7], 30 years had to pass before the gene for GFP was cloned and the 238 amino acids sequence determined by Prasher [8]. Douglas Prasher was the first person to realize the potential of GFP as a biomarker for proteins in cells [9]. The manufacture of proteins using the instructions from the gene is called protein expression. Prasher envisioned that it would be possible to use biomolecular techniques to insert the GFP gene at the end of the gene of any protein, right before the stop codon (Fig. 10.2, left). Encoded in the DNA is some type of index that directs the molecular machinery to the start of the gene of each necessary protein. When new protein is required, the gene is read, and the protein is manufactured. At the end of the gene is a message called a stop codon, which ends protein production. When the cell needs to make that protein, it would go to the specific gene, use the information encoded in the gene to make it, but instead of stopping when the protein was made, this cell would carry on making GFP until it reached the stop codon at the end of the GFP gene. As a result, the cell would produce a “chimeric” (e.g., modified) protein with a GFP attached to it. A variety of techniques have been developed to construct FP fusion prod- ucts and enhance their expression in mammalian and other systems. The primary vehicles for introducing FP chimeric gene sequences into cells are genetically-engineered bacterial plasmids and virus that act as vectors and transfer the genetic information into the cell either transiently or stably (Fig. 10.2, right). In transient, or temporary, gene transfer experiments (of- ten referred to as transient transfection), plasmid or viral DNA introduced into the host organism does not necessarily integrate into the chromosomes of the host cell, but can be expressed in the cytoplasm for a short |
period of plasmid vector endocytosis DNA endosome DNA-lipid gene released gene complex DNA for protein for protein lipofection reagent cell gene for GFP membrane stop code stop code for protein for protein Fig. 10.2. Left : Fusion Construct. Adapted from [10]. Right : Lipid-mediated trans- fection in mammalian cells 180 M. Zamai et al. time. Expression of gene fusion products usually takes place over a period of several hours after transfection and continues for 72–96 h after introduction of plasmid DNA into mammalian cells. In many cases, the plasmid DNA can be incorporated into the genome in a permanent state to form stably trans- formed cell lines. The choice of transient or stable transfection depends upon the target objectives of the investigation. Structure of GFP There are several reasons why GFP is a powerful biomarker. GFP is easy to detect by its fluorescence in cells; therefore, it is more versatile than most other bioluminescent molecules that require the addition of other substances before they glow. For example, firefly luciferase requires ATP, magnesium, and luciferin before it luminesces. It is a fairly small and compact protein (molecular weight, 27 kDa). The small size does not hinder the proper func- tion of the protein to which it is attached, and particularly its intracellular trafficking and translocation. Among the most important aspects of the GFP to appreciate is that the entire 27 kDa native peptide structure is essential to the development and maintenance of its fluorescence. It is remarkable that the fluorophore derives from a triplet of adjacent amino acids: the serine, tyrosine, and glycine residues at locations 65, 66, and 67 (referred to as Ser65, Tyr66, and Gly67; in Fig. 10.3). Ser 65 Gly 67 Tyr 66 + 2H+ − H2O 395 nm Imidazolinone − H+ Ring System Tyr 66 Prematuration Chromophore Cyclization Quinone 475 nm Reaction Sites Fig. 10.3. GFP-chromophore and the β-barrel structure. Within the hydrophobic environment in the center of the GFP, a reaction occurs between the carboxyl car- bon of Ser65 and the amino nitrogen of Gly67 that results in the formation of an imidazolin-5-one heterocyclic nitrogen ring system. Further oxidation results in con- jugation of the imidazoline ring with Tyr66 and maturation of a fluorescent species. It is important to note that the native GFP fluorophore exists in two states. A pro- tonated form that has an excitation maximum at 395 nm, and a unprotonated one that absorbs at approximately 475 nm. However, regardless of the excitation wave- length, fluorescence emission has a maximum peak wavelength at 507 nm, although the peak is broad and not well defined 10 Biomarkers and Luminescent Probes in Quantitative Biology 181 Although this simple amino acid motif is commonly found throughout in nature, it does not generally result in fluorescence. What is unique to the fluorescent protein is that the location of this peptide triplet resides in the center of a remarkably stable barrel structure consisting of 11 β-sheets folded into a tube (Fig. 10.3). Since 1992, the FPs have become widely used as non- invasive markers in living cells, and their successful integration into variety of living systems illustrates that the expression of these proteins in cells is well tolerated. Virtually, any protein can be tagged with GFP, the resulting chimera often retains parent-protein targeting and function when expressed in cells, and therefore can be used as a fluorescent reporter to study protein dynamics. The Future of Fluorescent Proteins The GFP has been engineered to produce a vast number of variously colored mutants, fusion proteins, and biosensors that are broadly referred to as FPs. The GFP sequence has been modified for optimizing the expression in different cell types as well as the generation of GFP variants with more favorable spectral properties, including increased brightness, relative resistance to the effects of pH variation on fluorescence, and photostability. References [1, 3] illustrate in detail the biochemical and fluorescent properties of GFP-variants. The scientist who mostly contributed to understand how GFP works and developed new techniques and mutants is Roger Tsien. His group has obtained mutants that start fluorescing faster than wild type GFP, are brighter and have different colors (Fig. 10.4) [3]. The latest generation of jellyfish variants has solved most of the deficien- cies of the first generation FPs. The search for a monomeric, bright, and fast-maturing red FP has resulted in several new and interesting classes of FPs, particularly those derived from coral species. Development of existing FPs, together with new technologies, such as insertion of unnatural amino acids, will further expand the color palette. The current trend in fluorescent probe technology is to expand the role of dyes that fluoresce into the far red and near infrared. In mammalian cells, both autofluorescence and the absorp- tion of light are greatly reduced at the red end of the spectrum. Thus, the development of far red fluorescent probes would be extremely useful for the examination of thick specimens and entire animals. Given the success of FPs as reporters in transgenic systems (Fig. 10.5), the use of far red FPs in whole organisms will become increasingly important in the coming years. Finally, the tremendous potential in FP applications for the engineering of biosensors is just now being realized. The success of these endeavors certainly suggests that almost any biological parameter will be measurable using the appropriate FP-based biosensor. 182 M. Zamai et al. Table 1 Properties of the best FP variants Excitation Emission Class Protein (nm) (nm) Brightness Photostability pKa Oligomerization Far-red mPlum 590 649 4.1 53 <4.5 Monomer Red mcherry 587 610 16 96 <4.5 Monomer tdTomato 554 581 95 98 4.7 Tandem dimer mStrawberry 574 596 26 15 <4.5 Monomer J-Red 584 610 8.8 13 5.0 Dimer DsRed-monomer 556 586 3.5 16 4.5 Monomer Orange mOrange 548 562 49 9.0 6.5 Monomer mKO 548 559 31 122 5.0 Monomer Yellow-green mCitrine 516 529 59 49 5.7 Monomer Venus 515 528 53 15 6.0 Weak dimer YPetg 517 530 80 49 5.6 Weak dimer EYFP 514 527 51 60 6.9 Weak dimer Green Emerald 487 509 39 0.69 6.0 Weak dimer EGFP 488 507 34 174 6.0 Weak dimer Cyan CyPet 435 477 18 59 5.0 Weak dimer mCFPm 433 475 13 64 4.7 Monomer Cerulean 433 475 27 36 4.7 Weak dimer UV-excitable green T-Sapphire 399 511 26 25 4.9 Weak dimer Fig. 10.4. Fluorescent protein palette. Engineered FPs cover the full visible spec- trum. Top: Protein samples were purified from E. coli expression systems, excited at wavelengths up to 560 nm and photographed by their fluorescence. Bottom: Prop- erties of the GFP variants (Adapted from [3]) Fig. 10.5. Past and present of green fluorescent protein (GFP). 1961: The jellyfish Aequorea and its light-emitting organs. 1992: The amino acids sequence of GFP. 2007: Mouse with brain tumor expressing the fluorescent protein variant DsRed; the zoomed area shows the tumor mass (grey) and the blood vessels expressing GFP (white) that are growing in the tumor mass mPlum mGrape2 mRaspberry mGrape1 mCherry mStrawberry mTangerine tdTomato mOrange mBanana mHoneydew YFP (Citrine) EGFP ECFP EBFP 10 Biomarkers and Luminescent Probes in Quantitative Biology 183 10.2 Microspectroscopy in Quantitative Biology: Where and How Static localization of the macromolecules and proteins in cells and tissues is not sufficient by itself to elucidate the key mechanisms that regulate funda- mental processes of the cell. The activity of a biological molecule is not just a function of its structure but also of its dynamic behavior in the cell i.e., localization and interactions and their modifications in time. Thus, great im- portance has to be given to the spatio-temporal dynamics of the molecular interactions within cells in situ and, in particular, in vivo. In the following sections, we describe two spectro-microscopy approaches that, combined with the FPs chimera technology, are of growing importance in modern biology. 10.2.1 Fluorescence Correlation Spectroscopy Fluorescence correlation spectroscopy (FCS) was introduced by Elson, Magde, and Webb in 1972 [11]. In 1990, Denk and Webb demonstrated a new type of microscope on the basis of two-photon excitation of molecules [12]. In 1995, Berland, So, and Gratton put together the two technologies, two-photon exci- tation microscopy and FCS, and demonstrated the potential of this method- ology in intracellular measurements [13]. FCS is now a widely used technique. The reader is invited to refer to the many good articles published on the physics of the two-photon excitation process [14–16] and on the FCS tech- nique [17–20]. Here, we discuss in specific the use of FCS in cell biology as it provides information about mobility (diffusion coefficients), concentration (number of particles), association (molecular brightness), and localization (im- age) of the target molecules. All this information helps understanding the complex molecular interaction networks, which are at the basis of cellular processes. Chemical reactions and diffusion of molecules in solutions can be followed by a variety of methods but when we need to study them in living cells, most methods fail abruptly. The appeal of FCS is that, especially using two-photon excitation, living cells can be explored anywhere by a subfemtoliter volume without disrupting the cell integrity. The most stringent requirement for FCS to work is the possibility to observe the fluorescence signal in a small volume and at very high sensitivity and dynamic range (Fig. 10.6). Only if the volume is so small at any instant of time, it might contain just one or few molecules. Because of these requirements, FCS is also known as a single molecule spectroscopy. If the number of fluorescent molecules in the volume does not change with time and if the quantum yield of the fluorophore is constant, then the average number of the emitted photon is constant. How- ever, the instantaneous number of detected photon is not constant, because of the nature of the emission/detection process, which follows the Poisson statistics (2). This added shot-noise is independent of time. 2 The Poisson distribution describes events that occur rarely in a short period 184 M. Zamai et al. 2-photon volume (< 10−15≈ 1µm3) 12000 cell A 10000 8000 FCS - τ 6000 4000 0.1 0.3 0.5 0.7 0.9 1.1 1.3 1.5 1.7 1.9 2.1 Time (s) 0.05 100 0.05 B C 10 0.04 0.04 0.03 1 0.03 0.02 0.1 0.02 0.01 0.01 0.00 0.0001 0.001 0.01 0.1 1 10 100 0.01 Delay time(s) 0.001 0.00 0.0001 0.0 0.5 1.0 1.5 2.0 0 1 2 3 4 5 6 Delay time(s) counts per Bin Fig. 10.6. Principle of fluorescence fluctuation spectroscopy. The number of mole- cules can change because of diffusion in and out of the volume, then fluorescence intensity fluctuates. (a) The time of the diffusion process causes characteristic fre- quencies to appear in the fluorescence intensity trace. (b) The time structure is analyzed by the autocorrelation function (ACF); Inset : original data are plotted in log scale. (c) The amplitude of the fluctuation depends on brightness (i.e., the aggregation state of a protein) and it can be analyzed by PCH Instead, if the number of molecules in the excitation volume changes, even if the quantum yield is constant, the fluorescence intensity will change with time (Fig. 10.6a). The number of molecules can change because they can dif- fuse out of the volume. The time of the diffusion process causes characteristic frequencies to appear in the fluorescence intensity trace. Assume that we have two molecules in the volume and one leaves, the relative change in intensity will be one-half. However, if there are 10 molecules in the excitation volume e−µµx P (x, µ) = , (10.1) X! where x = 0,1,2,3, (number of emitted photons) and µ = mean number of successes in the given time interval or region of space (number of counts). If µ is the average number of successes occurring in a given time interval or region in the Poisson distribution, then the mean and the variance of the Poisson distribution are both equal to µ: E(x) = m, and V (x) = σ e2 = µ. In a Poisson distribution, only one parameter, µ, is needed. Auto Correlation Auto Correlation CPS frequency PCH amplitude 10 Biomarkers and Luminescent Probes in Quantitative Biology 185 and one of them leaves, the relative change will be only 1/10. This means that the smaller the ratio fluctuation/average-signal, the larger the number of molecules in the volume. It can be shown that this |
ratio is exactly proportional to the inverse of the number of molecules in the volume of excitation [21]. This relationship allows the measurements of the number of molecules in a given volume in the interior of cells [13, 22]. Molecular heterogeneity in the cell or molecular reactions can also induce the simultaneous presence of molecules of different kind in the same volume [23]. One important case is when proteins bind each other forming a molecular aggregate. Let us consider two identi- cal proteins with one fluorescent probe each forming a molecular dimer. The dimer is different than the monomer because it carries twice the number of flu- orescent moieties (Fig. 10.6). When one of these aggregates enters the volume of excitation, it will cause a larger fluctuation of the intensity than a single monomer. Clearly, the amplitude of the fluctuation carries information on the brightness of the diffusing particle (i.e., monomer vs. dimer). Thus, both time and amplitude structures are affected by underlying molecular species, and the dynamic processes that cause the change of the fluorescence intensity. The statistical analysis of the fluctuations of the fluorescence signal has to recover both information (Fig. 10.6a). Analysis of the Time Structure of the Fluctuating Signal The analysis of the time structure of the fluctuating signal is typically done by autocorrelation analysis. The autocorrelation function, ACF = G(τ), char- acterizes the time-dependent decay of the fluorescence fluctuations to their equilibrium value (Fig. 10.6b). In simple terms, ACF calculates the similarity between a signal I(t), and a copy of the same signal shifted by a time lag τ , I(t + τ): 〈I(t)I(t + τ)〉 − 〈I(t)〉2 G(τ) = 〈I(t)〉2 . (10.2) The autocorrelation function yields two parameters: the diffusion coef- ficient (D) and the average number of particles in the observation volume < N > given by the inverse of G(0), multiplied by a constant that depends on the illumination profile. In the case of identical molecules undergoing random diffusion in a Gaussian illuminated volume, the characteristic autocorrelation function is given by the following expression [22]: ( ) ( ) γ 8 −1 Dτ 8 −1/2 Dτ G(τ) = 1 + 1 + , (10.3) N ω2 r ω2 a where D is the diffusion coefficient; ωr and ωa are the beam waist in the radial and in the axial directions; N is the number of molecules; γ is the numerical factor that accounts for the nonuniform illumination of the volume; and τ is the delay time. Other formulas have been derived for the Gaussian-Lorentzian illumination profile [13] and for molecules diffusing on a membrane [21]. 186 M. Zamai et al. Analysis of the Amplitude Structure of the Fluctuating Signal The expression for the statistics of the amplitude fluctuations is generally given under the form of the histogram of the photon counts for a given sam- pling time ∆t. This is known as the photon counting histogram (PCH) dis- tribution. The PCH analysis calculates the probability of detecting photons per sampling time [24, 25]. The probability is experimentally determined by the histogram of the detected photons. In principle, the Poisson distribution describes the occupation number of particles that can freely go in and out a small excitation volume. However, mainly due to the diffusion of molecules in the inhomogeneous excitation volume, the distribution of photon counts devi- ates from the Poisson distribution. The analysis of the distribution of photons is based on the deviation of the measured PCH from the expected Poisson distribution because of the molecule occupation number (Fig. 10.7). Two pa- rameters characterize the photon distribution: the number of molecules in the observation volume (N) and the molecular brightness (ε), which is defined as the average number of detected photons per molecule per second. The ana- lytical expression for the PCH distribution for a single molecular species of a given brightness has been derived for the 3D-Gaussian illumination profile [24] and is reported below: ∫∞ 1 πω2 ( ) 0z P3DG (k;V0; ε) = 0 γ k, εe−4x2 dx, (10.4) V0 2k! 0 where V0 is the volume of illumination; ε is the brightness of the molecules (counts per seconds per molecule); and k is the number of photons in a give time interval. The integral that contains the incomplete gamma function (γ) can be numerically evaluated. Similar expressions have been derived for other shapes of the illumination volume [24]. PCH is extremely useful for analysis fluctuations inside cells, where the target protein can associate in clusters or bind to some other preexisting ag- gregates. Diffusion coefficients for proteins in solution depend on their mole- cular weight (τdiff ∝ D−1 ∝ M1/3). However, in the case of monomer–dimer species, the difference between the diffusion coefficients of the two species is 100 position 1 Position 2 super-poisson position 2 Position 3 Position 1 10 poisson position 3 Position (in time) 0 1 2 3 4 5 6 7 8 9 photons counts/ time Fig. 10.7. Super-Poissonian distribution of detected photons from diffusing fluo- rophores due to (1) fluctuation of the particles number (Poissonian), (2) no uniform volume of the PSF, and (3) photon detection statistics (Poissonian) Intensity Frequency of events 10 Biomarkers and Luminescent Probes in Quantitative Biology 187 just 1.2. It can be shown that a mass ratio of at least 5 is necessary to detect significant differences in the diffusion coefficient. Instead the brightness can change by a factor of 2 when the equilibrium moves from all monomers to all dimers (Fig. 10.8). The all idea of FCS-PCH is the detection of fluorescence intensity fluctua- tions that can be achieved reducing the observation volume to subfemtoliters but also having few molecules in it. In practice this means that we need con- centrations of the fluorophore in the nanomolar range. The consequences are twofold: (1) To study binding events, the dissociation constant of the com- Fig. 10.8. Study of molecular association and diffusion at the cell membrane of the ∆D1 form of the GPI-anchored uPAR, fused to EGFP and transfected in hu- man kidney cells, HEK293. Top panels: Two-photon images of live cells showing the distribution of the receptor at the ventral, mid, and topside of the cell membrane; fluorescence in pseudo-color scale from low (blue) to high (red) intensity; (+): re- gions at which fluorescence fluctuation was recorded. Mid panel : A representative fluorescence fluctuation trace acquired in position (a). Bottom panels: Representa- tive ACF and PCH for two fluorescence fluctuation traces acquired in regions (a) and (b). PCH indicates that the mutant receptor associates in oligomers (brightness b = 3, 900 cpsm, brightness a = 9, 100 cpsm). The ACFs of the two forms, however, do not reveal detectable differences in diffusion coefficient. Control experiments in solution have shown that the EGFP variant, lacking the receptor uPAR domains, does not dimerise at the concentrations found in the cells 188 M. Zamai et al. plexes must be in the nanomolar range or below; (2) The expression in vivo of the FP-proteins systems must be low. This is not a trivial issue as many proteins are segregated in intracellular and organelle compartments often at local concentration higher than nanomolar. FCS-PCH criteria are opposite to those of classical confocal or multiphoton fluorescence microscopy and a systematic optimization of the expression levels of the FP-tagged proteins is necessary. 10.2.2 Fluorescence Lifetime Imaging (FLIM) The development of confocal and multiphoton fluorescence microscopy has introduced enormous prospectives in biomedical sciences. These microscopies permit a variety of high-contrast and multidimensional imaging. However, the fluorescence of organic molecules is not only characterized by the emission spectrum, but also possesses a distinctive lifetime. The fluorescence lifetime is defined as the mean amount of time a fluorophore spends in the excited state after the absorption of an excitation photon. Lifetime can be measured by the time domain or by the frequency domain techniques. In the time domain (Fig. 10.9a–b), a short pulse of light excites the sample, and the subsequent fluorescence emission is recorded as a function of time. This usually occurs on the nanosecond timescale. In the frequency domain (Fig. 10.9c–d), the sam- ple is excited by a modulated source of light. The fluorescence emitted by the sample has a similar waveform, but is modulated and phase-shifted from the excitation curve. Both modulation (M) and phase-shift φ are determined by the lifetime of the sample emission; that lifetime can be calculated from the observed demodulation ratio and phase-shift. Both of these domains yield equivalent data, and take advantage of the fluorescence decay law, which is based on the first-order kinetics. The decay law postulates that if a population of molecules is instantaneously excited when photons are absorbed, then the excited population, and hence the fluorescence intensity as a function of time, I(t), gradually decays to the ground state. Decay kinetics can be described −t by: I(t) = αe τf ; where α is the intensity at time t = 0, t is the time after the absorption, and τf is the lifetime, that is, when the fraction of the population of molecules in the excited state (and the fluorescence intensity) has decreased by a factor of 1/e. Note that before absorption, I(t) = 0. Time Domain Time-domain measurements are based on the assumption that, when photons are absorbed, the molecules can be excited in an infinitely brief moment. This idea is commonly known as the delta or delta-pulse. The delta-pulse idea is used to interpret data obtained with real-pulsed light sources with measur- able pulse-widths. In practice, the time-dependent profile of the light-pulse is reconvolved with the decay-law function. Reconvolution assumes that the delta-pulses are continuous functions, so that the observed decay is the con- volution integral of the decays from all delta-pulses initiated during the finite 10 Biomarkers and Luminescent Probes in Quantitative Biology 189 Fig. 10.9. Lifetime measurements in time and frequency domains. (a) Actual pulsed light-source (gray) and sample response (black), showing the gradual de- cay of fluorescence intensity with time. In this example, the decay could be fit- ted with a mono-exponential curve (dotted) giving a lifetime of 1.309 0.003 ns. (b) TCSPC-fluorometer: A pulsed light source excites the sample repetitively. The sample emission is observed by a detector, while the excitation flashes are detected by a synchronization module (SYNC). A constant fraction discriminator (CFD) responds to only the first photon detected (small arrows), independent of its ampli- tude, from the detector. This first photon from sample emission is the stop signal for the time-to-amplitude converter (TAC). The excitation pulses trigger the start signals. The multichannel analyzer (MCA) records repetitive start–stop signals of the single-photon events from the TAC, to generate a histogram of photon counts as a function of time channel units. The lifetime is calculated from this histogram. (c) Excitation (black) and sample response (gray), illustrating the phase-angle shift (φ) and demodulation ratio (M). (d) Multifrequency cross-correlation fluorometer. An unmodulated light source emits a spectrum of continuous-wave light. The excita- tion monochromator (Excit. Mono.) selects an excitation wavelength. An amplified (Amp 1) master synthesizer (Master) drives the Pockels cell (Pockels) at a base frequency, Rf , which modulates the excitation beam. The modulated beam excites the Sample, causing the sample to emit modulated fluorescence also at the base Rf . An emission monochromator (Emis. Mono.) selects one wavelength of modulated fluorescence. The photomultiplier tube (PMT) is modulated by an amplified (Amp 2) slave synthesizer (Slave) at the base Rf plus a low-frequency cross-correlation note (f). The sample emission at Rf cancels the slave Rf + f frequencies to yield the f signal containing the same phase-angle shift (φ) and demodulation ratio (M) as the Rf fluorescence 190 M. Zamai et al. pulse-width. The method of time-correlated single-photon counting (TCSPC) is, by far, a superior method for measuring time-domain decays (Fig. 10.9b). TCSPC uses a pulsed light-source and a circuit to detect single-photon events at a detector. In a repetitive series of many start–stop signals from the cir- cuitry, a binned histogram in time channels of single-photon counts is gradu- ally generated. TCSPC relies on a principle of Poissonian statistics that only one photon can be counted at a time and in any one channel, to avoid skewing the time-dependent statistics in photon-pile-up. Pile-up thus limits the data- acquisition rate of TCSPC to a few (typically 12) percent of the repetition |
rate. In practice, the single-photon limit is not a major hindrance because the pile-up limit can be monitored during the experiment, and decay times with sufficient photon counts in can be obtained in seconds to minutes with repetition rates in the megahertz range. In addition, the Poissonian nature of the statistics allows the data to be rigorously analyzed. Frequency Domain The fluorescence decay parameters in the decay law impulse function may be obtained on the basis of the relation of a sinusoidally modulated excitation beam to the fluorescence emission response (Fig. 10.9c). The emission occurs at the same frequency as the excitation. Because of the loss of electron en- ergy (Stokes shift) between excitation and emission, the emission waveform is demodulated and phase-shifted in comparison to the excitation. Thus the demodulation ratio (M) and phase-angle shift (φ) constitute two separate ob- servable parameters that are both directly related, via a Fourier transforma- tion, to the initial fluorescence intensity, α, and lifetime, τ for a population of fluorophores. Frequency-domain measurements are best performed using multi-frequency cross-correlation phase-and-modulation (MFCC), shown in Fig. 10.9d. A modulated beam excites the sample. The fluorescence emission is detected by a photomultiplier tube (PMT) modulated at the same base radio-frequency as the master plus a low cross-correlation frequency (a few hertz). The base-frequency signals are filtered to reveal the cross-correlation frequency signal, which contains all the same demodulation (M) and phase angle shift (φ) information as the fluorescence emission. Fluorescence Lifetime Imaging Microscopy (FLIM) and Förster Resonance Energy Transfer (FRET) Fluorescence lifetime imaging microscopy (FLIM) is a technique to map the spatial distribution of nanosecond excited state lifetimes within microscopic images. FLIM has proven to be a robust and established technique in modern cell biology for lifetime contrast in ion-imaging, quantitative imaging, tissue characterization, and medical applications [26–28], and it is especially suitable for in situ protein–protein interaction studies using Förster Resonance Energy Transfer (FRET) [29–31]. FRET is a photophysical phenomenon in which energy is transferred from the first excited electronic state (S1) of a fluorophore 10 Biomarkers and Luminescent Probes in Quantitative Biology 191 (called donor D) to another nearby absorbing (but not necessarily emitting) molecule (called acceptor A). Thus, there is a concerted quenching of D and activation of A fluorescence (Fig. 10.10). For this reason, the acronym FRET is often used to designate fluorescence resonance energy transfer. The process involves the resonant coupling of emission and absorption dipoles and is thus nonradiative. That is, it competes with other radiative (fluorescence) and nonradiative pathways for deactivation, resulting in a decrease of the donor lifetime. The energy transfer rate from the donor to the acceptor decreases with the sixth power of the distance and thus is apparent only at distances shorter than 10 nm [32]. At the critical distance where 50% of the donor energy is transferred to an acceptor, the Förster radius, the donor emission and fluorescent lifetime are each reduced by 50%, and sensitized emission (acceptor emission specifically under donor excitation) is increased. Because of its utility in reporting nanometer-scale interactions, FRET has become an important tool in cell biology [33–38]. FRET in cell biology is used commonly to verify whether labeled proteins physically interact: by measur- ing the FRET efficiency, distances on the nanometer scale (a scale within the globular radii of proteins) can thus be estimated using a light microscope. By measuring these effects, FRET microscopic imaging can verify close molecular associations between colocalized donor and acceptor-labeled fusion proteins that are far beyond the resolution of fluorescent microscopy. Obvious difficul- ties in intensity-based FRET measurements in cells is that the concentrations of the donor and acceptor are variable and unknown, the emission band on the donor extends into the absorption and emission band of the acceptor, and the absorption band of the acceptor commonly extends into the absorp- tion band of the donor. A further complication is that only a fraction of the Fig. 10.10. The principle of FRET and dependence of FRET efficiency on donor– acceptor distance 192 M. Zamai et al. donor molecules interacts with an acceptor molecule. These effects are hard to distinguish in intensity-based FRET measurements [39, 40]. In contrast, FLIM-based FRET techniques have the benefit that the results are obtained from a single-lifetime measurement of the donor. These approaches do not need calibration in different cells, and are nondestructive. The important dif- ference between TCSPC and frequency domain FLIM resides in the source of excitation. In frequency domain FLIM (Fig. 10.9c) as the sample is ex- cited by a sinusoidally modulated light, only single photon excitation can be applied, which in living cells induced major photobleaching effects during life- time scans. Alternatively TCSPC-FLIM takes advantage of the two-photon laser subnanosecond pulses (Fig. 10.9a). However, the principal strength of TCSPC−FLIM is the statistical accu- racy, and this is reflected in the ability to fit two or sometimes three expo- nential functions to a particular fluorescence decay. The advantage is that it is often possible (depending on the quality of the data acquired) to determine the ratio of FRET vs. non-FRET lifetimes contained within a single pixel of an image, and therefore to generate a map of interacting vs. noninteracting proteins in the cell (Fig. 10.11). These results may reflect the relative bound and unbound fractions within a particular protein–protein interaction reac- tion. It is difficult or impossible to obtain these results directly using any other FRET or FLIM approach. Importantly for biochemists, these parameters are directly available using TCSPC−FLIM, and if the concentrations of the in- teracting proteins can be estimated (this is no mean feat inside cells, but it can be done), then estimates of association and dissociation constants can be donor + acceptor tD+A pulse donor τD 1.0 EGFP emission 0.8 FRET 0.6 eff = 1_τD+A τD 0.4 mRFP1 0.2 excitation 0.0 450 500 550 600 Wavelength nm Fig. 10.11. FRET measured by TCSFC−FLIM. Representative fluorescence decays are shown for cells transfected with the donor (GPI−uPAR−EGFP) and cell cotransfected with donor and acceptor receptors (GPI−uPAR−EGFP, GPI−uPAR−mRGFP1). FRET efficiency is derived from the ratio between quenched and unqueched lifetimes Intensity (arb. units) 10 Biomarkers and Luminescent Probes in Quantitative Biology 193 made. The resolution of a biexponential FRET system relies in part on the correct selection of fluorophore, particularly for the donor. Many FPs have been shown to have complex fluorescence decays, often biexponential or even more complex, presumably due to protonation or some internal relaxation process, even in a non-FRET system. An example of this is ECFP (enhanced cyan fluorescent protein). This is commonly used as a FRET donor, because of its high quantum yield, long emission tail, and rel- atively blue emission. However, several fluorescence lifetime studies have re- vealed ECFP to have a biexponential lifetime [41, 42], making it difficult to resolve the interacting fraction from a noninteracting fraction in a FRET sys- tem (using FLIM); to do this, four lifetimes would need to be fitted to the data (i.e., two for the non-FRET system, and two for the FRET system), and it is beyond most FLIM measurements to acquire accurate enough data in a practical time period. However, there are FPs useful as FRET donors in FLIM measurements. EGFP (enhanced GFP) has a high quantum yield and a monoexponential decay in a non-FRET system, and has been shown to be a good donor for red FPs, such as dsRed and mRFP [monomeric RFP (red fluorescent protein)] [43] (Fig. 10.12). One advantage of EGFP for many cell top membrane ventral membrane Autocorrelation functions Fluorescence Intensity images 1.00 high 0.75 0.50 ventral membrane low 0.25 top membrane TCSPC-FLIM images 0.00 FRET 0.0001 0.001 0.01 0.1 1 10 tau (s) 0 +/− 8% FRET 18 +/− 8% Fig. 10.12. Study of molecular association and diffusion at the cell membrane of the active GPI-anchored uPAR, fused to EGFP and transfected in human kidney cells, HEK293. Top panels: two-photon images of live cells showing the distribution of the receptor at the ventral and topside of the cell membrane; Representative ACF acquired in two regions at the top and ventral membrane (+) showing that the receptor has longer diffusion time at the cell adhesion site (about 0.9 and 0.1 µm2/s respectively). Lower panels: FLIM analysis of cells cotransfected with the GFP- mRFP1 receptor pair showing that the regions where the receptor is slow diffusing are also regions of high FRET efficiency cps g(tau) norm 194 M. Zamai et al. biologists is that it has been in use for many years, so cDNA constructs are often already made. More recently, the drive for a mono-exponentially decay- ing fluorescent protein led to the development of Cerulean, a variant of ECFP with all the advantages of a cyan donor, but a monoexponential fluorescent decay [44]. FLIM is still an emerging technology, of great interest to the cell biologist. One reason for lack of use of FLIM in biology laboratories is the technical difficulty associated with making reliable measurements. Although there are a number of different approaches to FLIM, some more turn-key than others, TCSPC can resolve additional parameters that make the technical dif- ficulties worthwhile. In particular, the ability to make statistically accurate estimates of the bound vs. unbound fractions of fusion proteins inside living cells ought to be a very attractive prospect for cell biologists and biochemists. 10.2.3 Glossary of Molecular Biology Vector : The DNA “vehicle” used to carry experimental DNA and to clone it. The vector provides all sequences essential for replicating the test DNA. Typical vectors include plasmids, cosmids, phages, and YACs. Plasmid : A circular piece of DNA present in bacteria or isolated from bac- teria. Escherichia coli, the usual bacteria in molecular genetics experiments, has a large circular genome, but it will also replicate smaller circular DNAs as long as they have an “origin of replication”. Plasmids may also have other DNA inserted by the investigator. A bacterium carrying a plasmid and repli- cating a million-fold will produce a million identical copies of that plasmid. Common plasmids are pBR322, pGEM, pUC18. Transfection: A method by which experimental DNA may be put into a cultured mammalian cell. Such experiments are usually performed using cloned DNA containing coding sequences and control regions (promoters, etc.) to test whether the DNA will be expressed. As the cloned DNA may have been extensively modified (for example, protein-binding sites on the promoter may have been altered or removed), this procedure is often used to test whether a particular modification affects the function of a gene. Transient Transfection: When DNA is transfected into cultured cells, it is able to stay in those cells for about 2–3 days, but then will be lost (unless steps are taken to ensure that it is retained – see stable transfection). During those 2–3 days, the DNA is functional, and any functional genes it contains will be expressed. Investigators take advantage of this transient expression period to test gene function. Stable Transfection: A form of transfection experiment designed to produce permanent lines of cultured cells with a new gene inserted into their genome. Usually, this is done by linking the desired gene with a “selectable” gene, i.e., a gene that confers resistance to a toxin (like G418, aka Geneticin). 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Piston, An improved cyan flu- orescent protein variant useful for FRET. Nat. Biotechnol. 22(4), 445–449 (2004) 11 Fluorescence-Based Optical Biosensors F.S. Ligler 11.1 Introduction Biosensors integrate biological molecules with a signal transduction device to produce a signal when a molecular recognition event occurs. This tutorial will focus entirely on biosensors that employ optical devices and incorporate fluorescent mechanisms for signal transduction. Compared with the electro- chemical glucose sensors found in pharmacies world wide, optical biosensors are often more complex and more costly. However, optical biosensors are bet- ter suited for repetitive analysis or continuous monitoring, for interrogation of complex fluids, and for measuring binding events in real time. Optical imaging methods have also been widely adapted to measuring microarrays of recog- nition events; this experience provides a base for the development of highly multiplexed optical biosensors. Optical biosensors not included in the following discussion are, nonetheless, worth mentioning. This group of biosensors is primarily directed at detection of a target without the requirement for a label. Thus performing the assay is simplified by requiring only the exposure of the sample (containing target) to the biological recognition molecule. Noteworthy among these methods are interferometry, surface plasmon resonance, resonant and antiresonance reflec- tometry, and cantilever-based systems [1, 2]. All of these sensors measure a change in optical properties, usually refractive index, at the sensing surface where the recognition molecules are immobilized. Convenient to use, they are excellent tools for measuring reactions in well-defined fluids. They tend to be less sensitive to very small targets or to very large targets that have most of their mass outside the sensing region; improvements in waveguide technology are minimizing these problems. However, nonspecific adsorption of components from complex samples can reduce the sensitivity by creating a significant background signal that must be accurately subtracted. The following tutorial on fluorescence-based optical biosensors is orga- nized into five parts: (1) biological recognition molecules and assay formats, (2) displacement immunosensors, (3) fiber optic biosensors, (4) bead-based 200 F.S. Ligler biosensors, and (5) planar biosensors. These five groups are certainly not an exhaustive coverage of fluorescence-based biosensors, but should provide the reader with at least a general appreciation of the breadth of options available. All of these sensors include the same basic components: biological recognition molecules, a source of excitation light, and an optical readout device capa- ble of discriminating excitation light from emitted fluorescence. Most of the biosensors also include a fluid transfer system to move samples and reagents over the sensing surface. The components and configurations for these systems depend on the geometry of the sensing surface and the degree of automation of the biosensor. 11.2 Biological Recognition Molecules and Assay Formats Optical biosensors have utilized a wide variety of biological recognition mole- cules. With the exception of a few instances where enzyme catalysis is mea- sured, they employ affinity or binding functions. The most often employed molecules are antibodies and oligonucleotides because they are readily avail- able and the binding reactions have undergone extensive analysis at the mole- cular level. However, oligosaccharides, antibiotic peptides, siderophores, and combinatorial molecules have also been utilized. No matter what the binding molecule is, the method by which it is im- mobilized on the sensing surface is very important if that binding function is to be preserved. In general, the surface is first modified with a silane or thiol to provide reactive groups for subsequent attachment of the biomolecules; if possible, this layer also helps prevent subsequent nonspecific binding of the biorecognition molecule that would result in denaturation. Next a crosslinker is attached to the free end of the silane or thiol and binds the biorecogni- tion molecule [3]. For antibody immobilization, many studies have focused on orienting the antibody to avoid interference with the binding site. Although attachment through thiol groups in antibody fragments or the carbohydrate on the region of the molecule distal to the active sites achieves this goal, preventing secondary nonspecific adsorption is often even more critical. In a popular variation of the silane-based immobilization approach, avidin is immobilized instead of the biorecognition molecule, and the biorecognition molecule is biotinylated and bound to the avidin through a high affinity, non- covalent bridge. When plastic is used instead of glass or silicon, nonspecific adsorption of the avidin provides a fully functional surface [4, 5]. The avidin- based approach provides several advantages: (1) the avidin helps to prevent binding of the recognition molecule to the surface and subsequent denatura- tion, (2) the number of linkages of the recognition molecule to the surface can be limited by controlling the number (and possibly the position) of the biotins attached, and (3) the same surface can be used for immobilizing a wide variety of recognition molecules. This latter advantage is particularly useful 11 Fluorescence-Based Optical Biosensors 201 for making arrays of different recognition molecules to capture multiple tar- gets [6,7]. However, the biotin-avidin approach may have disadvantages if the recognition molecules used for capture are very small; in this case, a spacer may be required between the biotin and the capture molecule to make sure that it extends out of the binding pocket on the avidin and into the solution. Another situation where immobilization via an avidin bridge may not be the optimum approach is where the density of the capture molecule is critical for efficient binding – as has been documented with sugars [8] and antimicrobial peptides [9]. Assays with biological recognition molecules for fluorescence biosensors are configured in four basic formats: direct binding, sandwich, competition, and displacement assays. The direct binding format is by far the simplest to implement as it involves only the capture molecule and the target. However, it only works if either the target is inherently fluorescent or if it has somehow been prelabeled, as is the case in many DNA hybridization assays. Direct- binding assays are often used with prelabeled target standards for several reasons: to select the best capture molecules for a particular application by characterizing affinity and specificity of the binding function; to optimize the assay conditions for sensitivity, speed, and reproducibility; and to provide positive controls to monitor assay performance. A variation of the direct- binding assay utilized molecular beacons (reviewed by Yao et al. in [10]). In these assays, a fluorescence energy transfer donor is immobilized on one area of a capture molecule, while an acceptor is immobilized on an area that is adjacent only when no target is bound. When the target is bound, the two fluorophores separate, and a positive signal is generated. The sandwich assay is used for detection of |
targets with at least two bind- ing sites, including large molecules such as proteins, oligonucleotides, bacte- ria, and viruses. In the sandwich assay, the target binds the capture molecule (usually immobilized) and a fluorescent tracer molecule, usually in that order. The formation of the resultant fluorescent complex is measured, while free flu- orescent tracer molecules are either removed or optically excluded from the sensing region. Sandwich assays are described in the majority of publications on optical biosensors. For small molecules with only a single-binding site, the formation of such a sandwich is not possible and either a competitive or displacement assay must be utilized (Fig. 11.1). Two versions of the competitive assay are widely used. In one version, the capture molecules are immobilized, labeled target is added to the sample solution, and the labeled and unlabeled target molecules com- pete for the binding sites on the immobilized capture molecule. In the second version, a target molecule is immobilized, labeled antibody is added to the sample solution, and any target free in solution prevents the binding of the labeled antibody to the immobilized target. In both these versions, the fluo- rescent signal at the surface decreases with the increase in target in the sample solution. Although the fluorescence in solution can also be measured, the flu- orescent component in solution is generally in sufficiently high concentration 202 F.S. Ligler (A) Sandwich (B) Competitive (C) Displacement (D) Direct Binding Target Alexafluor or Cy5-Dye Fig. 11.1. Assay configurations. Four different types of assays are possible using fluorescence biosensors, with variations such as the binding of antibodies to the surface and labeling of antigen in competitive assays in a variation of the format shown in (b) or the use of molecular beacons to provide a signal upon direct binding of antigen in a variation of the format shown in (d). The figure was prepared by Kim Sapsford, George Mason University that the very small decreases caused by low concentrations of target are dif- ficult to measure. One way to avoid this problem is to use a displacement assay. In this configuration, the active sites of an immobilized monoclonal an- tibody are saturated with a fluorescent analog of the target molecule. Pulses of sample are introduced over the surface in a continually flowing stream. When the stream contains the target, the fluorescent analog is displaced from the immobilized capture molecule and measured downstream. Thus a signal is generated that increases in proportion to the amount of target present. Of the most commonly described recognition molecules, oligonucleotides provide the greatest sensitivity. The principle reason is that the target genes can be amplified prior to the detection reaction using a polymerase and se- lective primers. This amplification step not only produces more molecules for the detection reaction, but also increases the ratio of target to background molecules in complex samples. Fluorescence biosensors have been developed that simply measure the process of amplification using dyes that bind to in- creasing concentrations of double stranded DNA, assuming that only target oligonucleotides are replicated. For more selective analysis, the products of the amplification have been measured using capillary electrophoresis [11] or hy- bridization to selective probes in solution or on a surface (reviews in [1,2,10]). The biorecognition molecules most frequently used by the optical biosensor community have been antibodies. Antibodies have excellent selectivity and are relatively stable – unless they are exposed to too much heat or harsh chemicals. However, to find the most appropriate antibody for each appli- cation can be a time-consuming process. Genetic engineering techniques are being used to create a bigger range of specific-binding molecules. Advances in this area include the development of single-chain antibodies, in which the 11 Fluorescence-Based Optical Biosensors 203 heavy and light chain regions of the active site are cloned and produced as a single peptide, [8] and the formation of libraries of shark and camelid ac- tive site peptides, which are naturally composed of a single chain. The latter, in particular, are very stable and usually recover from chemical or thermal denaturation to recover binding function [12]. The disadvantage of using DNA or antibodies for biorecognition is that the user must know exactly what the target is prior to assay development. To expand the repertoire of detectable targets, particularly for toxins and pathogens, molecules that recognize families of targets have been exploited as capture molecules (reviewed in [8]). Sugars and gangliosides have been used with the idea of mimicking the surface of the mammalian cells targeted for infection by pathogen. Antimicrobial peptides, used by amphibians and other organisms for protection against pathogens, have been immobilized and proven to bind families of bacteria and toxins. Other candidates for broader spectrum target recognition include siderophores, phages, and combinatorial peptides. 11.3 Displacement Immunosensors Displacement immunosensors are some of the simplest optical biosensors in both form and function. However, why they work is not straight forward. The basic idea is as follows: First, a monoclonal antibody is immobilized on a solid surface (beads, porous membranes, capillary walls). Second, the active sites on the antibody are filled with a fluorescent version of the target of inter- est. Third, a continuous stream containing injected samples is passed over the immobilized antibodies. (This can continue for days.) When the target is present, it displaces a small proportion of the bound, fluorescent analog. The fluorescent molecules pass downstream and are measured using a sim- ple fluorimeter. Simple antibody kinetics do not explain the behavior: if the fluorescent analog were bound with the dissociation constants typically mea- sured in solution, it would come off in the flow within 20 or 30 min. If it is bound very tightly, it would not come off instantaneously in the presence of target – but it does. The general understanding is that the fluorescent analog is continually in the process of association and dissociation with the antibody. However, it tends to remain in the boundary layer at the solid surface, only diffusing very slowly into the bulk flow. When a target is present, it prevents the rebinding of the labeled analog that is dissociated at that point in time. The result is that, depending on the actual antibody–target pair selected, an operator may be able to add sample after sample and get a positive response for 50 or 60 target-containing samples in series (Fig. 11.2). The displacement immunosensor is particularly useful for the detection of low molecular weight targets such as explosives and drugs of abuse. Biosensors for these applications were commercially available for a time. Sensitivities of part per million even to part per billion have been obtained in assays that only require a minute or two 204 F.S. Ligler Fig. 11.2. Flow immunosensor using beads as the solid support. Target displaces labeled target analog from immobilized antibodies to generate positive fluorescence signal downstream. Reprinted from Ligler and Taitt (2002) with permission from Elsevier to perform. For a review, see Kusterbeck in [1, 2]. Kusterbeck and colleagues have recently developed a flow immunosensor to detect unexploded ordinance in the ocean that has been tested mounted on an unmanned, underwater vehicle (personal communication). 11.4 Fiber Optic Biosensors Fiber optic biosensors utilize two distinct assay configurations for signal gen- eration and measurement: the optrode configuration and the evanescent wave configuration. Both configurations rely on the same principle of total internal reflection (TIR) for light propagation and guiding. However, optrodes use the light shining out the end of the fiber to generate a signal either at the dis- tal face of the fiber or in the medium near the fiber’s end, while evanescent wave sensors rely on the electromagnetic component of the reflected light at the surface of the fiber core to excite only the signal events localized at that surface. The penetration depth of the light into the surrounding medium is much more restricted than for optrodes, while the surface area interrogated is much larger in comparison to optrodes of equal diameter. The result is that evanescent wave biosensors require immobilization of the biological recogni- tion molecules onto the longitudinal surface of the optical fiber core, primarily 11 Fluorescence-Based Optical Biosensors 205 measure binding events, and are relatively immune from interferents in the bulk solution. Optrode-type sensors are adaptable to a much wider variety of measurements and produce a higher power excitation light in the sensing area, but the assays must be formatted to accommodate excitation light in the bulk solution and signal collection from a very small surface, i.e. the fiber tip. A history of the earliest applications of fiber optics for biosensor applications can be found in [13]. 11.4.1 Fiber Optics for Biosensor Applications Total internal reflectance (TIR) is observed at the interface between two di- electric media with different indices of refraction as described by Snell’s law: n1 sinθ1 = sinθ2, (11.1) n2 where n1 and n2 are the refractive indices of the fiber optic core and the surrounding medium, respectively. The angle of light incident through the core of the optical fiber is represented by θ1 and the angle of either the light refracting into the surrounding medium or the internal reflection back into the core is represented by θ2. Total internal reflection requires that n1 > n2 and occurs when the angle of incidence is greater than the critical angle, θc, defined as: ( ) n θc = sin−1 2 . (11.2) n1 This parameter must be considered when designing any biosensor on the ba- sis of optical fibers. However, although Snell’s law describes the macroscopic optical properties of waveguides, it does not account for the electromagnetic component of the reflected light, known as the evanescent wave. The evanes- cent wave is an electric field that extends from the fiber surface into the lower index medium and decays exponentially with distance from the surface, gener- ally over a distance of hundred to several hundred nanometers. For multimode waveguides, the penetration depth, dp, the distance at which the strength of the evanescent wave is 1/e of its value at the surface, is approximated by: λ dp = (11.3) (4π[n2 1/2 1sin 2θ − n2 2] ) where n1 and n2 are refractive indices of the optical fiber and surrounding medium, respectively, and θ is the angle of incidence [14]. The importance of the evanescent wave is its ability to couple light out of the fiber into the surrounding medium, thereby providing excitation for fluorophores bound to or in proximity to the fiber core surface. This confined range of excitation is one of the major factors responsible for the relative im- munity of evanescent wave-based systems to the effects of matrix components or interferents beyond the reaction surface (Fig. 11.3). Love and Button [15] 206 F.S. Ligler Fig. 11.3. Evanescent illumination of fluorescent complexes at the surface of the core of a partially clad waveguide. Reprinted from Ligler and Taitt (2002) with permission from Elsevier Fig. 11.4. Strategies for separation of excitation and emission light paths (Ligler and Taitt, 2002). Figure reprinted with permission from Elsevier originally suggested that dipoles close to the surface could emit approximately 2% of their radiated power (fluorescence) into modes coupled back up the fiber; however, Polercký et al. (2000) have more recently analyzed films of dipoles and concluded that a much higher proportion of the radiation can couple into guided modes. A variety of configurations have been utilized with optical fibers for excit- ing fluorescence and collecting the emitted signal. In Fig. 11.4a, a traditional dichroic mirror is used to separate excitation and emission on the basis of the difference in wavelengths. The excitation light may pass through the mirror, while the emission light is reflected onto a detector (left) or, in a scheme that has generally proven more effective, the stronger excitation light is reflected onto the end of the fiber, while the emitted light passes straight through. In Fig. 11.4b, the excitation light passes through a hole in an off-axis parabolic 11 Fluorescence-Based Optical Biosensors 207 mirror, while the emission light is reflected by the mirror onto a detector [16]. In Fig. 11.4c, a fiber bundle is used between a high numerical aperture sens- ing probe and the optics. The 635 nm excitation light is channeled down the central silica fiber while the emitted higher wavelength fluorescence is coupled back up the surrounding plastic fibers [17]. In Fig. 11.4d, the fiber is |
illumi- nated using a light source normal to the fiber, and the fluorescence is detected at the distal end [18,19]. 11.4.2 Optrode Biosensors Optrode biosensors grew out of a wealth of applications for chemical sensing on the basis of absorbance, fluorescence, and even time-resolved fluorescence. The fibers were relatively easy to work with and facilitated the separation of excitation and emission light; frequently, the fibers themselves were used to transmit only excitation to the sensing region or only emission away from the sensing area as shown in the diagram below. Furthermore, a wide variety of light sources and detectors were used. These included lamps, LEDs, lasers, and diode lasers for excitation and photomultiplier tubes, photodiodes, and CCDs for detection. The optrode configuration is one of the most flexible in terms of the types of assays that can be performed. Optrodes have been used not only with immunoassays and DNA hybridization assays, but also with enzyme assays and for analysis of whole cell function. These assays and configurations have been reviewed by Walt [1, 2] (Fig. 11.5). Probably the most exciting application of optrode technology today is the fiber optic bundles developed by Walt [20] and currently marked by Illumina (www.Illumina.com). In this approach, the ends of the optical fibers in the bundle are etched out, leaving the surrounding cladding to form microwells. Coded beads are placed in the microwells so that hundreds of thousands of as- says can be conducted simultaneously. The assays are being used for genomics, proteomics, and biodefense. 11.4.3 Evanescent Fiber Optic Biosensors The first use of an optical fiber as a biosensor, and one of the first optical biosensors was described by Hirschfeld and Block [21, 22] in the mid 1980s. This type of sensor was also the first biosensor to be fully automated and used remotely. In 1996–1998, Ligler, Anderson, and colleagues developed a biosen- sor payload for a small, unmanned plane and tested it at Dugway Proving Ground, Utah. The payload included a biosensor with four fiber probes, a RAM-air driven cyclone for collecting aerosolized bacteria, an automated flu- idics system, batteries, and a radiotransmitter. They demonstrated that the airborne biosensor could collect a biothreat stimulant, identify it, and radio the data to an operator on the ground in 6–10 min [23,24]. The best known of the commercially available fiber optic biosensors is the RAPTOR-Plus, made by Research International (http://www.resrchintl.com) 208 F.S. Ligler (a) Light source Detector (b) Light source Detector Light source (c) Detector Sensing layer Fig. 11.5. Design principles for optrode biosensors. (a) Two fibers: one carries light to the sensing layer and one carries the signal to the detector. (b) Bifurcated fiber: the biosensing layer is placed on the fused end of the fiber. (c) The biosensing layer is placed on the central fiber, and the surrounding fibers are used to collect the light signals. Reprinted from [1] with permission from Elsevier Fig. 11.6. Schematic (a) and photograph (b) of injection-molded optical fiber probes and fluidics coupon for use with the RAPTOR-Plus. (a and b from [1], reprinted with permission from Elsevier). (c) Photograph of the Raptor Plus with keypad control and result window on the left and the sample port and reagent chamber on the right (Fig. 11.6). This system is proving to be very reliable in terms of long term operation (¿3 years to date, George Anderson, personal communication). In both the 4-probe Raptor and the more recently developed 8-fiber BioHawk, a disposable coupon contains the fiber probes, providing protection dur- ing long-term storage and fluidic channels for automated sample processing. 11 Fluorescence-Based Optical Biosensors 209 The polystyrene probes are inserted after being coated with the recognition biomolecule and dried. The probes are designed to integrate a combination tapered sensing region with a lens for signal [25]. The coupon automatically aligns the probe so that it is in the middle of the fluid channel [26]. In the Raptor, the light emitted from the end of the fiber is focused onto a collec- tion lens in the permanent portion of the biosensor, while in the BioHawk, the light is collected normal to the fiber. The Raptor has been used to assay for a wide variety of targets, including explosives, toxins, clinical markers, and pathogens. It has proven to have sensitivities comparable to traditional enzyme-linked immunoassays (ELISA) using the same recognition molecules, but with assay times in the 10–15 min range instead of 2–4 h. Furthermore, the assays can be performed in the presence of highly complex sample matri- ces, including blood, urine, food homogenates, beverages, ground water, and effluents from air samplers. Results of such assays have been reviewed in [1,2]. 11.5 Bead-Based Biosensors The use of beads as biosensors was first made popular by Raoul Kopelman with his PEBBLES (reviewed by Brasuel et al. in [1]). PEBBLES were small beads originally including ion- or pH-sensitive dyes that could be injected into cells to measure local microenvironments. Imaging was performed using a fluorescent microscope. The Kopelman group expanded the repertoire of PEBBLES to include the use of more specific recognition elements such as calcium ionophores for real-time interrogation of cell function. Building on this approach, other groups developed highly specialized nanoparticles to measure other intracellular functions. One of the more inter- esting particles is composed of quantum dots modified with binding protein and a quenching dye [27]. In this system, a dye molecule is positioned on the binding protein adjacent to the active site. The dye quenches the signal from the quantum dot unless the binding protein binds its target. At that point, the environment of the dye changes so that it can no longer quench the quantum dot, and a positive signal is generated. Medintz et al. used a broad-spectrum quenching dye coupled to genetically modified maltose-binding proteins and demonstrated the capacity of different colored dots to generate signals in re- sponse to different target analytes. The next major breakthrough in bead-based sensing was the development of coded beads that enabled simultaneous measurement of multiple analytes using beads with different amounts of two dyes. Originally developed for com- binatorial chemistry applications, the fluorescent coded beads enabled highly multiplexed assays to be performed in a single fluid compartment. The bun- dled optrode was perfect for making such measurements (see Yu and Walt in [2]) as was flow cytometry [28]. In both the cases, beads of each code or dye ratio are coated with a distinct biorecognition molecule. When the tar- get binds to the bead of that code, another label is introduced using either 210 F.S. Ligler Fig. 11.7. High-density multianalyte bio-optrode composed of microsphere array on an imaging fiber. (a) Scanning force micrograph (SFM) of microwell array fabricated by selectively etching the cores of the individual fibers composing the imaging fiber. (b) The sensing microspheres are distributed in the microwell. (c) Fluorescence im- age of a DNA sensor array with ∼13,000 DNA probe microspheres. (d) Small region of the array showing the different fluorescence responses obtained from the differ- ent sensing microspheres [20]. Reprinted from [1] with permission from American Association for the Advancement of Science a sandwich reaction or a third die, e.g., one that binds to double stranded DNA. Then only the beads with target have three colors and the identity can be read from the bead code (Fig. 11.7). 11.6 Planar Biosensors Planar waveguide biosensors also usually employ evanescent illumination from a coherent source to excite fluorophores at the surface of the waveguide. The resulting fluorescence emission can be measured either by a single photode- tector or by a system capable of imaging, usually a CCD or CMOS camera. A variety of focusing lenses have been used to improve detector response [7, 17,29–32]. The introduction of bandpass and longpass filters was found to improve the rejection of scattered laser light and hence reduce the background of the system [7] (Fig. 11.8). Unfortunately, a side effect of using bulk waveguides and collimated light is the production of sensing “hot spots” along the planar surface that occur where the light beam is reflected, illuminating only discrete regions. These 11 Fluorescence-Based Optical Biosensors 211 Flow Chamber Waveguide Diode Laser Glass Window GRIN Lens Array Emission Filter CCD Imaging Array Fig. 11.8. Detection of emitted fluorescence at right angles to the waveguide inter- face as described by Golden et al., 1999. Reprinted with permission of the Elsevier hot spots have been successfully utilized as sensing regions by Brecht and coworkers in the development of an immunofluorescence sensor for water analysis [33,34]. Feldstein et al. [7,45] overcame this problem by incorporating a line generator and a cylindrical lens to focus the beam into a multimode waveguide, which included a propagation and distribution region prior to the sensing surface. This resulted in uniform lateral and longitudinal excitation at the sensing region. Another method of achieving uniform longitudinal excitation of the sensing region is to decrease the waveguide thickness [30]. When the thickness of the waveguide is much greater than the wavelength of the reflected light, the waveguide is referred to as an internal reflection element (IRE). However, if the thickness of the waveguide is decreased such that it approaches the wavelength of the incident light, and the path-length between the points of total internal reflection become increasingly shorter. At the thickness where the standing waves, created at each point of reflection, overlap and interfere with one another, a continuous streak of light appears across the waveguide, and the IRE becomes known as an integrated optical waveguide (IOW) [35]. Integrated optical waveguides, used frequently in TIRF studies, are mono- mode and prepared by depositing a thin film of high refractive index material onto the surface of a glass substrate. These thin films are typically 80–160 nm in thickness and consist of inorganic metal oxide compounds such as tin oxide [29], indium tin oxide [36], silicon oxynitride [37], and tantalium pentoxide [38, 39]. The light is coupled into these IOWs via a prism or grating arrangement. Studies by Brecht and coworkers compared IRE and IOW-based waveguides and concluded that the integrated optics significantly improved the sensitivity of the system by a factor of 100 [33]. 212 F.S. Ligler 200 ng/ml Ricin 100 ng/ml Cholera toxin 7.3x106 cfu/ml F. tularensis LVS 1.4x105 cfu/ml killed B. abortus 7.1x104 cfu/ml B. anthracis Sterne 100 ng/ml SEB Direction of flow Fig. 11.9. Antibody array used to interrogate six samples for six different biothreat agents. Reprinted from [40] with permission of the Elsevier The planar geometry provides a surface for depositing arrays of detection molecules. This makes it ideal for multianalyte detection. An example of such an array is shown below. In numerous papers reviewed by Sapsford et al. in [1,2], the limits of detection are typically about an order of magnitude bet- ter with good array biosensors, based on planar waveguides than for standard ELISAs using the same reagents. In addition to antibodies and DNA, detec- tion molecules used with planar waveguide biosensors include siderophores, carbohydrates, antimicrobial peptides, and gangliosides [2] (Fig. 11.9). 11.7 Critical Issues and Future Opportunities Fluorescence-based optical biosensors have inherent advantages for discrimi- nating targets in complex samples and for increasing sensitivity compared with label-free methods. Limits of detection can be further reduced using amplifi- cation methods such as enzymes, polymerases, or multifluorophore complexes (e.g., labeled dendrimers as in [41], or virus particles carrying 60 carefully sep- arated fluorophores as in Sapsford et al., 2006). However, labeling and ampli- fication procedures can complicate assay processes, making automation more difficult and possibly increasing fluorescence background. Another method for increasing signal include preconcentration of target prior to analysis using anti-SEB anti-B.an a th n rt a i- c B is .ab a on rt t i u -F s .tula a rn e t n i- s C istx anti-Ricin 11 Fluorescence-Based Optical Biosensors 213 filters, dielectrophoreses, or magnetic bead separations. The latter approach has been very successfully implemented in commercial systems for chemilu- minescence assays (reviewed by Richter [1, 2]). Microfluidics are becoming increasingly used for three particular facets of optical biosensors: 1. Microfluidic devices facilitate methods for automated sample processing, including the mixture of the sample with the fluorescent reagents. 2. They provide an efficient mechanism for manipulating very small volumes, which saves reagent costs. 3. They can deliver the target more efficiently to the sensing surface. Examples of the latter are |
laminar fluid focusing as described by Hofmann et al. [42] and Munson et al. [43] and passive mixing to minimize target dep- letion at the sensing surface as described by Golden et al. [44]. However, care must be taken to make sure that fluidic channels are sufficiently large to avoid clogging by complex samples, and that they have the appropriate surface chemistry to prevent nonspecific adsorption of the target outside the sensing region. Furthermore, the issue of interrogating a sufficient volume to include targets present at low concentrations must never be neglected. Advances in optics offer new opportunities for increased sensitivity and reductions in size and cost. Silicon technology is producing better and better integrated optical waveguides for highly multiplexed analysis. Arrays of sin- gle photon detectors are described by Eduardo Charbon in this volume that may offer the opportunity to detect single targets if the affinity of the recog- nition molecule is sufficiently high and if the background can be sufficiently reduced. Both scattered excitation light and stray fluorescence from molecules not bound in the detection complex can generate “noise” beyond that inher- ent in the optical device itself. The production of devices based on organic polymers is also very exciting because they should be relatively simple to inte- grate biological recognition elements and polymer-based microfluidics to form monolithic, inexpensive, or disposable sensors. Organic LEDs, transistors, and photodiodes are described in this volume in the chapter by Peter Seitz. Finally, systems biology at the molecular level is identifying new targets for analysis that are of importance for medical, environmental, and defense applications. Furthermore, the understanding of how to make rationally de- signed molecules for sensing applications offers new approaches to perform the biorecognition function with appropriate specificity, increased sensitivity, and enhanced stability during sensor storage and use. New fluorophores also provide opportunities for interrogating new types of samples and systems and for generating multiple signals simultaneously. The future for fluorescence-based optical biosensors will be rich. If there is a real limitation, it is only on the ability to synthesize all the emerging technologies into the most useful system for each customer. That ability rests on the willingness of scientists and engineers in universities, government, and industry to work together in interdisciplinary teams and the longer term vision of those that support such efforts. 214 F.S. Ligler Acknowledgments The preparation of this chapter was supported by NRL 6.2 Work Unit 6006. 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Block: US Patent No. 4,447,546 (1984) 48. Ngundi, M.M., N.V. Kulagina, G.P. Anderson, and C.R. Taitt: Expert Rev. Proteonomics 3, 511 (2006). 49. Polerecký, L., J. Hamrie, and B.D. MacCraith: Appl. Opt. 39, 3968 (2000). 50. Sapsford, K.E., P.T. Charles, C.H. Patterson Jr., and F.S. Ligler: Anal. Chem. 74, 1061 (2002). 51. Sapsford, K.E. , C.M. Soto, A.S. Blum, A. Chatterji, T. Lin, J.E. Johnson, F.S. Ligler and B.R. Ratna: Biosens. Bioelectron. 21, 1668 (2006). 12 Optical Biochips P. Seitz The citizens of our modern society are exposed to an increasing number of chemical substances that are potentially harmful or that need to be monitored and kept under check. At the same time, everybody would like to know much earlier, with higher certainty and at lower cost, whether her or his health is affected in any way. For this reason there is a huge demand for very sensitive, highly specific, cost-effective, and rapid methods to detect the concentration of bio-active molecules. Because of the simplicity, efficiency, and speed with which light can be generated, manipulated, detected, and used to sense the ef- fects of many chemical reactions, optical techniques have become the method of choice for many sensing problems involving bio-active molecules. A par- ticularly attractive aspect is the possibility to miniaturize and to parallelize optical measurements, which has led to the very active field of integrated optical sensing [1]. Such miniaturized optical systems for the sensing of bio- active molecules are also known as biochips, although this name is used for many different levels of integration. Applications of these optical biochips in- clude medical diagnostics (for specialized laboratories and increasingly also for home use), contaminant detection in the food industry (e.g., hormones in milk or meat), pharmaceutical research and development (drug screening), environmental and pollution monitoring (e.g., pesticides in water), security and counter-terrorism (detection of chemical and biological warfare agents), as well as process and quality control in industry. The present contribution reviews the different principles and realizations of optical biochips, and it offers an outlook on the potential of monolithically in- tegrating complete optical measurement systems on one single, self-contained biochip of unprecedented complexity and functionality. 12.1 Taxonomy of Optical Biochips 12.1.1 Basic Architecture of Optical Biochips The principle architecture of an optical measurement system for bio-active molecules is illustrated in Fig. 12.1. Coordinated by an electronic control 218 P. Seitz Fig. 12.1. Principle architecture of an optical measurement system for chemical and biochemical sensing, as used for the different types of optical biochips system, which can be a single microcontroller chip in the simplest case, dif- ferent optoelectronic, optomechanic, and microfluidic components interact to detect the effects of a chemical reaction, which is sensed by an optical effect in the system’s transducer section [2]. A light source is emitting either modulated or unmodulated light, which is conditioned by input optics such as focusing, in-coupling, or angle-adjustment elements. In the transducer part, a chemical reaction takes place between the analyte (the target molecule) and a suitable receptor in the microfluidics part of the system. This reaction is observed by measuring a suitable change of any of the optical properties in the fluid vol- ume where the reaction is taking place. The light exiting the interaction region is carrying the information about the analyte concentration, and it needs to be conditioned by appropriate output optics, such as imaging, out-coupling, or angle-adjustment elements, so that the light can be efficiently detected by a photosensor. The resulting electric signal is acquired and converted by an electronic analog/digital circuit, and the digital signal is the processed to ex- tract and communicate the desired concentration information, for example, by displaying it for the user on a screen. Although it is possible to employ many optical effects (see Sect. 12.3 below) in an extended volume of the transducer part, much more control can be exer- cised if this interaction volume is restricted to the immediate vicinity (typically less than 1 µm) of a sensitized surface. This principle is illustrated in Fig. 12.2. The target molecules together with other possibly bio-active molecules are in solution, and the microfluidics system has the task to transport them to and from the transducer volume. There, receptor molecules are chemically bonded to a surface where light is passing to sense the effect of the chemical bonding. These receptors should react in a highly specific way only with the analyte, so that the analyte molecules remain attached to the surface, effectively changing the mass density close to the surface, which can be subsequently measured. Since no additional measure is necessary to determine the concentration of the analyte, this approach is called label-free sensing. A higher detection sensitivity can be obtained if the analytes are tagged with photoactive molecules. Most often, these labels are fluorescent molecules 12 Optical Biochips 219 Fig. 12.2. Illustration of the transducer principles in an optical biochip. Left : Label- free sensing principle. Right : Labeled |
sensing principle that emit light when excited with radiation of shorter wavelength. Alterna- tively, one can also make use of luminescent molecules, such as the famous luciferase, which are causing a biochemical enzymatic reaction as part of which (visible) light is emitted [3]. The term “biochip” is used very broadly in the field and does not describe the particular implementation of the principles described above precisely. Depending on the level of integration, one distin- guishes usually between three different types of biochips: Biochemical Microarray The simplest and still the dominating type of biochip consists of a one- or two- dimensional array of transducer regions, which are sensitized with different receptors on a common substrate [4]. Neither microfluidics transportation systems nor the light sources or the detectors are part of such a biochemical microarray. This type of a biochip can consist, therefore, of up to one million individual transducer regions, each with an area as small as 5× 5 µm2, on an optical biochip with an active surface of 1–2 cm2 [5]. Most often, biochemical microarrays employ fluorescence tagging as the optical transducer mechanism. If they are employed for DNA analysis, optical microarrays are known as DNA- chips or GenechipsTM, and such DNA-chips with half a million transducer regions are commercially available for about ¤200 [5]. Although DNA analysis (genomics) is still the predominant application of optical microarrays, they are increasingly used for protein (proteomics) and cell analysis [6]. Lab-on-a-Chip (µ-TAS) To simplify the practical use of biochips and to reduce the sample volumes required for an analysis, an increasing amount of functionality is placed on the same substrate. A lab-on-a-chip (LOC) combines the microfluidic sub-system, including all elements required to transport, mix, process, separate, and drain fluids, together with the optical transducer part on a single chip [7]. Such an 220 P. Seitz Fig. 12.3. Building blocks of an integrated diagnostic-therapeutic biochip LOC can analyze minute sample volumes measured in femtoliters (equivalent to µm3). In 1990, researchers at Ciba-Geigy (Basel, Switzerland) coined the alternate name Micro Total Analysis System (µ-TAS) for an LOC [8]. Since biochips are disposable items, many LOCs operate with external light sources, imaging optics, and 1D or 2D photosensor arrays (solid-state cameras). However, first LOCs have been realized making use of integrated light sources and/or photosensors, as, for example, in the OSAILS (optical sensor array and integrated light source) principle [9]. Diagnostic-Therapeutic Microsystems The ultimate biochip consists not only of a complete LOC to perform a com- prehensive analysis of biological, chemical, and physical parameters, it also includes a therapeutic BioMEMS (Micro-Electro-Mechanical System), such as a drug delivery or an electro-stimulation sub-system [10] (Fig. 12.3). Before such a vision of an integral biochip becomes reality, many practical problems have to be solved, such as the regeneration of biochemical sensing surfaces, bio-fouling resistance, robustness in the chemically aggressive environment of the body of animals and human beings, long-term power-supply using bio- fuel harvesting or inductive coupling, as well as reliable communications with other implanted systems or the outside world [11]. 12.2 Analyte Classes for Optical Biochips The analytes in our optical biochips were simply illustrated as generic mole- cules in Fig. 12.2. Depending on the actual biochemical function of the type of molecule, different classes of analytes are distinguished [4]. 12.2.1 DNA (DNA Fragments, mRNA, cDNA) To map or sequence the genetic information of a cell, DNA chips contain a large number (up to one million) of different single-stranded DNA fragments 12 Optical Biochips 221 (so-called oligonucleotides), which are immobilized on the substrate surface in discrete spots. Each spot contains several millions of identical oligonucleotides to increase sensitivity. The sample to be tested contains single-stranded ge- netic chains (DNA fragments, mRNA, or cDNA), and they are usually labeled with a fluorescent dye. The genetic chains that match the immobilized oligonu- cleotides bind to the spots on the substrate. The biochip is then illuminated with a suitable wavelength, so that the fluorescence light pattern of the dif- ferent spots allows the determination of the type and concentration of target genetic chains in the sample. 12.2.2 Proteins (Antigens) Since the genetic information contains the instructions that proteins are pro- duced in the body, the identification of proteins – so-called proteomics – is even more important than genomics. For this reason, the utilization of biochips for protein analysis is growing at a rate of about 29%, while the growth of DNA-chips is only about 18% [12]. A protein biochip is similar to a DNA-chip, but instead of oligonucleotides, protein probes (antibodies) for the target proteins (antigens) are immobilized on the individual spots on the biochip’s surface. However, protein biochips are more difficult to handle, less specific, and less sensitive than DNA-chips for several reasons [4]: Proteins are unstable, and they can easily be dena- tured at solid–liquid and liquid–air interfaces. Today’s production techniques for protein probes produce only low-affinity capture antibodies, making the coupling of the antigens to the antibodies not very specific. Since antibodies have usually rather large surface areas for interaction, they show important cross-reactivity between target proteins. DNA samples can readily be am- plified using PCR (polymerase chain reaction), while no such amplification process is known for proteins. The optical principles employed in protein biochips include fluorescence as well as evanescent wave sensing (see Sect. 12.4). 12.2.3 Specific Organic Molecules The same principle used for DNA and protein biochips can be employed, in principle, to determine the concentration of various organic molecules that are of biochemical importance. Suitable probe molecules are fixed on the surface of a biochip. Corresponding target molecules then attach to the probe molecules, and their presence can be measured using the same optical techniques as used for the other types of biochips. 12.2.4 Cell Gene Products (cDNA, Proteins) A cell microarray consists of a number of spots with different types of genetic information, such as DNA strands, plasmids, or adeno viruses. Mammalian 222 P. Seitz cells are grown on these spots, where they can absorb the prepared genetic information – the cells are being transfected – and they are expressing the cor- responding cDNA molecules. This genetic information, in turn, is employed by the cell to produce a specific protein. This protein can influence the func- tioning of the live cell, and various experiments can then be carried out to study the role of the particular protein in the cell’s metabolism. After the particular proteins are expressed in a cell, the cell can be in- cubated with fluorescently labeled antibodies, and the protein presence and concentration can be measured and imaged with fluorescence microscopy. Although cell biochips are only useful for cells that are easily transfected, they overcome many of the deficiencies of conventional protein biochips de- scribed earlier, and cell biochips are adding pertinent information about the cell’s metabolism to our knowledge [13]. 12.2.5 Tissue Instead of preparing a two-dimensional pattern of receptor molecules on a biochip, one can assemble a tissue microarray by combining tissue samples from a large number of specimens on the same substrate that can then be analyzed in a highly parallel fashion, for example, for specific protein expres- sion patterns. The typical diameter of an individual tissue spot on a tissue microarray is 600 µm and its thickness is about 5–8 µm. A particularly important application of such tissue microarrays is the high-throughput molecular profiling of tumors [14]. This technology is already offered commercially, and other applications are seen for large-scale epidemi- ology studies, for the development of new diagnostic and prognostics markers for tumors, for the investigation of biochemical pathways, for drug develop- ment, and for quality control of food, in particular, if genetically modified foods are involved [4]. 12.3 Optical Effects for Biochemical Sensors The many ways in which light interacts with matter can all be exploited for the detection of the concentration of an analyte by making use of suitable chemical reaction between the analyte and a receptor (or detector) molecule, either in the bulk or at the surface of an optical biochemical sensor. The taxonomy of these different optical effects that may be used for biochemical sensing is illustrated in Fig. 12.4, see also [1–4]. In the following, the most popular of these optical effects are briefly described. 12.3.1 Spectral Absorption For a long time, colorimetric sensing principles have been used very widely because the visual color perception of the observer could be exploited, and 12 Optical Biochips 223 Fig. 12.4. Taxonomy of optical effects potentially useful for biochemical sensing no additional instrument was necessary. These optical sensing methods were based on chemical reactions of the analyte with a receptor molecule that resulted in a chemical product with optical absorption properties in the visible spectral range. The resulting color change was then detected and evaluated by the observer or an instrument. Since optical measurement techniques based on spectral absorption are less sensitive than methods detecting phase changes in light, optical biochips do generally not make use of spectral absorption. 12.3.2 Phase Shift A change in the real part of the refractive index causes a phase change of the electromagnetic wave that is used to probe the detection volume of the sensor. This phase change can either be observed in an interferometer setup, it can manifest itself as a change of the polarization properties of incident linearly polarized light, it can change the optical field distribution close to an interface, or it can change the propagation characteristics of a traveling wave. This last effect is most commonly exploited in optical biochips, in particular, through the modification of in- or out-coupling conditions, because it works very well in the confining geometries at the surface of biochips, as illustrated in Fig. 12.2, and it does not require active labeling of the analyte molecule. 12.3.3 Fluorescence Because of the extremely high sensitivity of today’s solid-state image sensors, the detection of very low light levels down to individual photons is technically not difficult to achieve [15]. As a consequence, the use of fluorescent labels to tag analyte molecules leads to a highly sensitive optical measurement method for the concentration of the analyte. Incident light at a certain wavelength is absorbed by the fluorescent labels, and the optical energy is re-emitted at a longer wavelength where it is detected by a sensitive camera. For this reason, fluorescence is one of the most widely used sensing methods in the life sciences and, in particular, in optical biochips. 12.3.4 Luminescence Instead of attaching a fluorescent molecule to the analyte, it is also possible to bond a chemiluminescent molecule to it, such as the well-known luciferase [3]. 224 P. Seitz The resulting enzymatic chemical reaction leads to the emission of (visible) light, without any need for an optical excitation, and this light can be detected by a sensitive camera. Since it is often easier to label an analyte with a fluo- rescent molecule than to label it with a suitable chemiluminescent molecule, luminescence is not very commonly used in optical biochips. 12.3.5 Raman Scattering Raman scattering describes an optical process in which incident light of a certain energy is absorbed by a molecule, and it is re-emitted with a slightly different energy, where the energy difference is a characteristic function of the chemical structure of the molecule. Although Raman scattering is very specific and rich in the information it carries, its efficiency is very low: Typically, only about one in 105 photons is Raman scattered [4]. For this reason, Raman scattering is currently not used in optical biochips. 12.3.6 Nonlinear Optical (NLO) Effects If the electrical field strength is not too high, the optical polarization of a molecule is linearly proportional to the amplitude of the applied electric field. Under illumination with an intense light source, however, the response of the molecule is not any more sufficiently described by a linear effect, and nonlinear optical (NLO) effects come into play. Two NLO effects are of particular importance for biochemical sensing: higher harmonic generation and intensity-dependent multiphoton absorption. Higher harmonic generation involves the mixing of two or more light beams with frequency νi to produce a light beam with frequency ν = Σνi. Multipho- ton absorption describes the simultaneous absorption of two or more photons to reach a particular excited energy state [4]. Since the efficiency of both processes is a sensitive function of a molecule’s environment, these NLO effects can |
effectively be used for biochemical sensing. However, because intense laser light is required, NLO effects are rarely used in optical biochips. 12.4 Preferred Sensing Principles for Optical Biochips Because of the simplicity of the practical realization and the sensitivity of the methods, two basic sensing principles are preferred in optical biochips. (1) Evanescent wave sensing, where an electromagnetic wave is propagating along an optical interface, and a part of its energy is detecting changes in the refractive index in the sample volume. (2) Fluorescence sensing, where incident light is exciting the fluorescence labels on the analytes whose light emission is detected. 12 Optical Biochips 225 Fig. 12.5. Propagation of an electromagnetic wave in a dielectric slab waveguide (left) or as a surface plasmon at a metal–dielectric boundary (right) 12.4.1 Evanescent Wave Sensing Electromagnetic waves can be guided by geometrically confining the space where they should propagate. Two particular configurations are commonly employed in optical biochips, as illustrated in Fig. 12.5: slab waveguides, con- sisting of a stack of laterally extended thin films of dielectric materials, with a core layer whose refractive index is larger than that of the neighboring layers. Alternatively, boundary layers between a dielectric and a metallic medium are used, where the dielectric constant of the metal must be negative in the used wavelength range. At such a boundary, a special guided electromagnetic mode, a so-called surface plasmon, can propagate. In the case of a slab waveguide, the guided modes in most waveguide types can be classified in TE and in TM modes, where the electrical (TE), respectively, the magnetic (TM) field has a dominant component in the lateral direction, i.e., for the coordinate y in Fig. 12.5. Since three of the field compo- nents are zero, the TE modes consist of Ey,Hy, and Hz, and the TM modes consist of Hy, Ex, and Ez. Additionally, it is found that the field components in the propagation direction,Hz, respectively, Ez, are usually quite small [2]. Solving Maxwell’s equation for the slab waveguide geometry and for a given vacuum wavelength λ of the light, one obtains for the jth TE mode an electric field component Ej(x, y, z, t, λ) in y-direction, which is expressed as Ej(x, y, z, t, λ) = Ej(x, y, λ) ei(neff (λ)kz−ωt), (12.1) with the wave vector k = 2π/λ = ω/c. This equation describes a harmonic wave propagating in z-direction with the velocity v = c/neff , where the effec- tive refractive index neff is limited to the range nc > neff > (n1, n2). (12.2) The transverse electric field amplitude Ej(x, y, λ) is called the field profile of the electromagnetic mode number j, and the modes are numbered according to their neff values in descending order, i.e., the mode with the highest neff corresponds to the lowest mode number j. 226 P. Seitz The number of guided modes that can propagate in a dielectric slab wave- guide, their neff values and their field profiles Ej(x, y, λ) depend on the geom- etry of the slab waveguide and the actual distribution of refractive indices. Configurations exist in which only one TE mode (and often one TM mode) can propagate; such structures are called monomodal or mono-mode waveguides. If the dielectric slab waveguide is lossy, for example, through absorption or scattering, the effective refractive index neff in (12.1) has a nonzero imaginary component expressing these losses. High-quality dielectric slab waveguides with negligible losses can be fabricated, with absorption constants that are much lower than 1 cm−1. The surface plasmon, the special electromagnetic mode that can propagate along a metal–dielectric interface, is a single TM mode: At each wavelength only one surface plasmon can propagate, and it requires that the dielectric constant of the metal is negative in the used wavelength range. In the visible region, this is true for metals such as gold, silver, and copper. The propagation velocity in z-direction is given by v = c/neff , and the effective refractive index neff depends on the relative dielectric constants of the metal and of the dielectric material [4]. In contrast to the case of the dielectric slab waveguide, the propagation length of a plasmon is quite limited since the metal is strongly absorbing; the propagation length of a surface plasmon is usually smaller than 100 µm. The importance of the dielectric slab waveguide and of the metal–dielectric interface for optical biosensors is due to the fact that in both structures a part of the wave energy is propagating in the covering dielectric material with refractive index n1. The field strength in the dielectric materials with n1 and n2 is decaying exponentially as a function of the distance to the interface, with a decay length Di (i = 1, 2), for which the field strength is reduced to 1/e, given as Di = √ 1 . (12.3) k n2 eff − n2 i This exponentially decaying field is called evanescent field, and the covering dielectric material is actually the fluidic probe volume whose change in the refractive index is measured. Typical values of the decay length D1 in a dielectric slab waveguide used for optical biosensing are 0.1 µm and slightly larger, about 0.2–0.3 µm, for surface plasmons. Principles of Guided Wave Coupling Since guided waves are characterized by their property that they do not nor- mally leave the waveguide structure, special means are necessary to couple them into or out of the waveguide. The most common coupling approaches are prism coupling, grating coupling, and end-face coupling, as illustrated in Fig. 12.6. Plasmons are almost exclusively coupled in and out with a prism in 12 Optical Biochips 227 Fig. 12.6. Coupling of incident light into a waveguide using a prism (left), a grating coupler (middle), or a focusing lens at the waveguide’s end face (right) the so-called Kretschmann configuration [4]. The advantage is that all com- ponents are planar and easy to coat. Grating coupling is effectively used for dielectric slab waveguides, and innovative sensor configurations can be devised with grating couplers due to the freedom one has with selecting special grat- ing parameters [1]. End-face coupling, however, is not often employed, mostly due to the alignment and stability problems encountered in practice. Waveguides with Prism Coupler Snell’s law for the transmission and reflection of light at an optical interface states that the product of refractive index times the sine of the propagation angle with the surface normal must be identical for incident and for trans- mitted light [16]. Since we demand from a prism coupler that light incident under an angle Θ is guided into a waveguide parallel to the prism’s surface, the sine of the output angle equals unity, and the coupling condition for the prism coupler simply requires that neff(λ) = np(λ) sin θ, (12.4) where np is the refractive index of the prism; a standard glass prism has n ∼ p = 1.5. If a prism coupler is used for an optical biosensor using surface plasmons, it is important that the metal film on the prism is thin enough so that the evanescent wave on the metal’s other side is formed without too many losses. In practice, the thickness of the metal film is typically 40–50 nm. Waveguides with Grating Coupler The same reasoning as for the prism coupling condition applies to the case of a grating coupler with period Λ. The grating coupling condition is given by λ neff(λ) = n2(λ) sin θ − m . (12.5) Λ According to (12.2), the effective refractive index neff of the guided mode is larger than the refractive index of the substrate n2. It is immediately obvi- ous from (12.5) that light can be coupled in from any substrate, even from 228 P. Seitz air (n2 = 1), since the integer diffraction order m, which can also be a neg- ative number, gives us a large degree of freedom for our choice of coupling configuration. In practice, coupling gratings are very shallow and the core layer of the dielectric slab waveguide is rather thin. Typical values are 100–200 nm for the core thickness, 300–500 nm for the grating period, and 5–50 nm for the grating depth [1]. Waveguides with End-Face Coupler Since the core of the waveguide is so thin, the “obvious” end-face coupling of light with a focusing lens, as illustrated in Fig. 12.6, is very difficult to achieve. The alignment tolerances and the mechanical stability of the optical setup must be below 100 nm, which is quite difficult to achieve in practice, where optical biochips should be disposable, quick to read out, and mechanically very robust. Therefore, end-face coupling is not used in commercial optical biosensors. Resonance Condition for Evanescent Wave Sensing From (12.4) and (12.5) it becomes clear how measurements are carried out in optical biosensors: The presence of the analyte subtly changes the refractive index in the sample volume. This change is sensed by the evanescent wave, since it implies a change in neff of the guided wave. A change in neff alters the coupling condition, which can be adjusted either with the in- or out-coupling angle Θ or with the wavelength λ. For this reason, evanescent wave sensing in optical biochips as described above is a resonance method: The amount of light in the guided wave is maximum for the combination of Θ and λ that fulfills the coupling condition for the present value of neff which is dependent on the analyte concentration. The most sensitive methods for measuring this resonance condition are capable of resolving changes in the refractive index of about 10−7 [1, 2]. This corresponds to a mass detection sensitivity of nearly 100 fg mm−2, and the molar concentration of the analyte can be determined with a sensitivity of about 10−11, i.e., the concentration sensitivity is 10−11 mol l−1. Note that it is important to stabilize for temperature variations at these high measurement sensitivities, since ∂n/∂T ≈ 10−4 K−1 for water at room temperature [17]. 12.4.2 Fluorescence Sensing Since fluorescence (or luminescence) sensing requires the imaging of two- dimensional distributions of excited or self-luminous light patterns, the usual sensing technique is fluorescence (or luminescence) microscopy [4]. For this purpose, either conventional optical microscopes or scanning (confocal) op- tical microscopes are employed, where the sample is typically illuminated 12 Optical Biochips 229 through the same lens system through which the fluorescence light is collected (so-called epi-fluorescence excitation setup) [4]. The high demands on the collection and detection efficiency of the emitted light in fluorescence (and luminescence) sensing could also be met with an alternative, as yet little explored sensing approach. Instead of using an inactive substrate such as glass or plastic for the fixation of the receptor molecule, the receptors can be bonded directly to the surface of a solid-state image sensor. In this way, a large part of the light that is emitted close to the pixel surface, usually less than 1 µm from the photosensitive semiconductor material, can be collected and detected. Although this approach has been studied by several companies, no product has yet appeared on the market. This is probably due to the current cost of high-sensitivity image sensors. Since it is expected that the dropping prices and the increased sensitivity of cell phone cameras will soon have an influence on the use of these components in other areas, in particular in the life sciences, this alternate approach to fluorescence (and luminescence) sensing might become important in the future. 12.5 Readout Methods for Evanescent Wave Sensors As mentioned earlier, the analyte concentration is measured in surface plas- mon or in dielectric waveguide sensors by determining the maximum of a resonance effect. The following realizations of optical readout principles have been successfully employed in practice for this purpose: 12.5.1 Angular Scanning The most common methods to satisfy the in-coupling condition (12.4) or (12.5) is to work with a convergent beam of light or to mechanically adjust the angle of a parallel light beam. In both cases, it is assured that a prop- agating mode is created at the sensor surface. While it is much simpler to employ a convergent beam of light, the efficiency is much lower compared to a mechanically adjusted system because the in-coupling condition is fulfilled only for a small fraction of the incident light. No mechanical adjustment must be foreseen on the detection side, however, because |
one- or two-dimensional solid-state image sensors are employed, which can detect the resonance maximum without any loss of light. In Fig. 12.7, a complete such system based on prism coupling is schemat- ically illustrated, together with typical sensor signals acquired with a solid- state line-sensor. In this example, a convergent beam of light is employed, so that the complete system can be realized without any mechanically adjustable components. Such systems are commonly used with surface plasmon sensing. 230 P. Seitz Fig. 12.7. Schematic illustration of an optical biosensor based on prism coupling (left) and typical line-sensor signals acquired with the system (right) 12.5.2 Wavelength Tuning The light loss or the complexity of angular adjustment of the input light can be avoided if the wavelength of the incident light is modified instead. An efficient, all solid-state optical biosensor results, in which the wavelength tuning of the light source is realized by tuning the drive current of a VCSEL (vertical cavity surface emitting laser diode) [18]. 12.5.3 Grating Coupler Chirping Another approach to avoid the mechanical adjustment of the angles of the incident and the out-coupled light is to work with gratings whose period is varied laterally. The use of such chirped gratings ensures that there is always a part of the grating for which the in- or out-coupling condition (12.5) is ful- filled. This technique has the interesting property that the lateral propagation location of the guided wave is a direct measure of the resonance position. For this reason, the method has also been called the “light pointer” approach to optical biosensing [19]. A significant disadvantage of the chirped grating technique is the low effi- ciency with which the available light is used, similar to the convergent beam approach described in Sect. 12.5.1. Only the fraction of light for which the in- coupling condition is fulfilled can propagate in the waveguide and contributes to the sensing signal; the largest part of the optical energy is just transmitted through the grating. 12.6 Substrates for Optical Biochips Although optical biochips are most often disposable devices, the optical sens- ing techniques employed put high demands on the surface quality, the mechan- ical stability, and the robustness of the used materials. At the same time, the materials should be of low cost, easy to process, and of low environmental con- cern. For these reasons, the dominant substrate materials for optical biochips 12 Optical Biochips 231 are glass, fused silica (high purity synthetic amorphous silicon dioxide), or mono-crystalline silicon as used in large quantities for the semiconductor in- dustry. Since the interface between the coupling prism and the biochip does not need to be patterned – it must only be optically flat – glass or fused silica are the preferred substrate materials for the prism coupling approach. This is particularly true for surface plasmon resonance (SPR) biosensors. On the other hand, grating coupling requires the fabrication of coupling gratings on the substrate. Although glass is still the material of choice for this application, the use of plastic or polymeric materials such as PMMA (poly- methylmethacrylate), PDMS (polydimethylsiloxane) or OrmoceresTM is being investigated, since surface gratings can easily be replicated in these materials with nanometer precision. Before these materials are employed routinely in practice, however, several problems regarding their surface roughness, the me- chanical stability, and the physical/chemical inertness of the surfaces need to be resolved satisfactorily. An additional problem that needs to be addressed is the thermal expansion coefficient of polymeric materials, which is typically ten times as large as the one of glass or silicon [17]. Fluorescence and luminescence sensing, in particular, using the special types of microarrays discussed in Sect. 12.2, is most often realized on glass or silicon substrates. Since the materials must neither be transparent nor of particular optical quality, the choice of cost-effective substrates is larger than that for the other optical biosensors. Because of the ease with which the microfluidic functionality can be integrated into plastic or polymeric materials (e.g., by injection molding), these materials are also of interest for fluorescence biosensors [20]. 12.7 Realization Example of an Optical Biosensor/Biochip: WIOS As a practical realization example of an optical biochip and its associated readout system, the wavelength-interrogated optical sensor (“WIOS”) princi- ple described in [18] is explained in more detail. This label-free optical biosen- sor is based on a dielectric waveguide with grating couplers for coupling light into and out of the waveguide. The disposable biochip consists of a 12.5×12.5 mm2 AF45 glass substrate of 0.7 mm thickness covered with a 150 nm thick film of Ta2O5 (n = 2.13) with a grating structure that was previously etched into the glass using dry etching. This grating has a period of Λ = 360 nm and a thickness of 13.2 nm. To minimize interference effects between the input and the output grating, the Ta2O5 film thickness was fabricated 150 nm thicker at the output grating, resulting in a smaller out-coupling angle. Since the complete input/output grating structure measures only 0.8×1.0 mm2, several of them can be produced on the same biochip. The input grating of the sensing pads is selectively sensitized with suitable receptor molecules that are photo-bonded to the input 232 P. Seitz Fig. 12.8. Measurement principle of the WIOS optical biosensor [18] grating surface using OptoDexTM photolinker material. The sensor chip is then placed into a fluidic cell and sealed. The disposable biochip is read out with a compact optical system illus- trated schematically in Fig. 12.8. It consists of a VCSEL light source with cen- ter wavelength of 763 nm and an electrical wavelength-tuning range of 2 nm. The laser beam is collimated and expanded to cover the input pad of the biosensor with a suitable optical sub-system. Because of the limited wave- length tuning range, an additional deflection mirror has been placed into the input beam path, so that a deflection range of 6◦ is obtained. The light from the output grating is collected by an optical multi-mode fiber and transported to a silicon photosensor array for detection, amplification, and analysis. In op- eration, the VCSEL current is modulated with a sawtooth function, resulting in a sawtooth modulation of the VCSEL’s emission wavelength. The photode- tector’s signal is analyzed for the precise temporal position of the resonance peak, and this time shift is a direct measure for the refractive index change and the analyte concentration in the sample volume. As described in [18], the WIOS biochip and readout instrument reach an experimental uncertainty of the refractive index measurement of 3.1× 10−7, corresponding to a mass cov- erage detection standard deviation of about 100 fg mm−2. These values were obtained for the rather small Biotin molecule with a molecular weight of only 244 Da, using neutravidin as the receptor. 12.8 Outlook: Lab-on-a-Chip Using Organic Semiconductors In the future, optical biosensors will contain more and more functionality on the same substrate for increased ease-of-use, higher reliability, and lower cost. This will lead to highly integrated lab-on-a-chip systems that will see a large 12 Optical Biochips 233 number of additional uses at home, in industry, in safety/security, and for environmental applications. Current approaches employ many different mate- rials to perform the various required functions: Optical, mechanical, fluidic, optoelectronic (light generation and detection), signal processing, information display, etc. The resulting solutions are hybrid systems, consisting of many different components, whose integration into a final product is not very cost- effective. There is a promising class of materials, however, that might make it pos- sible to realize a monolithic lab-on-a-chip for low-cost optical biosensors: or- ganic semiconductors, in particular, polymer semiconductors. In the following, a few salient points of this material class and its potential for the fabrication of highly functional yet cost-effective optical biosensors are summarized. 12.8.1 Basics of Organic Semiconductors The scientific field of organic semiconductors is barely 30 years old, and it started with the development of organic light-emitting diodes (LED) with useful conversion efficiency by the American company Kodak. Since then, this field has progressed very rapidly, due to the promise of a new class of materials offering all the optoelectronic functionality required for the realiza- tion of generic photonic microsystems: light generation, light detection, analog and digital electronic signal processing, as well as photovoltaic power gener- ation [21]. In addition, efficient and fast production methods are available, which are often modified printing techniques such as inkjet, silk-screen, or gravure printing. Therefore, it is anticipated that the fabrication costs will be about 100 times lower than that for silicon-based semiconductor circuits in the near future (¤0.1 cm−2 compared to ¤10 cm−2 for standard CMOS chips). However, the performance of organic semiconductors can be significantly lower in several respects than the performance of their inorganic counterparts. The charge carrier mobility is about 1,000 times lower (about 1 cm2 V−1 s−1 compared to 103 cm2 V−1 s−1), the diffusion length of good material is several orders of magnitude smaller (typically 10 nm compared to several 100 µm), the electrical resistivity is significantly higher (since it is much more difficult to dope organic semiconductors), and organic semiconductors deteriorate much more rapidly if exposed to ultraviolet light, to oxygen or to water vapor. For these reasons, organic semiconductors are not a cheaper and simpler replacement of inorganic semiconductors, and it is rather necessary to evaluate the performance of each device individually for the intended application. 12.8.2 Organic LEDs Since a large variety of organic semiconductors are available, organic LEDs (oLED) of many different colors can be fabricated [22]. Their external quan- tum efficiency can exceed 10%, and their power efficiency already surpasses 234 P. Seitz the performance of conventional light sources. The highest luminous efficacy of a white oLED reported in mid 2006 by the Japanese company Konica Minolta is 64 lm W−1, which compares very favorably with the luminous effi- cacy of conventional light bulbs (15 lm W−1), halogen lamps (25 lm W−1), or compact fluorescent lamps (60 lm W−1). Because of the simplicity and the efficiency with which oLEDs – also of different color – can be produced on large surfaces, they are increasingly used for the fabrication of displays in cameras, for PCs, and for TV screens. 12.8.3 Organic Lasers The first optically pumped organic (polymer) laser has been demonstrated already in 1996 [23]. However, because of the high electrical resistivity of organic semiconductors, the electrically pumped organic CW laser working at room temperature remains still elusive. The fact that no intense, narrow-band or even monochromatic light sources can currently be fabricated with organic semiconductors is one of the major shortcomings for the realization of monolithic optical biochips and labs-on-a- chip using solely organic materials. 12.8.4 Organic Photodetectors and Image Sensors Once the problem of dissociation of photogenerated, tightly bound charge pairs (Frenkel excitons) had been solved using finely dispersed polymer blend heterojunctions, the quantum efficiency of photodetectors fabricated with or- ganic semiconductors approached that of inorganic semiconductors [24]. Total external quantum efficiencies exceeding 50% are routinely obtained today in photosensors realized with organic semiconductors. By using thin multilayer structures, it is also possible to produce high-speed photodetectors despite the low mobility of charge carriers in organic semiconductors: Maximum detection frequencies of about 500 MHz have already been demonstrated. A particular advantage of photodetectors fabricated with organic semicon- ductors is the high number of sensors (pixels) that can be realized simultane- ously on a very large substrate. As an example, the cost-effective fabrication of page-sized image sensors has already been demonstrated. Since different organic semiconductors with various spectral responses can be easily integrated on the same substrate, the fabrication of color photo- sensors with good colorimetric performance has been achieved already early in [24]. 12.8.5 Organic Photovoltaic Cells One of the major problems of a practical, disposable optical biochip is electri- cal power supply. If it were possible to replace the conventional electrochemical 12 Optical Biochips 235 batteries of environmental concern with another, less harmful source of electri- cal power, this would add considerably to the attractiveness of an all-organic optical biochip solution. Since it is possible to fabricate efficient photodetectors using organic semi- conductors, one immediately thinks of the monolithic cointegration of photo- voltaic cells on an optical biochip. This is indeed feasible, and all-organic solar cells have been successfully demonstrated. Because of the relatively large frac- tion of power contained in the near infrared spectral |
range of sunlight, where organic semiconductors are notoriously insensitive, the total quantum effi- ciency of organic photovoltaic cells is rather limited. The maximum quantum efficiency demonstrated up to date does not exceed 5%. 12.8.6 Organic Field Effect Transistors and Circuits The first field-effect transistors (FETs) and electronic circuits based on or- ganic semiconductors were demonstrated in 1995 [25]. The electrical charac- teristics of these organic FETs are similar, in principle, to the one of inorganic FETs. However, because of the small charge carrier mobility and low electri- cal conductivity of organic semiconductors, the speed and the current density of organic FETs are much lower than those in their inorganic counterparts. The switching speed is currently limited to a few megahertz, and the high- est frequency at which an organic circuit has worked to date is 13.56 MHz, a frequency of importance for RF-ID electronic tags and inductive data com- munications. An additional problem is the difficulty of cointegrating both n-MOS and p-MOS transistors with comparable performance using compatible organic semiconductors. For this reason, power-efficient CMOS circuits cannot yet be routinely integrated with organic semiconductors, and ultra-low-power elec- tronics still remains in the realm of inorganic semiconductors. 12.8.7 Monolithic Photonic Microsystems Using Organic Semiconductors Organic semiconductors allow the fabrication of similar electronic and opto- electronic components as produced with inorganic semiconductors. In almost no respect are organic components superior in their performance compared to their inorganic counterparts. The huge appeal of organic semiconductors is in the ease, rapidity, and low cost of their production using modified printing techniques on large areas (several square meters) with very little material: only 1 g of organic semiconductor is sufficient to produce about 10 m2 of “plastic optoelectronics.” Another unique property offered by organic semiconductors is the possibility of monolithic fabrication of complete photonic microsystems on a single substrate: Analog and digital electronics, light generation, light detection, and even a photovoltaic power supply can all be integrated on the 236 P. Seitz same chip [26]. Once the diffusion barrier and sealing problem is solved, it will even become possible to produce these complete photonic microsystems on flexible substrates with a lifetime of many years. 12.9 Conclusions and Summary Optical biochips of various integration levels have become important tools in many different areas of the life sciences: Genomics, proteomics, medical diagnostics, pharmaceutical drug screening, contamination detection in the food industry, environmental and pollution monitoring, as well as counter- terrorism all make substantial use of this technology, due to its simplicity, reliability, sensitivity, and low cost. Increasing levels of integration will soon lead to complete, disposable labs-on-a-chip with even higher functionality and performance. The availability of organic semiconductors makes it possible to fabricate complete monolithic photonic microsystems on a single substrate, for example, on an injection-molded piece of polymer containing the entire microfluidics part of a lab-on-a-chip. This will open up many more appli- cation areas to optical biochips, and it will make them highly versatile and indispensable tools of our everyday lives. Acknowledgments This contribution could not have been prepared without the invaluable help of M. Wiki and R.E. Kunz, both at CSEM SA, whose generous support I gratefully acknowledge. References 1. R.E. Kunz, in Integrated Optical Circuits and Components, ed. by E.J. Murphy (Marcel Dekker, New York, 1999), p. 335 2. P.V. Lambeck, Meas. Sci. Technol. 17, 93 (2006) 3. J. Tschmelak et al., Talanta, 65, 313 (2005) 4. P.N. Prasad, Introduction to Biophotonics (Wiley, Hoboken, NJ, 2003) 5. Affymetrix Inc., 3380 Central Expressway, Santa Clara, CA 95051, USA, http://www.affymetrix.com 6. M. Schena (ed.), Microarray Biochip Technology (Eaton Publishing, Natick, MA, 2000) 7. E. Oosterbroek, A. van den Berg (eds.), Lab-on-a-Chip (Elsevier, Amsterdam, Netherlands, 2003) 8. A. Manz, N. Graber, H.M. Widmer, Sens. Actuators B 1, 244 (1990) 9. E.J. Cho, F.V. Bright, Anal. Chem. 73, 3289 (2001) 10. A.C.R. Grayson et al., Proc. IEEE 92, 6 (2004) 11. R. Bahir, Adv. Drug Deliv. Rev. 56, 1565 (2004) 12 Optical Biochips 237 12. H.J. Fecht et al., Nanotechnology Market and Company Report 2003 (WMtech and University of Ulm Publishing, D-Ulm, 2003) 13. J. Ziauddin, D.M. Sabatini, Nature 411, 107 (2001) 14. J.A.B. Kononen et al., Nat. Med. 4, 844 (1998) 15. P. Seitz, in Computer Vision and Applications – A Guide for Students and Practicioners (Academic Press, San Diego, CA, 2000) 16. J.D. Jackson, Classical Electrodynamics, 3rd edn. (Wiley, New York, 1998) 17. D.R. Lide (ed.), CRC Handbook of Chemistry and Physics, 87th edn. (Taylor & Francis, Bota Raton, FL, 2006) 18. K. Cottier et al., Sens. Actuators B 91, 241 (2003) 19. R.E. Kunz et al., Sens. Actuators A 47, 482 (1995) 20. F. Fixe et al., Nucleic Acids Res. 32, e9 (2004) 21. S.R. Forrest, IEEE J. Sel. Top. Quantum Electron. 6, 1072 (2000) 22. R.H. Friend et al., Nature 397, 121 (1999) 23. M.D. McGehee, A.J. Heeger, Adv. Mater. 12, 1655 (2000) 24. G. Yu et al., Synth. Met. 111–112, 133 (2000) 25. E. Cantatore, in Proceedings of ESSDERC 2001, (Nürnberg, Germany, 11–13 Sept. 2001) 26. P. Seitz, U.S. Patent 7,038,235, 2 May 2006 13 CMOS Single-Photon Systems for Bioimaging Applications E. Charbon 13.1 Introduction Recent advances in neurobiology and medical imaging have put an increasing burden on conventional sensor technology. This trend is especially pronounced in time-correlated imaging and other high precision techniques, where timing accuracy and sensitivity is critical. Techniques have been proposed to achieve high speed in conventional charge-coupled devices (CCDs) and CMOS active pixel sensor (APS) archi- tectures [1–4]. Among some of the most successful techniques are ultra-fast low-noise electronic readout circuitries, on-pixel A/D conversion, and local analog electrical storage. However, in general a significant design effort and considerable experience is needed to achieve satisfactory results. Alternatives to conventional CCDs and CMOS APS sensors are single pho- ton counters (SPCs). Several types of SPCs have been known for decades. Among the most successful devices in this class are microchannel plates (MCPs) and photomultiplier tubes (PMTs) that have become the sensors of choice in many applications [5]. Even though they have been studied since the 1960s [6], silicon avalanche photodiodes (SiAPDs) have become a serious competitor to MCPs and PMTs only recently. In SiAPDs, carriers generated by the absorption of a photon in the p-n junction are multiplied by impact ionization; thus producing an avalanche. The resulting optical gain is usually in the hundreds. The main drawback of these devices, however, is a relatively complex amplification scheme and/or complex ancillary electronics. In addi- tion, specific technologies are often required. If biased above breakdown, a p-n junction can operate in so-called Geiger mode. Such a device is known as single photon avalanche diode (SPAD). In Geiger mode of operation, SPADs exhibit a virtually infinite optical gain; however, a mechanism must be provided to quench the avalanche. There ex- ist several techniques to accomplish quenching, classified in active and pas- sive quenching. The simplest approach is the use of a ballast resistance. The 240 E. Charbon avalanche current causes the diode reverse bias voltage to drop below break- down; thus pushing the junction to linear avalanching and even pure accumu- lation mode. After quenching, the device requires a certain recovery time to return to the initial state. The quenching and recovery times are collectively known as dead time. Recently, SPADs have been integrated in CMOS achieving timing res- olutions comparable to those of PMTs [7]. Current developments in more advanced CMOS technologies have demonstrated full scalability of SPAD devices, a 25 µm pitch, and dead time as low as 32 ns. The sensitivity, char- acterized in SPADs as photon detection probability (PDP), can exceed 25– 50%. The noise, measured in SPADs as dark count rate (DCR), can be as low as a few hertz [8, 9]. Thanks to these properties, SPCs based on SPADs have been proposed for imaging where speed and/or event timing accuracy are critical. Such applications range from fluorescence-based imaging, such as Förster Resonance Energy Transfer (FRET), fluorescence lifetime imaging mi- croscopy (FLIM) [10], and fluorescence correlation spectroscopy (FCS) [11], to voltage sensitive dye (VSD) based imaging [12,13], particle image velocimetry (PIV) [14], instantaneous gas imaging [15,16], etc. In the following sections we explore some applications of SPCs in com- parison to conventional sensors, including potential fields of imaging where SPADs can be a compelling implementation aspect of SPCs. We also look at performance and architectural issues that the designer needs to take into account when approaching real problems involving the use of SPCs. 13.2 Spectroscopy Spectroscopy-based imaging has many incarnations. One, very successful one, is known as fluorescence correlation spectroscopy (FCS); a technique used to measure transitional diffusion coefficients of macromolecules to count fluores- cent transient molecules or to determine the molecular composition of a fluid being forced through a bottleneck or a gap. In FCS, a femtoliter volume is exposed to a highly focused laser beam that causes the molecules in it to emit light in a well-defined spectrum. Figure 13.1 shows an example of optical molecular response depending on the size and diffusion pattern of the mole- cule. The physical causes of this behavior are related to the mobility of the ligands. In the first case, free fluorescent ligands are continuously entering and leaving the detection volume. In the second case, macromolecule ligands are less mobile; thus producing slower but highly correlated intensity fluctuations. Figure 13.2 shows an example of typical autocorrelation functions simulated for different molecules [17]. A tighter correlation is observable in the case of low molecular weight ligands. A macromolecule ligand generates a much more relaxed correlation. A mixture of free and bound ligands is shown in the middle curve. 13 CMOS Single-Photon Systems for Bioimaging Applications 241 Time Time Fig. 13.1. Optical response of molecules bombarded by highly focused laser beam. Rapidly diffusing small molecule (left); slow, large molecule, with its large well- defined bursts of optical energy (right) Fig. 13.2. Autocorrelation of fluorescence response. High time resolution is espe- cially useful for sub-nanosecond response dynamics Dual-color cross-correlation FCS measures the cross-correlation of the flu- orescence intensities of two distinct dyes [18]. Thus, it becomes possible to detect different molecules without reference to their diffusion characteristics. More recently, a combination of the two methods has been reported to re- duce the need for a distinct, often rather large mass ratio between the two molecule types [19]. A typical FCS setup is shown in Fig. 13.3. A highly fo- cused laser beam is directed towards the detection volume. The reflected light is applied to the high sensitivity optical sensor through a prism. A digital correlator matches the autocorrelation function with a database of known re- sponses. Generally, in FCS time resolutions of a few tens of picoseconds and sensitivities equivalent to a few hundred photons are needed. Fluorescence Fluorescence 242 E. Charbon Fig. 13.3. Basic FCS setup (gap volume not to scale). Single or multipixel detectors can be used in this setup. With multipixel imaging devices information can be gained from the emissions from the surroundings of the molecule 13.3 Lifetime Imaging Among time-correlated imaging methods, time-correlated single photon counting (TCSPC) is perhaps one of the most used in bioimaging. Mul- tiple exposures are employed to reconstruct the statistical response of matter to sharp and powerful light pulses. The statistics are generally represented in form of a histogram, while light source repetition frequencies may vary from kilohertz to hundreds of megahertz. From the response statistics, biologists generally extract parameters that can be used to characterize the molecule under observation and/or its environment, e.g., the calcium concentration. The study of calcium at the cellular level has made intensive use of flu- orescent Ca2+ indicator dyes. Examples of heavily used dyes or fluorophores are Oregon Green Bapta-1 (OGB-1), Green Fluorescent Protein (GFP), and many others. Calcium concentration can be determined precisely by measur- ing the lifetime of the response of the corresponding fluorophore, when excited at a given wavelength. Using FLIM, for example, one can determine the variation of calcium con- centration in neural cells as a function of a given activity. Figure 13.4 shows a conceptual setup where a neural cell soma is being exposed to light via, for example, a fiber. The fluorophore molecules, previously injected into the cell, exhibit different lifetimes depending upon the calcium concentration in the vicinity of ion channels. There exist several flavors of FLIM based |
on how lifetime is characterized or based on the excitation mode. In one-photon FLIM, for example, only one photon is required to force a state change in the fluorophore molecule [10]. In this case, only a small shift in wavelength is observed between the excitation and response. As a consequence, filtering the excitation pulse from the measurement response may be challenging. In addition, any scattering occurring along the observation path induces pho- tonic noise (usually at the same or close wavelength of fluorescence emissions) 13 CMOS Single-Photon Systems for Bioimaging Applications 243 Fig. 13.4. Calcium environment in and around a cell membrane. Calcium moves through the membrane via so-called ion channels. A fluorophore can detect small variations of calcium concentration into the measurement. Nonetheless, the electro-optical setup required by this method is generally less critical and relatively easy to build. Multiphoton FLIM, even though conceptually known for decades, only re- cently has become an essential tool for neurobiology and other disciplines. Two-photon FLIM has been the method of choice, thanks to the recent ad- vances in laser technology that can now concentrate kilowatts of light power in micrometric volumes of matter. There are several advantages to two- photon FLIM. First, fundamental spatial confinement for the excitation can be achieved; thus allowing one to isolate a single molecule or cluster of mole- cules. Second, because of the reduced average optical powers in play, effects such as photobleaching and photoxicity can be mitigated; thereby enhancing the suitability of the approach. Third, a better penetration in turbid media can be achieved, due to reduced scattering. Fourth, the large spectral differ- ence between in- and out-going radiation simplifies the separation of response from excitation [10]. The main components of typical two-photon FLIM setups are a high-power femtosecond source, generally a mode-locked Ti:Sapphire laser; a SPC; time discrimination hardware; a standard laser-scanning microscope. An example of laser-scanner based FLIM image is shown in Fig. 13.5. In this experiment it was possible to detect intra-cellular chemical waves to help identify inner- workings of pathogens or the impact of certain pharmaceuticals [20]. The binding of neutrophil at the interface of two endothelial cells was monitored at high speed via calcium-triggered chemical waves propagating through the cells. 244 E. Charbon Fig. 13.5. High-speed image sequence of the binding of neutrophil. (Courtesy of H.R. Petty [20]) Fig. 13.6. Coplanar gamma emission where a positron annihilates with an electron. The synchronicity of the emission may be used to infer the loci of the emission using multiple synchronized detectors 13.4 Time-of-Flight in Bio- and Medical Imaging Time-of-flight (TOF) is the time a light ray takes to propagate between two points in the three-dimensional space. There exist several applications requir- ing a precise measurement of TOF to image particular properties of targets and environments. Let us consider two examples of such applications: positron emission tomography (PET) and depth map imaging. In a PET system, a positron is emitted in the tissue being imaged by a variety of substances. An example of one such substance is fluorodeoxyglucose (FDG). This compound is absorbed by human cells, in the brain for example, at different rates based on the operation – whether normal or abnormal – of the cell. After positron emission, a annihilation occurs followed by gamma ray emission. Gamma emission is well defined spatially and temporally. In fact, anni- hilation results in a pair of gamma photons in opposite directions (coplanar property) at exactly the same time. Figure 13.6 shows schematically how emission occurs. To detect gamma emission one usually utilizes a crystal, e.g., lutetium oxy-orthosilicate (LSO), which converts gamma into visible radia- tion. Typically several thousands photons may be released in this fashion. The timing resolution of detection via LSO, however, is generally worsened due to the properties of these crystals. The lifetime of a typical LSO crystal may be as much as 39 ns; hence statistics must be used in combination with detector with high timing resolution to derive the first non-noise photon to be detected (Fig. 13.7). Such photon will give an approximation to the actual moment in time when the gamma radiation is released. 13 CMOS Single-Photon Systems for Bioimaging Applications 245 Fig. 13.7. Typical crystal photoemission probability as a function of time since gamma absorption Fig. 13.8. Result of time correlation that enables the detection of the source of gamma radiation To find the exact location of positron emission, one must monitor all gamma radiation reaching a pair of detectors on an axis at exactly the same time and then cross-correlate all estimated arrival times. The emission loci may be derived by measuring the TOF of the particle with respect to a refer- ence point of known coordinates. The tomography of emissions may be con- structed using conventional Fourier transform techniques in combination with a mechanical system where a detector pair rotates around an axis longitudinal to the cylindrical volume being probed (Fig. 13.8). The pair may be further translated after each rotation cycle to complete a cylindrical scan. The litera- ture on TOF imaging and gated sensors (both CCD and CMOS APS) is very extensive, a few examples are found in [8, 21–23]. 13.5 System Considerations A SPC may be implemented in a number of ways. If the application requires an array of simultaneously operating single photon detectors (SPDs), then a SPAD array is a desirable alternative to PMT or SiAPD arrays in terms of cost, power consumption, and miniaturization. Several demonstrations of CMOS SPAD arrays exist in various technologies [8, 9, 24, 25]. The methods 246 E. Charbon Table 13.1. Performance parameters of a generic time discriminator implemented in a conventional IC technology Measurement Value Unit Timing resolution 1 ∼ 100 ps Timing accuracy < 30 ps Temperature stability 10 ∼ 1, 000 ppm/◦C Dead time 1 ∼ 1, 000 ns to read the output of every SPD pixel to the external world range from pixel random access, similar to APS architectures, to event-driven approaches [9,25] to pipelined readout methods [26]. Time-correlated counting can be performed either via a fast clock or dedi- cated time discrimination circuit (Table 13.1). Time-uncorrelated counting on the contrary requires only a counter whose bandwidth is determined by the inverse of the dead time of the measurement. If counting is performed on-pixel, the operation of the sensor becomes relatively straightforward, but the sili- con real estate may not be utilized efficiently. In principle, this configuration enables the best time utilization since it maximizes parallelism. If counting is performed on-column, SPDs may be implemented in a smaller area and a smaller pitch may be obtained; however, a mechanism must be devised for pixels to share the column counter. As a consequence, the time utilization efficiency is reduced and the readout complexity increases. A good trade-off has been shown to be an event-driven readout system, which works best with low photon fluxes [9, 25]. In this approach a SPD uses the column as a bus, accessing it only when it absorbs a photon. The saturation of the device is thus determined by the bandwidth of the column divided by the number of rows. A chip-wide counting is the technique that allows the simplest architecture, while it is the least time-efficient, since only one SPD may be counting at any point in time. Hence the maximum achievable frame rate is limited by the minimum exposure time per pixel, the total number of pixels, and the speed of the switching electronics. Time-correlated counting may be implemented using fast time discrimina- tion circuits running at lower speed, such as time-to-digital converters (TDCs), time-to-amplitude-converters (TACs), or correlators. A TAC is a device that converts a time interval into a voltage difference. A TDC converts a time interval directly into a digital code. A correlator relates a given impulse to another time-wise, determining in effect their phase difference. There exist a wide variety of implementations for time discriminators, differentiated in terms of performance, size, cost, and power dissipation. Figure 13.9 shows some of the main parameters. In the time-correlated mode of operation, a laser trigger may be used as a reference signal, call it START. The output of a SPD may be used to terminate the time measurement, call that STOP. The main drawback of these devices is their complexity, often requiring hundreds of transistors. 13 CMOS Single-Photon Systems for Bioimaging Applications 247 Fig. 13.9. Detector pair rotating around an axis and longitudinally translating through it On-column and on-chip approaches are, seemingly, the sole option, along with off-chip solutions. An example of an off-chip approach was proposed in [26]. In this design, two technologies are used to independently implement SPADs and time discriminators that are subsequently connected electrically using specific techniques. The development of architectures that support time-correlated modes with some degree of resource sharing is currently underway in many research groups. The main trade-off is at the architectural level, due to the nature of the signal generated by SPDs. In its most general implementation, an SPD generates a digital pulse when it detects a photon. Application-specific optimal architectures are possible, provided a model of the application is built to characterize the performance of the sensor a priori. The sharing of resources may involve a number of pixels, say 4 or 16, or on-demand sharing based upon the reaction of SPDs may be used. Other trade-offs may include the complexity of the time discriminator itself. 13.6 Conclusions With the introduction of CMOS single-photon avalanche diodes, it is pos- sible today to achieve great levels of miniaturization. Not only large arrays of photon counters are now possible, but also very high dynamic range and timing accuracy have become feasible. Thanks to these advances, applications requiring time-resolved single photon detection have become possible. Other applications have reached unprecedented levels of accuracy. We have outlined some of these applications and we have discussed system issues related to these and novel applications in the field of bio- and medical imaging. 248 E. Charbon Acknowledgments The author is grateful to his graduate students and to David Stoppa of FBK. This research was supported by a grant of the Swiss National Science Foun- dation and the Center for Integrated Systems, Lausanne. References 1. T.G. Etoh, Proc. SPIE 3173, 57 (1997) 2. T.G. Etoh et al., IEEE Trans. Electron Devices 50, 144 (2003) 3. S. Kleinfelder, S. Lim, X. Liu, A. El Gamal, J. Solid-State Circuits, 36, 2049 (2001) 4. G. Patounakis, K. Shepard, R. Levicky, in IEEE Symposium on VLSI Circuits, Kyoto, Japan, June 2005, p. 68 5. J. McPhate, J. Vallerga, A. Tremsin, O. Siegmund, B. Mikulec, A. Clark, Proc. SPIE 5881, 88 (2004) 6. R.H. 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Aull et al., Proc. ISSCC, p. 1179, 2006 14 Optical Trapping and Manipulation for Biomedical Applications A. Chiou, M.-T. Wei, Y.-Q. Chen, T.-Y. Tseng, S.-L. Liu, A. Karmenyan, and C.-H. Lin 14.1 Introduction The field of optical trapping and manipulation was started in 1970 by Ashkin et al. [1, 2], when the group at Bell Laboratory demonstrated the following: • A mildly focused laser beam in a sample chamber containing micron-size polystyrene beads suspended in water could attract the bead transversely towards the beam axis and drive the bead to move along the beam axis in the direction of beam propagation (Fig. 14.1a). • A pair of mildly focused laser beams aligned coaxially and propagating in opposite direction with approximately equal optical power could stably trap a bead (suspended in water) in a three-dimensional space (Fig. 14.1b). • A mildly focused laser beam directed vertically upward can stably levitate a bead by balancing the radiation pressure against the weight of the bead in water (Fig. 14.1c). The tremendous growth of the field was further stimulated by another mon- umental paper by Ashkin et al. in 1986 [3], when the group reported that a single laser beam alone when strongly focused with high numerical aper- ture (NA > 0.6) could stably trap micron-sized polystyrene bead in water (Fig. 14.1d). This strongly focused single-beam configuration, known as single- beam gradient-force optical trap or optical tweezers, has since become one of the most widely used optical trapping configurations mainly due to its relative simplicity in experimental setup. Furthermore, potential biomedical applica- tions were soon envisioned when noncontact and noninvasive optical trapping and manipulation of biological samples such as living cells, cell organelles, and bacteria by optical tweezers with a near infrared (NIR) laser beam were successfully demonstrated [4]. In addition to optical trapping and manipu- lation of a single particle outlined above, simultaneous optical trapping of multiple particles as well as independent manipulation of each particle has also been demonstrated by several methods, including, interferometric optical 250 A. Chiou et al. (a) (b) (c) (d) Fig. 14.1. A schematic illustration of optical trapping and manipulation of a mi- croparticle suspended in water. (a) Transverse confinement and axial driving by a mildly focused laser beam; (b) three-dimensional trapping in a counter-propagating dual-beam trap; (c) optical levitation by a mildly focused laser beam pointing up- ward balancing against the weight of the particle; (d) three-dimensional trapping by a strongly focused (numerical aperture NA > 0.6) laser beam patterning [5], interferometric optical tweezers [6], time-multiplexing with a scanning laser beam [7], and holographic optical tweezers [8–10]. In holo- graphic optical tweezers, a set of predesigned computer generated holograms (CGH) in a spatial light modulator is often used to create dynamic multiple focal spots in a three-dimensional space to trap several particles and ma- nipulate each independently. Likewise, a method known as generalized phase contrast (GPC) has been developed by Gluckstad et al. [11,12] for the manip- ulation of multiple particles. Several very entertaining and impressive video demonstrations of multiple particle trapping and manipulation can be viewed at the website of Prof. Miles Padgett at the University of Glasgow in UK (http://www.physics.gla.ac.uk/Optics/). Since the demonstration of optical tweezers (or single-beam gradient-force optical trap), the counter-propagating dual-beam trap has received much less attention until the demonstrations that (1) the counter-propagating dual- beam trap can be implemented fairly simply, without any focusing lens, by aligning a pair of single-mode fibers face-to-face in close proximity (ap- proximately tens of microns to a few hundred microns) inside a sample 14 Optical Trapping and Manipulation for Biomedical Applications 251 chamber containing microparticles suspended in water [13,14], (2) the counter- propagating dual-beam trap can not only trap a red blood cell (RBC), osmot- ically swollen into spherical shape, but also stretch the spherical RBC to deform into an ellipsoid [15–17]. This technique has since been investigated for the measurement of the visco-elastic properties of various cells to correlate with their physiological conditions, including the feasibility of identifying a cancer cell against a normal cell [18]. Although a self-aligned counter-propagating dual-beam trap can be imple- mented, in principle, with a single input beam in conjunction with a nonlinear optical phase-conjugate mirror [19], it has not been further developed due to some practical limitation in the nonlinear optical properties of the materi- als available for optical phase-conjugation. More recently, three-dimensional stable trapping of a microparticle from a single laser beam emitting from a single-mode fiber with different structure fabricated at the tip of the fiber and without any external lens has also been successfully demonstrated [20,21]. Another critical factor that further expands the potential biomedical ap- plications of optical trapping is its capability to measure forces on the order of tens of femto-Newton to hundreds of pico-Newton. This topic will be discussed in greater details in Sect. 14.3 along with potential biomedical applications in Sect. 14.4, which together form the core of this chapter. Other ramifications of optical trapping involve the integration of opti- cal trap with one or more other optical techniques, such as near-field mi- croscopy [22,23], Raman spectroscopy [24,25], and second and third harmonic generation microscopy [26], as well as with microfluidics for a wide range of particles or cells sorting [27–29]. In this chapter, we focus mainly on the measurement of optical forces in op- tical traps and potential applications of optical trapping as a force transducer for the measurement of biomolecular forces in the range of sub-pico-Newton to hundreds of pico-Newton. Partly because of the limited size of this chap- ter, and partly because of our lack of experiences with some ramifications of the subjects, several important topics such as those dealing with integrated photo-voltaic optical tweezers [30,31] and optically driven rotation [32–34] of micronsized samples in optical traps are left out. Interested readers are en- couraged to consult excellent accounts of these topics in the references cited above. For those who want to learn the subject in greater depth and in more details, two excellent review papers [35, 36] each with an impressive list of references can be consulted. In addition, many leading research groups all over the world have posted incredible amount of information with very entertaining and informative video demonstrations of a wide range of fea- tures of optical trapping and manipulation. A few selected video demonstra- tions of various forms of optical trapping can also be viewed at our website (http://photoms.ym.edu.tw). 252 A. Chiou et al. 14.2 Theoretical Models for the Calculation of Optical Forces Optical forces on microparticles in optical traps have been calculated theoreti- cally mainly with the aids of two theoretical models: (1) Ray-Optics Model (or RO Model) [35–38] and (2) Electromagnetic Model (or EM Model) [35,36,39]. The former provides a good approximation for cases where the particle size is larger than the wavelength of the trapping light, whereas the latter rep- resents a better model for cases where the particle size is smaller than the wavelength of the trapping light (often known as Rayleigh particles). For par- ticle size comparable to the wavelength of the trapping light (often known as Mie particles), the calculation is much more involved and the results often less accurate. The key concepts of the RO Model and the EM Model are briefly outlined below further in this section. 14.2.1 The Ray-Optics (RO) Model In the RO Model [35–38], each trapping beam is decomposed into a superpo- sition of light rays, each with optical power proportional to the laser beam intensity distribution. For each specific light ray with optical power P in a medium with refractive index n1, propagating along a direction represented by a unit vector ki, the photon momentum per second is given by (Pn1/c)ki, where c is the speed of light in vacuum. When this light ray enter from a medium with refractive index n1 into another medium with refractive in- dex n2, part of the optical power (RP ) is reflected and the remaining part (1 − R)P is refracted (or transmitted) as is prescribed by the Fresnel equa- tions, where R is the optical power reflectance at the interface due to Fresnel reflection. The photon momentum per second associated with the reflected light ray is thus (RPn1/c)kr, and that associated with the refracted light ray is [(1 − R)Pn2/c]kt, where kr and kt are the unit vectors representing the propagation direction of the reflected and the transmitted rays, respectively. The net optical force imparted on the interface due to the photon momentum change associated with Fresnel reflection (and refraction) of this ray is given by F = (Pn1/c)ki − {(RPn1/c)kr + [(1 − R)Pn2/c]kt} (14.1) or, equivalently, F = (Pn1/c){ki − [Rkr + (1 − R)(n2/n1)kt]}. (14.2) In the RO Model, the contribution of the optical force from each constituent ray of the trapping beam, as is prescribed by (14.1) or (14.2) above, is added vectorially to obtain the net optical force on the particle by the beam at each interface. For a dielectric microsphere (such as a polystyrene bead or a biolog- ical cell) in water, the difference in refractive indices of the two media is often fairly small such that only the momentum change due to the Fresnel reflection 14 Optical Trapping and Manipulation for Biomedical Applications 253 Fig. 14.2. Fresnel reflection and refraction of a light ray at the interface of a dielectric microsphere and the surrounding medium in the Ray-Optics (RO) model of optical trapping Reflected light Surface force α α Transmitted Laser Incident light F F β net net light n1 n2 (>n1) Laser (a) Laser Laser Laser Laser Fnet (b) Fig. 14.3. Optical force distribution at the surface of a dielectric microsphere (adapted from Guck et al. [17]) (a) in a single Gaussian beam; (b) in a pair of counter-propagating Gaussian beams with equal optical power at the first (or front) interface and that due to the transmission at the sec- ond (or back) interface are considered to simplify the calculation (Fig. 14.2). In general, optical forces originate from photon momentum changes due to reflection, giving rise to a net force, which mainly pushes the particle along the direction of the beam propagation; such a force is often referred to as the scattering force. In contrast, optical forces originating from photon momen- tum changes due to refraction often give rise to a net force pointing towards the direction of the gradient of the optical field; such a force is often referred to as the gradient force. Example of theoretical results for the optical force distribution on the surface of a dielectric microsphere due to a Gaussian beam and due to a pair of counter-propagating Gaussian beams with equal optical power are depicted in Fig. 14.3. Theoretical results from a more recent refined 254 A. Chiou et al. 90 2 90 a) 120 60 b) 2 120 60 1 1 150 30 150 30 180 z 0 180 z 0 210 330 210 330 240 300 240 300 270 270 w/R=1 w/R=2 Fig. 14.4. Non-uniform force distribution with four distinguished peaks at the surface of a dielectric microsphere in a pair of counter-propagating Gaussian beams with equal optical power. Optical power P = 100mW, particle radius R = 3.30 µm, refractive index of surrounding medium n1 = 1.334, refractive index of the particle n2 = 1.378, beam radius = w. (a) w/R = 1; (b) w/R = 2. (adapted from Bareil et al. [38]) model [38], however, indicate several distinguished spikes in the force distri- bution on the surface of a dielectric microsphere in a counter-propagating dual-beam trap (Fig. 14.4). In the counter-propagating dual-beam trap, the particle is stably trapped along the axial direction by the balanced scattering forces from the two beams, and in the transverse direction by the co-operative action of the transverse gradient forces from the two beams. In the single-beam gradient-force optical trap, the particle is stably trapped axially, balanced by the axial scattering force against the axial gradient force, and transversely by the transverse gradient force. To compare the efficiency of different optical trapping configurations, the optical force as given in (14.2) above is often written as F = (Pn1/c)Q, (14.3) where the parameter Q, known as the trapping efficiency, represents the frac- tion of photon momentum per second associated with the trapping beam, which is converted into the net trapping |
force. In most of the optical trapping configurations that have been demonstrated to date, the transverse trap- ping efficiency is typically on the order of 0.1–0.001, whereas the axial trapping efficiency is typically a factor of 3–10 smaller than the transverse trapping efficiency. The exact value of the trapping efficiency depends on many fac- tors, including the numerical aperture (NA) of the trapping beam, the size of the particle, the refractive index of the particle and that of the surrounding fluid, etc. 14 Optical Trapping and Manipulation for Biomedical Applications 255 Fig. 14.5. (a) The EM model of optical trapping of a dielectric microsphere in a Gaussian beam in terms of the minimum potential energy analogous to (b) the force that pulls a dielectric block partially filling a parallel plate capacitor connected to a constant voltage source 14.2.2 Electromagnetic (EM) Model In the EM model [35, 36, 39], stable trapping of a dielectric microparticle in one or more laser beams can be understood in terms of the potential energy minimum of the dielectric particle in the electric field associated with the opti- cal beam. The physical mechanism that a dielectric microparticle is attracted towards the region with high optical intensity is analogous to the electrostatic force on a dielectric block partially filling a parallel plate capacitor connected to a constant voltage source (Fig. 14.5). In cases where the dielectric constant of the particle (or the block in the example of the capacitor) is smaller than that of the surrounding medium, the direction of the force is reversed, i.e., the particle is repelled away from the region of high optical intensity and attracted towards the region of lower optical intensity. For detailed mathematics of the EM model, please consult the references cited above. 14.3 Experimental Measurements of Optical Forces Optical forces on microparticles can be measured by several methods. In gen- eral, under identical experimental condition, optical forces scale linearly with optical power. For a particle in the vicinity of the trap center of a stable three-dimensional optical trap, the net optical force along the direction of each orthogonal axis can be approximated by a Hookean optical spring; a set of optical force constants (or spring constants, kx, ky, and kz) can thus be used as a convenient set of parameters that specifies the three-dimensional optical force field within a small volume surrounding each stable equilibrium position of the particle in the trap. Techniques to measure the optical forces and optical force constants are described below in this section. 14.3.1 Axial Optical Force as a Function of Position along the Optical Axis One way to measure the axial optical force on a particle in a counter- propagating dual-beam trap as a function of position along the optical axis 256 A. Chiou et al. is to trap a particle, drive the particle to move back-and-forth along the op- tical axis (by alternately turning on and off one of the trapping beams), and record the position of the particle as a function of time z(t) via a precalibrated CCD camera or any other position sensing device. By taking the first and the second derivatives of the experimental data z(t) either numerically (or ana- lytically after curve-fitting z(t) with an appropriate polynomial), one obtains the velocity v(t) and the acceleration a(t) of the particle. The axial optical force Fao(t) can then be determined from the following equation of motion: ma(t) = Fao(t) − 6πηrv(t) (14.4) or, equivalently, Fao(t) = ma(t) + 6πηrv(t), (14.5) where r is the radius of the particle, η is the coefficient of viscosity of the fluid surrounding the particle, and the mass of the particle m = 4πr3ρ/3, (ρ = the density of the particle). The axial optical force as a function of position Fao(z) can be deduced directly from Fao(t) and z(t) by eliminating the parameter t from these data. If we assume a symmetric counter-propagating dual-beam trap with iden- tical optical power from each beam, the axial optical force from each beam will be identical (except for a sign-change in both the direction of force and the direction of the relative position of the particle). The net axial optical force, when both beams simultaneously act on the particle, is simply the vector sum of the contribution from each beam. As an example, a set of experimental data of z(t) is given in Fig. 14.6a and the axial force deduced by the prescrip- tion described above is given in Fig. 14.6b. In this specific example, the linear 130.0 4.0 120.0 110.0 3.0 100.0 2.0 90.0 80.0 1.0 70.0 60.0 0.0 50.0 40.0 −1.0 30.0 −2.0 20.0 10.0 −3.0 0.0 −10.0 −4.0 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 −60.0 −40.0 −20.0 0.0 20.0 40.0 60.0 Time (S) Position (mm) (a) (b) Fig. 14.6. (a) Experimental data showing the axial position of a particle as a function of time in a counter-propagating dual-beam trap as the particle was driven to move along the optical axis by switching off one of the trapping beams; (b) optical forces on the particle from each beam (represented by the upper and the lower curves) and the net force when both beams were on (represented by the middle curve) as a function of its relative position along the optical axis. The curves in (b) were deduced from (a) as is described in the text Position (m m) Force (pN) 14 Optical Trapping and Manipulation for Biomedical Applications 257 regime where the axial optical force can be approximated by a linear optical spring is approximately 80 µm, and the axial optical force constant (given by the slope of the curve in the linear regime) is approximately 0.05 pN µm−1. Since the total trapping power is approximately 25 mW, the maximum axial trapping efficiency (at the edge of the linear region) is estimated to be approx- imately 0.02. Although the optically driven motion method described above is relatively simple and the experiment can be easily repeated under identical experimental condition so that the measurement error can be minimized by averaging over several sets of data from repeated experiments, it suffers from several shortages: (1) it can not be used for the measurement of transverse trapping force, (2) it assumes that the particle moves linearly along the op- tical axis through out the course of motion, which is a good approximation only within a certain range ∼100 µm; besides, the linear motion of the parti- cle will be perturbed randomly (and unavoidably) by the thermal fluctuation (Brownian force) and systematically by the gravitation force. 14.3.2 Transverse Trapping Force Measured by Viscous Drag An earlier method to measure the transverse optical trapping force on a par- ticle in a single-beam gradient-force optical trap (or optical tweezers) is by trapping a particle and dragging it off transversely with increasing fluid flow speed until the particle escape from the trap [40]. The fluid flow can be imple- mented either by translating the sample chamber (mounted on a piezoelectric- translational stage) along with the fluid surrounding the trapped particle or with the aid of a microfluidic pump. At the threshold speed (vth) of the fluid flow when the particle escapes from the optical trap, the transverse op- tical trapping force (Fto) is maximum and is equal to the viscous dragging force (Fdrag). If the particle radius (r), the coefficient of viscosity (η) of the fluid, and the threshold flow speed (vth) are known, the maximum trans- verse optical trapping force is given by the viscous dragging force; hence, Fto = Fdrag = 6πηrvth (the Stokes’ Law). A systematic error introduced by the presence of the side wall of the sample chamber (which is ignored in the equation above) can be corrected if the distance of the trapped particle from the wall is known [35, 36]. Even though the random error introduced by the thermal fluctuation of the particle can be reduced in principle by statistically averaging over repeated measurements, in practice, the standard deviations associated with the experimental results are relatively high (∼50% or higher). A schematic diagram illustrating the underlying principle of this method is given in Fig. 14.7, and a video demonstration of this method can be viewed at our website (http://photoms.ym.edu.tw/). 14.3.3 Three-Dimensional Optical Force Field Probed by Particle Brownian Motion In the previous sections on the measurement of optical forces, particle fluctu- ation due to Brownian motion was regarded as an unavoidable nuisance that 258 A. Chiou et al. Fig. 14.7. A schematic illustration of the measurement of transverse optical force by balancing the optical force against a viscous fluid drag leads to random errors in the experimental results. Interestingly, the three- dimensional Brownian motion of a particle in an optical trap, when tracked and measured with high precision, can be used to probe the three-dimensional optical force field on the particle with relative ease and with fairly high preci- sion. This method, also known as photonics force microscopy [41,42], is briefly described below in this section. Tracking and the measurement of three-dimensional Brownian motion of a particle in an optical trap are often accomplished with the aid of one or more position sensing devices such as a quadrant photo-diode (QPD). From the projection of the particle position fluctuation as a function of time [x(t), y(t), and z(t)] along each orthogonal axis, the corresponding optical force constant (kx, ky, and kz) along each axis can be deduced by any one of the three methods: (1) root-mean-square (rms) fluctuation of the particle position [36], (2) the Boltzmann distribution of the particle in a parabolic potential well [41,42], (3) the temporal frequency analysis of particle position fluctuation [43,44]. To limit the length of this chapter, only the second approach based on Boltzmann distribution of the particle in a parabolic potential well is discussed below in this section. An experimental setup used by the authors to probe the three-dimensional optical force field on a particle trapped in a fiber-optical counter-propagating dual-beam trap is shown in Fig. 14.8 [14]. A laser beam (cw, λ = 532 nm from a Nd:YVO4 laser) was expanded and collimated via a 3X beam-expander (3X BE) through a half-wave plate (λ/2) and a polarizing beam splitter (PBS) cube to split into two beams with equal optical power, and each coupled into a single-mode fiber (NA = 0.11) via a single-mode fiber coupler (SMFC). The output ends of the two fibers were aligned so that the two laser beams exiting from the fibers formed a pair of counter-propagating beams along a common optical axis inside a sample chamber where microparticles or living cells were trapped in PBS solution. The distance between the two fiber end-faces was kept at 125 µm. Portions of the trapping beams scattered by the trapped par- ticle were collected by a pair of orthogonally oriented long-working distant objectives (LOBI and LOBII, ×50, NA = 0.42) and projected onto a pair of quadrant photodiodes (QPDI and QPDII). LOBI/QPDI and LOBII /QPDII collected the scattered lights and tracked the position of the trapped particle 14 Optical Trapping and Manipulation for Biomedical Applications 259 y z CCD x QPD BS Nd : YVO4 λ=532nm LOB SMFC 3X BE /2 PBS SMFC LOB λ QPD Lamp Fig. 14.8. A schematic illustration of the experimental setup for the mapping of three-dimensional optical force field on a particle in a fiber-optical dual-beam trap projected on the xz-plane and the yz-plane, respectively (the xyz co-ordinate system is depicted in the upper left corner in Fig. 14.8). Besides, the LOBI was also used for imaging the trapped particle on a CCD camera (752×582 pixels) via incoherent illumination from a lamp. To obtain the conversion factor that converts the output voltage of the QPD into the particle displacement, we trapped the microparticle of interest on the optical axis in the middle of the end-faces of two optical fibers via equal laser power from the two fibers, and momentarily turned off one of the laser beams to drive the particle (by the remaining single beam) along the optical axis towards the opposite end-face of the optical fiber. The second beam was subsequently turned on to drive the particle back along the optical axis towards the original equilibrium po- sition at the middle of the fiber end-faces. As the microparticle was driven back along the optical axis under the illumination |
of both beams, the corre- sponding output voltage of the quadrant photodiode and the position of the microparticle on the CCD camera were recorded simultaneously. An illustra- tive example of such a calibration curve (i.e., QPD output voltage vs. particle position recorded on the CCD) is depicted in Fig. 14.9, which shows a linear dependence with a slope of approximately 0.986 µm V−1 within the region of approximately 3.4 µm. Besides the Brownian fluctuation, the accuracy of this approach is mainly limited by the determination of the particle position from the incoherent image of the illuminated particle on the pixelated dig- ital CCD camera. The calibration method described above was carried out during the return trip of the particle (from a point offset from the center to the center equilibrium position) when both laser beams were on to ensure that the particle was evenly illuminated from both sides. Recently the authors have tracked the three-dimensional Brownian motion of polystyrene and silica 260 A. Chiou et al. Fig. 14.9. An example of a calibration curve for the conversion of the QPD output voltage into the axial position of a particle in a fiber-optical dual-beam trap beads of different sizes as well as Chinese hamster ovary cells trapped in a fiber-optical dual-beam trap, and analyzed their position distribution to ob- tain the optical force field approximated by a three-dimensional parabolic potential well [14, 15]. Under the parabolic potential approximation and the classical Boltzmann statistics, the associated optical force constant along each axis can be calculated from the following two equations [41]: ρ (z) = C exp [−E (z)/KBT ] , (14.6) / E (z) = −KBT ln ρ (z) + KBT lnC = kzZ 2 2, (14.7) where ρ(z) is the probability function of the trapped particle position along the z-axis, C is the normalization constant, E(z) is the potential energy function along the z-axis, KB is the Boltzmann constant, and kz is the optical force constant along the z-axis. Identical set of equations also apply for the x-axis and the y-axis. As a specific example, a set of experimental data representing the parabolic potential E(x) and E(z) of the optical force field on a 2.58 µm diameter silica particle in a fiber-optical dual-beam trap (total trapping power = 22 mW, distance between the fiber end-faces = 125 µm,) is depicted in Fig. 14.10. The solid lines represent the theoretical curves based on (14.7) given above; the corresponding optical force constants, defined by (14.7), were kx = 0.16 pN µm−1 and kz = 0.04 pN µm−1. The force constant along the optical axis is weaker than those along the transverse axes, which is consistent with the theoretical results reported earlier [45]. This is also true in the case of optical tweezers [46]. For clarity sake, the experimental data for y-axis along 14 Optical Trapping and Manipulation for Biomedical Applications 261 0.0E+0 -5.0E-21 -1.0E-20 -1.5E-20 -2.0E-20 -2.5E-20 -3.0E-20 -3.5E-20 -1.3E-7 -5.0E-8 0.0E+0 5.0E-8 1.3E-7 Distance from the trapping center (m) Fig. 14.10. Experimental data representing the parabolic optical potentials E(x) (the inner set of data points with a steeper slope) and E(z) (the outer set of data points) on a 2.58 µm silica particle. The solid lines represent the theoretical fits based on (14.7) with the theoretical fit with ky = 0.15 pN µm−1 are not shown in Fig. 14.10; the data and the theoretical curve for y-axis essentially overlap with those for the x-axis. 14.3.4 Optical Forced Oscillation Optical forced oscillation of a trapped particle is another powerful technique for the measurement of optical force and biological forces such as those as- sociated with protein–protein interaction. Optical forced oscillation (OFO) refers to the trapping and forced oscillation of a microparticle in an optical trap [47, 48]. Transverse force constants of the optical trap can be measured with fairly high precision by measuring the oscillation amplitude and the rel- ative phase (with respective to that of the driving source) of the particle as a function of oscillation frequency. OFO can be used as a convenient tool for the measurement of protein–protein interaction [49, 50] and of protein–DNA interaction with the aid of a microparticle coated with appropriate protein of interest. In this section, we introduce the basic principle and the experimental implementation of optical forced oscillation. Optical forced oscillation has been implemented by one of the two follow- ing methods: (1) by trapping a microparticle in a conventional single-beam gradient-force optical trap (or optical tweezers) and scanning the trapping beam sinusoidally with a constant amplitude and frequency (Fig. 14.11a), (2) by trapping a microparticle in a set of twin optical tweezers and chop- ping one of the trapping beams on-and-off at a constant chopping frequency Optical Potential Energy (J) 262 A. Chiou et al. Fig. 14.11. A schematic diagram of optical forced oscillation via (a) oscillatory optical tweezers and (b) a set of twin optical tweezers (Fig. 14.11b). In both cases, the steady-state oscillation amplitude and the relative phase (with respect to that of the driving source) of the oscillating particle can be conveniently measured with the aid of a quadrant photo-diode in conjunction with a lock-in amplifier. By changing the driving frequency, typically in the range of approximately a few hertz to a few hundred hertz, the oscillation amplitude and the relative phase of the oscillating particle can be plotted as a function of frequency. In general, the experimental data fit fairly nicely with the theoretical results deduced from a simple theoretical model of forced-oscillation with damping [48, 50]. By coating a microparticle with a protein of interest and allowing the oscillating particle to interact with protein receptors on a cellular membrane, the method described above can be used to measure the protein–protein interaction, which can be modeled as an- other linear spring in parallel to the optical spring (Fig. 14.12). The equation of motion of a particle, suspended in a viscous fluid, trapped, and forced to oscillate in oscillatory optical tweezers, can be written as mẍ = −βẋ − [ ( k x (t) − )] A eiωt , (14.8) where m is the mass of the particle, βẋ = 6πηrẋ is the viscous drag prescribed by Stokes’ law, η is the coefficient of viscosity of the surrounding fluid, r is the radius of the particle, k is the optical spring constant in the linear spring model, and A and ω are the amplitude and the frequency of the focal spot of the oscillatory optical tweezers, respectively. In the case of a set of twin optical tweezers with the particle interacting with a cell as is illustrated schematically in Fig. 14.12, the equation of motion of the particle can be written as [48] mẍ = −βẋ − k(eiωt + 1) (x − x1) − (k + kint)(x − x2), (14.9) 2 where x1 and x2 represent the position of the force center of optical tweezers 1 and 2, respectively; ω is the fundamental harmonics of the chopping frequency; 14 Optical Trapping and Manipulation for Biomedical Applications 263 k k m int 1 k2 B1 B2 x1 x2 Fig. 14.12. A simplified linear spring model of a particle simultaneously acted upon by optical forces from a set of twin optical tweezers and a cellular interactive force when a cell is in contact with the particle in the equilibrium trapping position of the tweezers on the right kint is force constant of the protein–protein interaction modeled as a linear spring; and the rest of the symbols are the same as those described earlier in association with (14.8). In (14.9), we have assumed that the equilibrium position of the protein–protein interaction at the cellular membrane coincides with that of the second optical tweezers; this was accomplished experimentally by bringing the cell to touch the particle when it was stably trapped in the second tweezers as is illustrated schematically in Fig. 14.12. The steady-state solution at the fundamental frequency of (14.8) and (14.9) above can be expressed as [ ] x (t) = D ei(ωt−φ) (14.10) and [ ] x (t) = D ei(ωt−φ) − x0, (14.11) respectively, where D is the amplitude, φ the phase lag (with respective to that of the driving source), and x0 the equilibrium position of the oscillating particle. Solving (14.8) with an assumed solution in the form of (14.10) gives D (ω) √ k = (14.12) D (0) k2 + ( 2 βω) − mω2 Y YY 264 A. Chiou et al. and ( ) φ (ω) = tan−1 βω − . (14.13) k mω2 Likewise, solving (14.9) with an assumed solution in the form of (14.11) gives D(ω) √ 3 2k′ = D(0) (k′ − (14.14) mω2)2 + (βω)2 and { /[ ]} φ(ω) = tan−1 βω k′ − mω2 , (14.15) where k′ = (3/2)k + kint. Experimental data representing the amplitude and the relative phase of the oscillation of a trapped 1.5 µm polystyrene particle in water as a function of frequency along with the corresponding theoretical fits based on (14.12) and (14.13) given above are depicted in Fig. 14.13a,b, respectively. The op- tical spring constant kx was deduced from the best fit to be 7.69 pN µm−1, from the amplitude data, and 7.61 pN µm−1, from the phase data. The opti- cal spring constants measured by different methods described above agree to within approximately 8%. In contrast to the Brownian motion method, the re- sults obtained by optical forced oscillation method depend only on the relative amplitude of particle oscillation and not on its absolute value. Besides, the signal-to-noise ratio is significantly enhanced by the phase locking technique. Although the transverse optical spring constant along any specific direction can be measured via oscillatory optical tweezers with reasonable accuracy by scanning the trapping beam along the selected direction, the two-dimensional 1.1 50.0 45.0 1.0 40.0 35.0 0.9 30.0 25.0 0.8 20.0 0.7 15.0 10.0 0.6 5.0 0.0 0.5 −5.0 1.0 10.0 100.0 1.0 10.0 100.0 Oscillation Frequency (Hz) Oscillation Frequency (Hz) (a) (b) Fig. 14.13. (a) The normalized amplitude, (b) the relative phase of the opti- cal forced oscillation of a polystyrene particle (diameter = 1.5 µm) suspended in deionized water as a function of the oscillation frequency. The solid curves are the- oretical fits Normalized Amplitude Relative Phasse (degree) 14 Optical Trapping and Manipulation for Biomedical Applications 265 Table 14.1. A comparison of optical spring constants measured by different methods Method Analysis kOT(x) kOT(y) (pN µm−1) (pN µm−1) Brownian motion Displacement variance 7.07 6.31 Potential well 7.28 6.84 Power spectrum 7.21 6.88 Optical forced oscillation Amplitude 7.69 Phase 7.61 optical force field E(x, y) can be simultaneously mapped, and the associated spring constants kx and ky conveniently measured in stationary optical tweez- ers via the Brownian motion method. The optical spring constants kx and ky on the transverse plane obtained from the Brownian motion analysis agree with each other to within approximately ±6%, which is consistent with the earlier theoretical and experimental results [46, 51, 52] and also with those reported previously for the case of a fiber-optical dual-beam trap [14,45]. As an example, transverse optical spring constants, kx and ky, for a polystyrene particle (diameter = 1.5 µm) suspended in deionized water and trapped in optical tweezers (characterized by λ = 1064 nm, NA = 1.0, trap- ping optical power = 2 mW) obtained by different methods are compared and summarized in Table 14.1. 14.4 Potential Biomedical Applications Potential biomedical applications of optical trapping and manipulation in- clude (1) trapping and stretching a cell to measure its visco-elastic property and to correlate with its physiological condition, (2) trapping two cells to mea- sure cell–cell interaction, (3) trapping two protein-coated microparticles to measure protein–protein interaction, (4) trapping one protein-coated particle to interact with membrane proteins on a cell to measure the protein–protein interaction at the cellular membrane, (5) trapping two beads with a segment of DNA molecule stretched in between to measure the interaction dynamics of the DNA with proteins injected into the sample chamber. Selected video demonstrations of some of the features listed above can be viewed at the website of the authors’ lab (http://photoms.ym.edu.tw). In general, optical trapping and manipulation promise to provide one or more of the following unique features in biomedical applications: 1. Micropositioning : One can guide cell–cell, cell–molecule, or molecule– molecule interactions in terms of where and |
when the interactions take place. The interaction can therefore be measured within a time point on the order of “second” after the initiation of the molecular interaction. 266 A. Chiou et al. 2. Living cell capability : The setup is readily adaptable for living cell imaging and manipulation. 3. The technique can be easily integrated with laser spectroscopy and microscopy for single cell measurement with subcellular resolution. 4. Single molecule resolution: Several protocols have been developed to measure the force exerted by a single biomolecule or the interaction between a molecular pair. New methods and algorithms have been developed enabling the extraction of single molecular results from the bulk measurements or other population data. 5. The assay is sensitive to the functions of the biomolecules: One can monitor in real-time the dynamics of molecular binding or of biological forces associated with molecular interactions to examine the functions of the biomolecules. 6. The assay is sensitive to the conformation of the biomolecules: One may obtain structural information of the biomolecule by integrat- ing optical tweezers with other photonics modalities (such as time- resolved fluorescence microscopy and Raman spectro-microscopy). 7. The possibility of noncontact and noninvasive cell compliance mea- surements may lead to a new paradigm for assessing the physiology and the pathology of living cells with potential clinical applications. In this section, we outline a few selected examples of the applications of optical trapping for protein–protein interactions, protein–DNA interactions, and cellular trapping and stretching. 14.4.1 Optical Forced Oscillation for the Measurement of Protein–Protein Interactions As an example of protein–protein interaction, we present in this section the application of a set of twin optical tweezers to trap and oscillate a ConA (lectin)-coated polystyrene bead and to measure its interaction with glycopro- tein receptors at the cellular plasma membrane of a Chinese hamster ovary (CHO) cell [50]. The bead was trapped between two quadratic potential wells defined by a set of twin optical tweezers and was forced to oscillate by chop- ping on-and-off one of the trapping beams. We tracked the oscillatory motion of the bead via a quadrant photodiode and measured with a lock-in amplifier the amplitude of the oscillation as a function of frequency at the fundamen- tal component of the chopping frequency over a frequency range from 10 to 600 Hz. By analyzing the amplitude as a function of frequency for a free bead suspended in buffer solution without the presence of the CHO cell and compared with the corresponding data when the bead was interacting with the CHO cell, we deduced the transverse force constant associated with the optical trap and that associated with the interaction by treating both the optical trap and the interaction as linear springs. The force constants were determined to be approximately 2.15 pN µm−1 for the trap and 2.53 pN µm−1 14 Optical Trapping and Manipulation for Biomedical Applications 267 6 Con A coated bead BSA coated bead 5 4 3 2 1 0 0 5 10 15 20 25 30 35 40 45 50 55 Time (s) Fig. 14.14. The time-dependence of the relative amplitude of a polystyrene bead (diameter = 2.83 µm) executing optical forced oscillation at 50Hz in the vicinity of a CHO cell. The experimental data for ConA-coated bead are denoted by solid squares and the data for a BSA-coated bead are denoted by “*” for the lectin–glycoprotein interaction. When the CHO cell was treated with lantrunculin A, a drug that is known to destroy the cytoskeleton of the cell, the oscillation amplitude increased with time, indicating the softening of the cellular membrane, until a steady state with a smaller force constant was reached. The steady state value of the force constant depended on the drug concentration. As an illustrative example, the time dependence of the relative amplitude of a polystyrene bead (diameter = 2.83 µm) oscillating at 50 Hz in the vicinity of a Chinese hamster ovary cell is depicted in Fig. 14.14 for the case of a BSA- coated bead and that of a ConA-coated bead. The decay of the oscillation amplitude in the case of the ConA-coated bead is a manifestation of the interaction of ConA protein with the glycol-protein on the cellular membrane of the CHO cell. 14.4.2 Protein–DNA Interaction The interaction of proteins with DNA plays a pivotal role on a number of important biological processes, including DNA repair, replication, recombina- tion, and segregation. Many different proteins can be used as models for such investigations. For example, one can optically trap-and-stretch a segment of dsDNA to analyze the dynamics of homologous DNA search and strand ex- change reaction mediated by RecA protein. In the study of protein–DNA interaction, micron-size polystyrene beads are often attached to the ends of each DNA sample, one at each end, to serve as handles for optical tweezers to trap and stretch the DNA sample between the beads. A schematic illustration of a dsDNA segment stretched between a fixed (large) bead (with diameter ∼20 µm) and an optically trapped small Relative Oscillation Amplitude (V) 268 A. Chiou et al. Optical tweezers Unwinding dsDNA polystyrene particle ~20µm d(t) RecA binding ssDNA polystyrene particle ~2µm Fig. 14.15. A schematic illustration of a segment of a DNA molecule stretched between two polystyrene beads (with one bead fixed to the bottom of the sample chamber and the other bead trapped in optical tweezers) for the measurement of the dynamics of the interaction of the DNA sample with RecA proteins injected into the sample chamber bead (with diameter ∼1 µm) for the study of the dynamic of the interaction with RecA-proteins carrying complementary segment of ssDNA is shown in Fig. 14.15. During the interaction, the stretched length and the elastic con- stant of the DNA can be measured in real-time by monitoring either the po- sition of the trapped particle as a function of time (in the case of stationary optical tweezers) or the amplitude and the phase of the oscillating particle as function of frequency and time (in the case of oscillatory optical tweezers) (Fig. 14.16). One of the technical challenges of this approach is the lack of an effective method to avoid the adherence of multiple-strings of DNA molecules in parallel between the two beads. Although this can be achieved in principle by optimizing the ratio of the concentration of the DNA samples and that of the beads during the sample preparation, it is very tedious and inefficient in practice. In the steady state, the bead is expected to wonder around an equilibrium position (slightly displaced from the optical axis) dictated by the balancing of the transverse gradient optical force and the elastic force of the stretched DNA molecule. The position fluctuation of the bead is a manifestation of the Brownian force acting on the bead and on the DNA molecule as well as the conformational change of the DNA molecule. Specifically, the stretching or the contraction as well as the winding or the unwinding of the double-strand DNA molecule is revealed by the translational and the rotational motion of the bead of which the former can be measured via a quadrant photodetector (QPD) or any other optical position sensing detector, while the later can also be mea- sured optically by using a bead with optical birefringence in conjunction with 14 Optical Trapping and Manipulation for Biomedical Applications 269 Fig. 14.16. A schematic illustration of the measurement of the dynamics of DNA– protein interaction by stretching the DNA between two beads, trapping one of the bead, and tracking its Brownian motion with a quadrant-photodiode any polarization sensitive detection scheme. Instead of a bead with optical birefringence, any bead lacking axial symmetry illuminated by a laser beam is also expected to generate an optical scattering signal modulated (in intensity) with a frequency component at that of the rotational frequency of the bead. The basic principle of this approach is to detect (by analyzing the transla- tional and the rotational motion of the trapped bead) the dynamic of the conformational change of a stretched double-strand DNA molecule interact- ing with RecA-ssDNA filaments injected into the surrounding buffer solution. The generic goal of the experiments, such as the one outlined above, is to un- derstand the relationship between the physical properties and the biochemical (or functional) properties of DNA at a fundamental level. 14.4.3 Optical Trapping and Stretching of Red Blood Cells As mentioned earlier, a red blood cell (RBC) can be trapped and stretched with the aid of a fiber optical dual-beam trap-and-stretch to measure its visco- elastic property. A schematic diagram to illustrate the application concept of a fiber-optical dual-beam trap-and-stretch in conjunction with a microfluidic flow chamber, fabricated with poly dimthylsiloxane (PDMS), to inject the RBC one at a time for such measurement is shown in Fig. 14.17. Photographs of an experimental set up in our laboratory are depicted in Fig. 14.18. Pre- liminary experimental results illustrating the morphological change of human RBC samples, osmotically swollen into spherical shape, as a function of the optical power are shown in Fig. 14.19 along with the experimental data on the fractional change in length of the major axis and the minor axis of a 270 A. Chiou et al. Optical Coupler splitter Objective Laser Lamp CCD 60X NA=0.85 = 1064nm Filter Single Model fiber Flow Chamber Top view of the flow chamber; material: Poly Dimthylsiloxane (PDMS) Fig. 14.17. A schematic illustration of the experimental setup for simultaneous trapping, stretching, and morphological deformation measurement of red blood cells in a fiber-optical dual-beam trap Fig. 14.18. Photographs of the experimental setup illustrated schematically in Fig. 14.17 human RBC sample as a function optical power. From these experimental data, the product (Eh) of the elasticity (E) and the thickness (h) of the RBC cell membrane was estimated to be Eh = 2.4× 10−4 N m−1, with the aid of a simple theoretical model [15–17]. 14 Optical Trapping and Manipulation for Biomedical Applications 271 19mw 43mw 66mw 92mw 118mw 146mw 174mw 0.15 y=0.0164x−0.0069 0.1 2 R =0.9808 fit line 0.05 fit line ρ (0)/ρ (0) ρ(π/2)/ρ (π/2) 0 RBC Diameter : 6.5±0.5mm −0.05 −0.1 y=−0.0157x−0.0077 Eh=2.4¥10−4 N/m 2 R =0.9782 −0.15 0 0.1 0.2 0.3 0.4 20.5 0.6 0.7 peak stress (N/m ) Fig. 14.19. Photographs of a human red blood cell (RBC), osmotically swollen into spherical shape, trapped and stretched at different laser power in a fiber-optical dual-beam trap, along with the experimental data showing the relative change in length along the major and the minor axes of the RBC sample as a function of laser power 14.5 Summary and Conclusion In this chapter, we give a brief historical overview of optical trapping and manipulation followed by an outline of two theoretical models, namely the ray optics model and the electromagnetic model, along with several methods for the experimental measurement of optical forces with sub-pico-Newton res- olution. Unique features of optical trapping and manipulation for biomedical applications are outlined and examples including the measurement of the dy- namics of protein–protein interaction and protein–DNA interaction as well as the simultaneous trapping, stretching, and morphological deformation mea- surement of human red blood cells are highlighted. Acknowledgment Research in optical trapping and manipulation carried out by the authors is supported by the National Science Council of the Republic of China Grants NSC 95-2752-E010-001-PAE, NSC 94-2120-M-010-002, NSC 94-2627-B-010- 004, NSC 94-2120-M-007-006, NSC 94-2120-M-010-002, and NSC 93-2314- B-010-003, and the Grant 95A-C-D01-PPG-01 from the Aim for the Top University Plan supported by the Ministry of Education of the Republic of China. References 1. A. Ashkin, Phys. Rev. Lett. 24, 156 (1970) 2. A. Ashkin, J.M. Dziedzic, Appl. Phys. Lett. 19, 283 (1971) 3. A. Ashkin, J. Dziedzic, J. Bjorkholm, S. Chu, Opt. 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Rossi, P. Matteini, and F. Ratto 15.1 Introduction Lasers have become already a widespread tool in many operative and ther- apeutic applications in the surgical field. The so-called “minimally invasive” laser techniques provide remarkable improvements: in these, laser surgery is performed inside the human body through small incisions by means of optical fibre probes and endoscopes, or laser tools are proposed as a replacement for conventional tools to minimize the surgical trauma, such as in the case of laser-induced suturing of biological tissues. The aim of these procedures is to improve the quality of life of patients, by decreasing healing times and the risk of postoperative complications. Joining tissue by applying laser irradiation was first reported at the end of the 1970s, when a neodymium/yttrium–aluminium–garnet (Nd:YAG) laser was used for the microvascular anastomosis of rat carotid and femoral arteries. Ever since, laser tissue welding has been evaluated in several experimental models including blood vessels, skin, nerve, intestine, uterine tube, and so on [1, 2]. Laser welding has progressively assumed increased relevance in the clinical setting, where it appears to be a valid alternative to standard surgical techniques. At present, there are many applications of tissue welding that are beginning to achieve widespread acceptance. Many types of lasers have been proposed for laser tissue welding. Infrared and near-infrared sources include carbon dioxide (CO2), thulium–holmium– chromium, holmium, thulium, and neodymium rare-earth-doped-garnets (THC:YAG, Ho:YAG, Tm:YAG, and Nd:YAG, respectively), and gallium aluminium arsenide diode (GaAlAs) lasers. Visible sources include potassium- titanyl phosphate (KTP) frequency-doubled Nd:YAG, and argon lasers. The laser energy is absorbed by water at the infrared wavelengths and by haemoglobin and melanin at the visible wavelengths, thereby producing heat within the target tissue. As the temperature rises, the extracellular matrix of the connective tissue undergoes thermal changes that lead to the welding of the wound (Fig. 15.1). 276 R. Pini et al. Fig. 15.1. Structural modifications induced in fibrillar collagen of connective tissue by temperature rise. Normal triple helix collagen molecules are packed in a quarter- staggered manner and connected by covalent bonds to form a collagen fibril (left). When heat is applied, hydrolysis of intramolecular hydrogen bonds occurs, which results in the unwinding of the triple helices (middle). The first step (1) leads to a shrinkage effect parallel to the axis of the fibrils. At higher temperatures, covalent cross-links connecting collagen strands break, resulting in a complete destruction of the fibrillar structure (right) and causing relaxation of the tissue (step 2) Laser tissue welding has been shown to possess several advantages com- pared with conventional closure methods, such as reduced operation times, fewer skill requirements, decreased foreign-body reaction and therefore reduced inflammatory response, faster healing, increased ability to induce regeneration, and an improved cosmetic appearance. Laser welding also has the potential to form complete closures, thus making possible an immediate watertight anastomosis, which is particularly important in the case of vas- cular, genito-urinary tract, and gastrointestinal repairs. A watertight closure also discourages the exit of regenerating axons and the entry of fibroblasts. Lastly, laser welding can be used endoscopically and laparoscopically to ex- tend the range of its applications to cases in which sutures or staples cannot be used. However, despite the large number of experimental studies reported in the literature, very few of them have reached the clinical phase. This is mainly be- cause of the lack of clear evidence of the advantages of laser-assisted suturing against conventional methods, and because of a low reproducibility of results. The damage induced in tissues by direct laser heating and heat diffusion and the poor strength of the resulting welding are the main problems as far as 15 Laser Tissue Welding in Minimally Invasive Surgery and Microsurgery 277 future clinical applications of the laser-assisted procedure are concerned. In fact, as water, haemoglobin, and melanin are the main absorbers of laser light within tissue, the heating effect is not selectively limited to a target area, and all irradiated tissues are heated. For instance, the CO2 laser has been used for laser repairs of thin tissues because of its short penetration depth (<20 µm). However, for thicker tissues, welding has been achieved only by irradiating with high laser power and longer exposure times, thus inducing high levels of heat damage [3]. The emissions of other near-infrared lasers, such as Nd:YAG and diode lasers, are more suited to the welding of thicker tissues. In any case, control of the dosimetry of laser irradiation and of corresponding temperature rise is crucial to minimize the risk of heat damage to the tissue and to generate strong welds. Two advances have been useful in addressing the issues associated with laser tissue welding: the application of laser-wavelength-specific chromophores and the addition of endogenous and exogenous material to be used as solder. The use of wavelength-specific chromophores enables differential absorp- tion between the stained region and the surrounding tissue. The advantage is primarily a selective absorption of laser radiation by the target, without the need for a precise focusing of the laser beam. Moreover, lower laser ir- radiances can be used because of the increased absorption of stained tissues. Various chromophores have been employed as laser absorbers, including in- docyanine green (ICG) [4], fluorescein [5], basic fuchsin, and fen 6 [6]. The use of a near-infrared laser – which is poorly absorbed by biological tissues – in conjunction with the topical application of a dye with an absorption peak overlapping the laser emission, is a very popular setting for the laser welding technique. Diode lasers emitting around 800 nm and ICG have been used in corneal tissue welding in cataract surgery and corneal transplant [7,8], vascu- lar tissue welding [9–11], skin welding [4, 12], and in laryngotracheal mucosa transplant [13]. Laser welding by means of solders, namely “laser soldering,” makes use of exogenous solders as topical protein preparations. This makes possible a bonding of the adjoining and underlying tissues when activated by laser light. The extrinsic agents provide a large surface area over which fusion with the tissue can occur, thus favoring the approximation of the wound edges that eventually heal together in the postoperative period. Useful welding materials include blood [14], plasma [15], fibrinogen [16] and albumin, which is the one most frequently employed [5,17]. Several studies have demonstrated that the addition of an albumin solder to reinforce laser tissue repairs significantly improves postoperative results [5,18]. Moreover, incorporation into the protein solder of a laser-absorbing chromophore makes it possible to confine the heat into the area of solder application, which reduces the extent of collateral heat damage to adjacent tissues. ICG-doped albumin has become an increasingly popular choice in the last decade [17]. The laser is used to denature the protein immediately after application of the protein solder to the wound site, thus yielding a bond at the solder–tissue interface. 278 R. Pini et al. Another improvement of traditional protein solders relies on the use of syn- thetic polymers mixed with serum albumin. This provides better flexibility, as well as improved repair strength over albumin-protein solders alone [19]. Poly(lactic-co-glycolic acid) (PLGA) is a class of synthetic biodegradable polymers that are easily degraded in vivo and are eliminated through normal metabolic pathways. These materials can be employed also as drug-delivery systems by adding a range of dopants including antibiotics, anaesthetics, and various growing factors that may enhance the rate and quality of wound heal- ing [20]. A more recent development in laser tissue welding is the use of an infrared temperature feedback control of the laser device. Diagnostic feedback via sur- face temperature measurements has been used to control laser power delivery to achieve well-defined welding protocols [21]. Although surface temperature monitoring may be important for a primary control of the energy delivery, it does not reveal dynamic changes occurring below the tissue surface, which is where the weld actually occurs. This problem has been addressed by em- ploying numerical models of laser-tissue interactions on the basis of directly measured surface-temperature data [21,22]. The combination of experimental and simulated data made it possible to characterize surface and bulk tissue heating, thus allowing for a prediction of the temperature rise within the irradiated tissues. Photochemical welding of tissues has also been investigated as an alterna- tive method for tissue repair without direct use of heat. This technique utilizes chemical cross-linking agents applied to the cut that, when light-activated, produce covalent cross-links between collagen fibres of the native tissue struc- ture. Agents used for photochemical welding include 1,8-naphthalimide [23], Rose Bengal, riboflavin-5-phosphate, fluorescein, and methylene blue [24]. Studies of photochemical tissue bonding have been conducted for articular cartilage bonding [23], cornea repair [24], skin graft adhesion [25] and for repairing severed tendons [26]. As far as the mechanism of tissue welding by means of laser light is con- cerned, a first distinction must be made between: (1) laser tissue welding with or without the addition of exogenous chromophores, (2) laser tissue soldering, and (3) photochemical tissue bonding. For all three cases, the exact under- lying mechanism is not fully understood. Conversely, several hypotheses do exist that are based on a few electron and optical |
microscopy observations and on some in vitro studies. It is widely accepted that laser tissue welding is primarily a thermal process [27]. Thermal modifications of biological components within the connective tissue have been mainly monitored by means of microscopy. The microscopic data reported in the literature on laser-welded tissues can be schematically split into two groups on the basis of different modifications of the collagen matrix observed upon laser welding. In certain studies, a full homogenization of the tissue was observed, in which the loose structure of the collagen fibrils was lost following laser welding 15 Laser Tissue Welding in Minimally Invasive Surgery and Microsurgery 279 Fig. 15.2. “Hard” laser welding approach resulting in a complete homogenization of the tissue. The extracellular matrix is schematically represented by an array of black dots simulating cross-sectioned collagen fibrils embedded in the ground substance, as in a typical connective tissue (left). Laser irradiation leads to hardly recognizable collagen structures completely (C) or partially (P) coagulated (right). Temperatures above 75◦C are usually recorded in these cases. The mechanism proposed relies on the adhesion upon cooling between proteins degradated by laser heat, which, acting as microsolders, seal the wound [4,10,28,29] (Fig. 15.2). In this context, fibrils fused together and morpholog- ically altered revealed a complete denaturation of the collagen matrix [3, 30]. As a result of laser irradiation, cell membranes were also disrupted causing leakage of the cellular proteins. In these cases, operative temperatures of over 75◦C were induced at the welded area [31]. The wound sealing mechanism has been attributed to the photocoagulation of collagen and other intracellular proteins, which act like micro-solders or endogenous (biological) glue, which form new molecular bonds upon cooling [28]. In other studies, less severe modifications of the collagen matrix have been observed (Fig. 15.3). In these cases, the collagen fibrils were still recog- nizable and not or only partially swollen [3, 32, 33]. Operative temperatures around 60–65◦C were generated at the welded site inducing no (or only par- tial) denaturation of fibrillar collagen [33–35]. In water-bath experiments, op- posed tendon specimens were thermally bonded, obtaining maximum tensile strength at similar temperature values [36, 37]. The most common interpre- tation of the working mechanism is an unravelling of the collagen triple-helix followed by “interdigitation” between fibers upon cooling, with generation of new bonds [9,32]. Some researchers have suggested the formation of a certain type of noncovalent bonding between collagen strands on both sides of the weld [38], while others have found that new covalent cross-links were created upon laser irradiation [39]. A secondary role of fibrillar type I collagen in the laser welding mechanism has been pointed out in some recent studies [33,40], suggesting the involvement of some other extracellular matrix components, and being in agreement with earlier studies on welded tissue extracts ana- lyzed by gel-electrophoresis [34,41]. 280 R. Pini et al. Fig. 15.3. “Mild” laser welding approach resulting in a partial modification of the tissue morphology. Native connective tissue (left) subjected to laser irradiation is characterized by collagen fibrils still recognizable and not or only partially swollen (right). Temperature values in the range 60–70◦C are usually induced at the weld site. The welding mechanism hypothesized is based on “interdigitation” upon cooling between collagen fibres unraveled by laser heat Photothermal soldering relies on the coagulation of a protein solder by means of a laser-induced temperature increase in the tissue. Upon cooling, noncovalent interactions between the solder and the collagen matrix within the tissue are supposed to be responsible for the strength of the weld. Evi- dence of albumin intertwining within the collagen matrix was found during scanning electron microscopy analyzes of specimens irradiated at tempera- tures above 70◦C [17]. Such a threshold value is fully in agreement with the threshold temperature of albumin coagulation (around 65◦C), as reported in several spectroscopic and calorimetric studies. Evidence of extracellular ma- trix infiltration of solder within the tissue was also found by using standard histological analysis [10,42]. In photochemical tissue bonding photosensitive dyes applied to the wound edges behave as reactive species when irradiated by laser light. They react with potential electron donors and acceptors such as amino acids (e.g. tryptophan, tyrosine, cysteine) of proteins. Strong covalent bonds are produced between the approximated surfaces of the wound, forming instantaneous protein cross- links [26]. The formation of cross-links in collagen type I molecules by means of photochemical activation has been confirmed by using gel electrophoresis [43]. Here as follows, we will review some of the main surgical applications of laser tissue welding, in particular with regard to the fields of ophthalmology and microvascular surgery, in which the clinical use of this technique seems closer to being accepted. 15 Laser Tissue Welding in Minimally Invasive Surgery and Microsurgery 281 15.2 Laser Welding in Ophthalmology The first attempt to join biological tissues were proposed in ophthalmology, for the treatment of retinal, corneal, and scleral samples [44–46]. The first studies were unsuccessful, resulting in no tissue fusion, while with the improvement of laser technique several research groups adopted different approaches to induce welding of scleral and corneal tissue. Retina fusion was achieved by inducing photocoagulation of the tissue, while the technique used to treat other ocular tissues is based on a soft thermal treatment, properly defined as laser welding. Successful experimental studies of laser-induced suturing of ocular tissues on animal models have been reported since 1992 by different authors [35, 42, 47–51] on the basis of the use of near and far-infrared lasers, directly absorbed by the water content of the cornea. Various laser types with wavelengths exhibiting high optical absorption in water have been used, such as CO2 (emitting at 10.6 µm) [42, 50, 51], Erbium:YAG (1455 nm) [35], and diode lasers (1,900 nm) [48,49]. The main problem with such laser wavelengths is that, without an adequate control of the laser dosimetry, the direct absorp- tion of laser light in a short penetration depth of the tissue outer portion caused a high temperature rise at the irradiated surface, followed by colla- gen shrinkage and denaturation; on the contrary, the deeper layers are hardly heated at all, resulting in a weak bonding, as the full thickness of the tissue is not involved in the welding process. Improved results in tissue welding were observed by using exogenous chromophores to absorb laser light, sometimes in association with protein solders. Addition of highly absorbing dyes allowed fusion of wounds at lower irradiation fluences, thus avoiding excessive thermal damage to surrounding tissues. In fact, the usage of a chromophore was found to induce a controllable temperature rise only in the area where it had been previously applied, resulting in a selective thermal effect. Recently a new approach was proposed and tested by some of us for the closure of corneal wounds [8, 52–54] and for the treatment of anterior lens capsule bags [55]. It is based on the use of a near-infrared diode laser, in asso- ciation with the topical application of a water solution of ICG. The welding procedure, which was optimized so that it could be used in ophthalmic surgery applications, has been proposed as a valid alternative to, or as a supporting tool for, the traditional suturing technique used for the closure of corneal wounds, such as in cataract surgery, penetrating keratoplasty (i.e., transplant of the cornea), and in the treatment of accidental corneal perforations. It can also be used for closure of the lens capsule (to repair capsular breaks caused by accidental traumas or produced intraoperatively), as well as to provide closure of the capsulorhexis in lens refilling procedures. 15.2.1 Laser Welding of the Cornea The cornea is an avascularised connective tissue on the outer surface of the eye, and forms the outer shell of the eyeball together with the sclera. It acts as one 282 R. Pini et al. of the main refractive components, assuring good vision with its clarity and shape. It also acts as a mechanical barrier and as a biological defence system. The cornea is composed of different layers: epithelium, stroma, and endothe- lium are the principal ones, proceeding from the external surface toward the inner part of the eye. More than 90% of the cornea is stroma, and consists of extracellular matrices (mainly type I collagen and glycosaminoglycans), keratocytes, and nerve fibers. The collagen is regularly arranged in fibers, thus contributing to corneal transparency; the collagen fibers are organized in lamellae, i.e., in planes running parallel to the corneal surface. These par- ticular structures and architecture confer unique properties to corneal tissue, e.g., in the reaction process to external injuries, such as incidental traumas, surgical incisions, or ulcers. The injured tissues heal by repair and do not recover the “normal” configuration, because of an induced disorganization in the ordered array of the collagen fibers. This results in an opaque scar with less tensile strength than that of an unwounded cornea, and in a subsequent impairment of the main corneal functions. Moreover, the healing of corneal stroma is slower than that of other connective tissues because of the lack of blood vessels. Clinical changes in scar formation may, in fact, be detected years after surgery has taken place [56]. For all these reasons, the characteristics of laser welding procedures may be very useful in practical surgery, offering the possibility of avoiding many post- operative complications. The immediate watertight closure of wound edges provides protection from external inflammation, and may prevent endoph- thalmitis, which sometimes occurs after cataract surgery. The position of the apposed margins has been found to be stable in time, thus assuring optimal results in terms of postoperative induced astigmatism after cataract [54] and keratoplasty surgery. The absence or the reduction in the number of stitches does not induce foreign body reaction, thus improving the healing process. Histological analyzes performed on animals and morphological observations on treated patients have shown that, in a laser-welded wound, tissue regains an architecture similar to that of the intact tissue, thus supporting its main functions (clarity and good mechanical load resistance). The technique recently proposed for welding corneal tissue [7, 8, 52–54] has been tested and optimized on animal models. Experimental analyzes were first performed ex vivo on pig eyes; the healing process was then studied in vivo in rabbits. When a satisfactory result was achieved, it was proposed clinically and is currently being used for the closure of corneal tissue after penetrating keratoplasty, supporting the application of eight stitches instead of a continuous suturing. The main advantages of this clinical practice are an increased patient comfort during the healing process and a reduction of hospitalization costs. The technique is based on the use of near-infrared continuous-wave AlGaAs diode laser radiation at 810 nm, in association with the topical application of a sterile water solution (10% weight/weight) of ICG to the corneal wound to be repaired. This dye is characterized by high optical absorption around 800 nm 15 Laser Tissue Welding in Minimally Invasive Surgery and Microsurgery 283 [57], while the stroma is almost transparent at this wavelength. ICG is a frequently used ophthalmic dye, with a history of safety in humans. It is being used increasingly as an intraocular tissue stain in cataract and vitreoretinal surgery, as well as in staining of the retinal internal-limiting membrane [58–60]. Furthermore, ICG is commonly used as a chromophore in laser welding or laser soldering [1], to induce differential absorption between the dyed region and the surrounding tissue. Photothermal activation of stromal collagen is thus induced by laser radiation only in the presence of ICG, resulting in a selective welding effect, which produces an immediate sealing of the wound edges and good mechanical strength. In addition, with the use of ICG, very low laser power is required (below 100 mW), and this generally means much safer operation with respect to the use of other laser types without the association of dyes. The procedure used to weld human corneal tissue is as follows: the chro- mophore solution is placed inside the corneal cut, using an anterior chamber cannula, in an attempt to stain the walls of the cut in depth. A bubble of air is injected into the anterior chamber prior to the application of the staining solution, so as to avoid perfusion of the dye. A few minutes after application, the solution is washed out with abundant water. The stained walls of the cut |
appear greenish, indicating that the concentration of ICG absorbed by the stroma is much lower than that of the applied solution. Lastly, the whole length of the cut is subjected to laser treatment. Laser energy is transferred to the tissue in a noncontact configuration, through a 300-µm core diameter fibre optic terminating in a hand piece, which enables easy handling under a surgical microscope. A typical value of the laser energy density is about 13 W cm−2 in humans, which results in a good welding effect. During irradia- tion, the fiber tip is kept at a working distance of about 1 mm, and at a small angle with respect to the corneal surface (side irradiation technique). This par- ticular position provides in-depth homogenous irradiation of the wound and prevents from accidental irradiation of deeper ocular structures. The fiber tip is continuously moved over the tissue to be welded, with an overall laser irra- diation time of about 120 s for a 25-mm cut length (the typical perimeter of a transplanted corneal button). Experimental studies on laser-induced heating effects on ocular tissues were performed both ex vivo on animal models [21] and in vivo during surgery. An IR thermo-camera provided information on the heating of the external surface of the irradiated tissues and on heat confinement, during the treat- ment. A partial differential equation modeling of the process enabled us to investigate temperature dynamics inside the tissue. From this study, it was possible to point out that the optimal welding temperature is about 60◦C inside the treated wound, i.e., in correspondence with a thermally induced phase transition of stromal collagen. The heating effect was found to be se- lectively localized within the cut, with no heat damage to the adjacent tis- sue, and to induce a controlled welding of the stromal collagen. After laser welding, collagen fibrils appeared differently oriented among themselves in 284 R. Pini et al. Fig. 15.4. Transmission electron microscopy images of the fibrillar arrangement observed in a control (left) and in a laser-welded (right) corneal stroma. Differently oriented and interwoven fibrils across the cut are visible at the weld site upon laser irradiation (×13,500) comparison with those of the untreated samples, but with similar mean fibril diameters [33] (Fig. 15.4). Moreover, thanks to the characteristics of the ICG solution, it seems that laser welding is a self-terminating process, which thus avoids accidental excessive temperature rises inside the tissue. A follow up study on animal models [7] and clinical application of the technique provided evidence that laser-welded tissues exhibits good adhesion and good mechanical resistance (Fig. 15.5). A thorough study of the healing process – on the basis of morphological observations, standard histology, and multispectral imaging auto-fluorescence microscopy (MIAM) [61] – proved ex- perimentally that this takes place in shorter time and with lower inflammatory reaction, when compared with conventionally sutured wounds. Objective ob- servations 2 weeks after surgery showed a good morphology of laser-treated corneas, with almost restored cuts, generally characterized by better adhesion and less edematous appearance compared with sutured ones. These features were confirmed by means of histological examinations of rabbit corneas, which revealed a well-developed repair process involving the epithelium, which al- most regained its physiological continuity and thickness and a partially reor- ganized architecture of the stroma. Histological analyzes on longer follow-up times indicated that the healing of laser-welded wounds was completed in about 30–60 days, while in sutured wounds the healing process was still in progress. This result is particularly important as far as corneal tissue is con- cerned, as it typically requires much longer times to be repaired than do other types of tissue. Furthermore, the restored tissue regains a stromal architecture that is very close to the native one, which is crucial to regaining of correct vision. 15 Laser Tissue Welding in Minimally Invasive Surgery and Microsurgery 285 Fig. 15.5. Left : histological section of a rabbit cornea after laser welding on post- operative day 30 (Hematoxillin and Eosin, ×50); the architecture of the cornea regained an almost physiological appearance (the original cut is indicated by the dashed line). Right : histological section of a rabbit cornea after conventional stay suturing on postoperative day 30; the cut is still clearly detectable and large lacunae are evident in the corneal stroma 15.2.2 Combing Femtosecond Laser Microsculpturing of the Cornea with Laser Welding In recent decades, femtosecond (FS) lasers have been developed, tested, and optimized for miniinvasive intratissue surgery and cell manipulation [62–64]. In particular, eye tissue characteristics are well suited to FS lasers applica- tions, as they provide precise intrastromal cuts that are the basis of the new corneal and refractive surgery techniques. With the term FS lasers, we indi- cate lasers that emit pulses in the near-infrared spectral region with durations ranging between a few femtoseconds and hundreds of femtoseconds. With the use of these ultra-short pulses, it is possible to induce photo-disruption inside semitransparent and transparent media, such as corneal tissue. To induce this effect, the laser beam is focused in a focal region of a few micrometers in diam- eter. High intensities ∼1013 W cm−2 are achieved, which induce multiphoton nonlinear absorption, followed by plasma formation. The laser-induced plasma expands rapidly with high pressure (GPa), and this is followed by development and further collapse of cavitation bubbles, accompanied by formation of de- structive shock waves. To obtain a highly-localized destructive effect without significant collateral damage, small bubble diameters and low photomechani- cal effects in the surroundings are required. These features are satisfied in FS laser-induced photo-disruption, first because initiation of nonlinear absorp- tion of laser radiation requires tight focusing of the light, which assures effect confinement in the central portion of the focus volume (where really high 286 R. Pini et al. intensities are achieved). Second, the multiphoton absorption coefficient has a high value when compared with the linear absorption value: the disruptive effect may be achieved with very low intensity levels (µJ cm−2 or nJ cm−2). These nonlinear effects have been used to ablate and to modify corneal tissue, with very high spatial precision and minimal side effects. Currently, FS laser cutting cells are characterized by high numerical aper- tures (NA) (>0.9 in water and glass) that make possible submicrometrical precision. Long series of pulses from FS lasers at 80 MHz repetition rate im- ply accumulative effects, which may cause tissue ablation at pulse energy be- low the optical breakdown threshold in presence of low density plasmas [64]. The advantages are that nonlinear propagation effects are reduced, highly lo- calized energy deposition occurs, and subsequently nanosurgery on a cellular and subcellular level is possible. Moreover, the optical breakdown threshold weakly depends on the target absorption coefficient. Thus, any arbitrary cel- lular structure may be manipulated. For these reasons, near-infrared FS lasers have been considered the innovation for overcoming problems associated with the use of UV nanosecond lasers in refractive surgery: UV mutation effects on cells, low light penetration depth, collateral damage outside the focal vol- ume, the risk of photon-damage to living cells due to absorption, and prob- able induction of oxidative stress leading to apoptosis. Moreover, a focused nanosecond-pulsed laser beam can cause thermal damage and denaturation around the laser focus. Typically, the systems proposed for performing corneal manipulation against refractive problems are based on mode-locked diode-pumped FS Nd:Glass lasers providing pulses of 500–800 fs duration and a few µJ energy, at repetition rates of some tens of kilohertz. Each individual laser pulse is fo- cused on a specific location inside the cornea, which is fully transparent at the laser wavelength. A micro-plasma is created, and this generates a microcav- itation bubble of 5–15 µm in diameter, which separates the corneal lamellae. Thus, a resection plane can be created by delivering, in a prescribed pattern, thousands of laser pulses connected together. The cut can be performed with micrometrical precision at different depths inside the stroma, thus allowing for corneal flaps with a preset, constant thickness. Laser pulses can also be stacked on the top of each other, to create a vertical or angled cleavage plane to precisely sculpture the border of the lamellar flap. The possibility of per- forming the same resection procedure on the donor cornea as well as on the patient’s recipient eye, allows to match the transplanted flap precisely with the recipient corneal bed. By exploiting previous clinical experiences in diode laser welding of corneal wounds, some of us have recently designed a new laser-assisted technique for lamellar keratoplasty (i.e., a corneal transplant involving replacement of only the anterior corneal stroma). It is performed by using a FS laser to prepare donor button and recipient corneal bed, and then suturing the edges of the wound by means of diode laser-induced corneal welding, without the application of conventional suture material. This minimally invasive procedure 15 Laser Tissue Welding in Minimally Invasive Surgery and Microsurgery 287 Fig. 15.6. Aspect of a human cornea affected by leucoma (left): before surgery, and (right): three days after corneal transplant, performed according to the so-called ALSL-LK, which combines the use of a femtosecond laser for corneal sculpturing and a diode laser for corneal welding was called “all-laser” sutureless lamellar keratoplasty (ALSL-LK) (Fig. 15.6). Intraoperative observations and follow-up results for up to 6 months indicated the formation of a smooth stromal interface, the total absence of edema and/or inflammation, and a reduction in postoperative astigmatism, when compared with conventional suturing procedures [65,66]. 15.2.3 Laser Closure of Capsular Tissue The lens, or crystalline lens, is a transparent, biconvex structure in the eye: after the cornea, it is the second refractive component in the eye. The lens is flexible and its curvature is controlled by ciliary muscles through the zonules. It is included within the capsular bag, maintained by the zonules of Zinn, which are filament structures connected to the ciliary muscles fulfilling lens accommodation (i.e., focusing of light rays into the retina in order to assure good vision). This capsule is a very thin (about 10 µm thick [67]), transpar- ent acellular membrane that maintains the shape of the lens. This tissue is a collagenous meshwork mainly composed of type IV collagen and other non- collagenous components such as laminin and fibronectin. Type I and type III collagen are also present [68]. The function of the lens capsule is primarily mechanical: in the accommodation process it has load-transmitting function. With ageing, the lens loses its ability to accommodate, thus requiring cataract surgery, which consists of replacing the native lens with a nonaccommodat- ing plastic prosthetic one. The ultimate goal of this surgery is ideally the restoration of the accommodative function by refilling the capsular bag with an artificial polymer [69], after the endocapsular aspiration of nuclear and cor- tical material. This technique may become a viable lens-replacing procedure, 288 R. Pini et al. as soon as experimental tests are able to prove preservation of capsular me- chanical functions and clarity of refilled lens. Moreover, it would be important for the feasibility of this technique, to demonstrate that a biocompatible valve on the anterior lens capsule tissue could be set up to facilitate lens-refilling operations. To improve cataract surgery, thus providing a surgical solution to presby- opia, some of us proposed a solution for performing a flap valve with the use of a patch of capsular tissue obtained from a donor lens, to be laser-welded onto the recipient capsule. The procedure may also be used to repair acciden- tal traumas, such as capsular breaks or perforations during intraocular lenses implantation [70]. Because of its particular fragility and elasticity, it is quite impossible to suture capsular tissue using standard techniques; however, at present there are no alternative methods. Laser welding could be used to ac- complish this goal. The study is in progress, and preliminary evidence of the feasibility of this technique has recently been obtained [55,70]. Experimental tests were carried out ex vivo, on freshly-enucleated porcine eyes (Fig. 15.7). Closure tests were performed by means of patches of donor capsulae (mean diameter: 3 mm). The inner side of the patch was stained with an ICG-saturated solution in sterile water (7% weight/weight). The staining solution was left in place for 5 min. The sample was then washed with abun- dant water, to remove any excess of ICG. The stained patch was then applied to the anterior lens capsule, through a previously |
performed corneal incision. Fig. 15.7. An ICG-stained capsular patch was welded onto the anterior lens capsule of a pig eye. Laser spots are clearly evident at the periphery of the patch 15 Laser Tissue Welding in Minimally Invasive Surgery and Microsurgery 289 The stained inner side of the patches were positioned on the exterior surface of the recipient capsule, so as to maintain the original orientation and curva- ture. This procedure facilitated adhesion between the tissues to be welded. The patch was then irradiated along its external perimeter by means of contiguous laser spots emitted by a 200-µm-core fiber, whose tip was gently pressed onto the patch surface (contact welding technique). Exposure times were found to be critical to avoid heat damage. Continuous wave irradiation, which is typi- cally employed in other laser welding applications, was unsuitable, while pulses around 100 ms (with energies of 30–50 mJ) provided the best results. Once welded, the capsular patch showed good adhesion to the recipient anterior capsular surface. Preliminary biomechanical tests performed on laser-welded anterior capsule flaps showed that the load resistance of welded specimens was comparable to that of healthy tissues. Standard histology analysis indicated good adhesion between the apposed samples and thermal damage localized in the treated area. 15.3 Applications in Microvascular Surgery Microvascular anastomosis is a surgical technique for the connection of two small-calibre blood vessels (both arteries and veins) with typical diameters of a few hundreds of micrometers. It is commonly used in various surgical fields, such as plastic and reconstructive surgery to restore traumatized or thrombotic vessels, as well as in neurosurgery in the treatment of cerebral ischemia, vascular malformations, or skull base tumors [71,72]. In this regard, conventional suturing methods are associated with various degrees of vascular wall damage, which can ultimately predispose to thrombosis and occlusion at the anastomotic site [73]. To minimize vascular wall damage and improve long- term patency, various alternative nonsuture methods have been investigated experimentally and, in some cases, in the clinical practice [74–76]. Laser welding of arteries was reported in 1979 by Jain and Gorisch [77] to perform vascular anastomosis by the use of a Nd:YAG laser. Then, other lasers (e.g., argon [78] and CO2 [79]) were used experimentally with questionable re- sults. Further improvements came with the introduction of low-energy diode lasers in association with exogenous chromophores and solders [80, 81]. More recently, in vitro and in vivo acute studies have better defined some technical aspects of end-to-end arterial laser welding [82,83]. Despite the large number of experimental studies reported in the literature, very few have reached the clinical phase, mainly due to the lack of clear evidence of the advantages of- fered by laser-assisted suturing (when compared with conventional methods), and of reproducibility of results. Experimental studies on diode laser-assisted end-to-end microvascular anastomosis (LAMA) in association with ICG topical application in femoral arteries and veins of rats were reported by some of us since 1993 [80, 81]. In the design and development phases of these studies [80], the number of suture 290 R. Pini et al. stays supporting the anastomosis was progressively reduced, to minimize for- eign body reaction, and eventually to achieve simpler and faster operation. In conventional suturing procedures of microvascular anastomosis, a number of suture stays from 8 to 12 are applied to support the anastomosis. On the other hand, laser-assisted procedures to weld the vessel edges were first accom- plished with the support of four conventional stays; then successful LAMAs were performed with only two sutures stays, and lastly with no permanent stays at all. These results demonstrated experimentally that diode laser weld- ing alone could withstand both blood pressure and vessel tensile strength, providing mechanical and functional support to the anastomosis. Moreover, the most significant result of these studies emerged from histological exami- nation on longer follow-up times (up to three months), by comparison of the healing of laser-welded and conventionally sutured vessels. Specimens from both arteries and veins yielded unquestionable evidence of faster and superior healing induced by the laser treatment, very similar to a “restitutio ad inte- grum” of vessel structures (Fig. 15.8). The repair mechanism of vessel walls in LAMAs seemed significantly favored by reduction or absence of suture ma- terial, which limited the occurrence of foreign body reaction, granulomas and inflammation, and ultimately resulted in a more effective and faster restora- tion of the architecture of the vessel walls. In a more recent study, diode laser welding was experimentally tested to perform end-to-side anastomosis in carotid bypass surgery [11]. This appli- cation may be particularly important in neurosurgery in order to minimize the occlusion time of the carotid artery during the application of a venous bypass graft, thus reducing the risk of post-operative brain damages. With regards to technical aspects of the laser-assisted welding procedure, the study Fig. 15.8. Left : aspect of femorary artery of a Wistar rat after end-to-end LAMA performed by staining the vessel edges with ICG and then welding them by using a low power diode laser emitting at 810 nm, without conventional stitches. Right : his- tological section of the artery wall 3 months after the surgery showing the complete restoration of the wall architecture (Wiegert elastica-Van Gieson stain, ×250) 15 Laser Tissue Welding in Minimally Invasive Surgery and Microsurgery 291 demonstrated the feasibility of laser-assisted end-to-side anastomosis in bypass surgery, which – to our knowledge – had never been tested before in vivo and presents peculiar technical features. In fact, beside a more complex geometry of the junction, it requires the accomplishment of an effective welding between the artery and the vein graft, which exhibit different wall structures. More- over, surgical advantages were observed in laser-assisted when compared with conventionally sutured anastomoses, such as a simplification of the procedure, a reduction or suppression of bleeding, and a shortening of the operative time, which may be potentially reduced by up to a factor of three. Again, histolog- ical, ultrastructural and immunohistochemical analyzes confirmed the occur- rence of lesser inflammation and of better preservation of the endothelium and of the inner wall structures of both artery and vein in laser-treated segments. This is expected to reduce the occurrence of thrombosis and favor an optimal restoration process. 15.4 Potentials in Other Surgical Fields 15.4.1 Laser Welding of the Gastrointestinal Tract Repair of the gastrointestinal tract by means of laser welding has been found to be a technique that is much easier, faster, and yielding better healing response and no stone formation, when compared with suturing closure techniques. The first published study regarding laser bowel welding was reported in 1986 by Sauer et al. They used a CO2 laser to repair longitudinal transmural incisions in an otherwise-intact rabbit ileum, producing strong hermetic closures [84]. The same authors later proposed the use of a biocompatible, water-soluble, in- traluminal stent in conjunction with India ink as an exogenous chromophore, to perform a suture-free end-to-end small bowel anastomosis [85]. In 1994 Rabau et al. described the healing process of CO2 laser intestinal welding in a rat model, which evidenced a higher probability of dehiscence in the first 10 postoperative days compared with control sutured repairs [86]. More- over, comparable healing response among argon and Ho:YAG laser-welded and control-sutured anastomoses have been found by using a temperature- controlled laser system [87]. Oz et al. investigated the feasibility of using a pulsed THC:YAG and a cw argon laser for the welding of biliary tissue. They found better histological response and higher pressure prior to breaking in the latter case [30]. Lastly, laser soldering with ICG-doped liquid albumin-solder in conjunction with a diode laser was successfully evaluated for the purpose of sealing liver injuries with minimal heat damage [88]. 15.4.2 Laser Welding in Gynaecology Laser welding was experimented in 1978 for reconstructive surgery in the fallopian tubes, using a CO2 laser with good results [89]. Contrasting results 292 R. Pini et al. appeared in the 1980s up until the study by Vilos et al., in which a series of intramural-isthmic fallopian-tube anastomoses were performed with a patency rate of 100% [90]. The main problem claimed in these studies appeared to be the nonuniform heating of the tissue. Improved results were obtained by introduction of a low-power diode laser in conjunction with a protein solder in a rabbit model [91]. In this study, no thermal damage was detected, while a significant reduction in the operative time was evidenced. 15.4.3 Laser Welding in Neurosurgery The major advantage of using laser welding on nerves is the much shorter time- consumption for operation when compared with conventional microsuturing techniques. However, as high power densities are required to coapt the nerve edges, thermal damage in the epineurium is easily induced [92]. In contrast, low power does not result in sufficient tensile strength. To improve nerve welding, usage of sealing material was proposed. The most popular solders are dye-enhanced protein solders, which were employed either with CO2 or diode laser [28,93]. The bonding rate and the functional recovery of the nerves subject to laser soldering are superior to those of manual suturing techniques, while the risk of inflammation and foreign body reaction are minimized [93]. 15.4.4 Laser Welding in Orthopaedic Surgery Other investigations of the laser tissue welding technique have been carried out in the case of orthopaedic surgery. The welding of tendinous tissue was investigated using a Nd:YAG laser alone [94] or in conjunction with an albu- min solder, and an argon laser in conjunction with a fluorescein-dye-doped albumin solder [95]. These studies pointed out the inability of the laser tech- nique to produce a weld of sufficient strength to withstand the significant tensile loads, which tendons are subjected to from immediately after the re- pair. Laser welding of meniscus tears using an argon laser in conjunction with a fibrinogen solder gave similar results [16]. 15.4.5 Laser Welding of the Skin The principal advantage claimed for the laser welding of skin is a superior cos- metic result. However, control of the temperature enhancement at the weld site is challenging. It is nearly impossible to obtain full-thickness welds and limit thermal denaturation laterally around the weld site in the epidermis and papillary dermis. Although thermal denaturation of tissue is necessary in order to produce a strong weld, excessive thermal damage may result in scarring and dehiscence [96]. The use of chromophores has been proposed to minimize unwanted collateral thermal injury ensuring selective laser absorp- tion at the same time [4, 96]. The application of a variety of solders has also 15 Laser Tissue Welding in Minimally Invasive Surgery and Microsurgery 293 been investigated for improving the strength of cutaneous repairs [19,97]. To minimize the effects of collateral thermal damage dynamic cryogen cooling was successfully employed [98]. A more recent proposal was the use of gold nanoparticles as exogenous absorbers, which allowed for the application of light sources undergoing minimal absorption from tissue components, thereby minimizing the damage to surrounding tissues [99]. Previously, the ability of laser welding to accelerate and improve the skin wound healing process was convincingly demonstrated [100]. However, it should be emphasized that – until now – clinical work on laser skin welding has never been reported. 15.4.6 Laser Welding in Urology Urological surgery requires watertight closures, because of the continuous flow of urine. As urine lacks the clotting features of blood, producing effective closures of the urinary tract using traditional suturing techniques tends to be technically demanding and time-consuming. Moreover, suture material itself can induce the formation of stones. Conversely, laser welding can provide immediate watertight, non-lithogenic anastomoses, yielding a tensile strength that is superior to that of conventional closure techniques [5]. Laser tissue welding has been tested on several tissues of the genitourinary tract and for different applications, including urologic applications, which gave the best clinical achievements. Laser vasal anastomosis using CO2 and noncontact Nd:YAG laser was ini- tially investigated in several experimental studies, followed by clinical trials with good postoperative results [101]. Afterwards, laser soldering with ICG- doped albumin in conjunction with an 808-nm diode laser was investigated for hypospadia repair on 138 children [18]. Results were compared with conven- tional suturing. The study emphasized the occurrence of fewer postsurgical complications in the laser group compared with the sutured one, and an easier operation in the laser set-up. 15.5 Perspectives of Nanostructured Chromophores for Laser Welding Laser welding of biological tissues has |
received substantial momentum from coupling with exogenous chromophores with enhanced absorbance in the near- infrared, applied topically at the edges of the wounds prior to irradiation. As mentioned earlier, suitable exogenous chromophores absorb efficiently and se- lectively the near-infrared light from a laser, which immediately translates into well-localized hyperthermia and an overall decrease of the power thresh- olds required to achieve closure of wounds. This in turn minimizes collateral thermal damage to healthy tissues, which has ultimately backed the emer- gence of laser-welding as a minimally invasive and convenient alternative to traditional suturing or grafting. The ultimate exogenous chromophore should 294 R. Pini et al. display high absorption coefficient in the near-infrared and enable high local- ization of power deposition, ideally down to the scale of individual biological structures. Further desirable features include good chemical and thermal sta- bility and high photo-bleaching threshold. Conventional chromophores of common use in laser-welding are organic molecules such as ICG. These have given outstanding experimental and clini- cal achievements in a number of medical fields, and that in spite of relatively poor performances with respect to the aforementioned criteria [102,103]. The absorption efficiency and photo-stability of organic molecules are limited. Their optical properties depend strongly on biochemical environment and temperature, and generally deteriorate rapidly with time [104]. The range of chemical functionalities accessible is narrow, which is incompatible with flexible and selective targeting of distinct biological structures. Overcoming of these limitations would represent a real breakthrough in the practice of laser-welding. Possible ways forward are disclosed by the advent of nanotechnology, as a powerful paradigm to develop new functionalities, by manipulation of self- organization processes at the nanoscale. Here, we mention the introduction of a new class of nanostructured chromophores, which is attracting much attention in view of many applications, including the laser-welding of bio- logical tissues: colloidal gold nanoparticles (nano-gold). Whereas the opti- cal response of organic molecules stems from electronic transitions between molecular states, light absorption, and scattering in nano-gold originates from excitation of collective oscillations of mobile electrons, i.e., surface plasmon resonances [102]. This translates into molar extinction coefficients higher by 4–5 orders of magnitude with respect to those of organic chromophores [102], enhanced thermal stability and photo-bleaching threshold, lower dependence on surrounding chemical environment (although surface plasmons shift with variations in dielectric constant and electron donating/withdrawing tendency of embedding tissues [105,106]). Inspiring perspectives arise from the surface chemistry of nano-gold. As a traditional material for implants, gold is be- lieved to ensure good biocompatibility [107], which is a critical prerequisite in front of clinical applications. The possibility of flexible conjugation of gold surfaces with biochemical functionalities opens a wealth of novel opportuni- ties [108], such as selective targeting against desired and well-defined biological structures. In summary nano-gold may become the ideal substitute of organic chromophores. The utilization of nano-gold dates back to the ancient Romans, when employed for decorative purposes in the staining of glass artifacts (e.g., the Lycurgus cup). Synthesis of stable aqueous colloidal preparations of nano-gold was first achieved by M. Faraday toward the 1850s by use of phosphorous to reduce a solution of gold chloride. Subsequent developments led to a number of variants especially based on reduction of chloroauric acid in sodium cit- rate (Turkevich method) [109, 110]. Conventional nano-gold is composed of spherical nanoparticles of variable and controllable radii [110]. Their optical 15 Laser Tissue Welding in Minimally Invasive Surgery and Microsurgery 295 properties are well-understood in the framework of classical Mie theory. The plasmon resonance of spherical nano-gold in aqueous environment is found within 500–600 nm, almost independent of geometrical volume [102]. Because of absorption in the visible, conventional nano-gold is not regarded as a candidate ideal chromophore for laser-welding of biological tissues. Lo- calization of power deposition requires preferential use of near-infrared radia- tion. Theoretical calculations based on different approaches (including Gans theory [102], dipolar approximations [102, 105], or hybridisation of Mie reso- nances [111]) agree on the possibility to steer the absorption of nano-gold to well within the near-infrared by introduction of nonspherical morphologies. The experimental synthesis of gold nanoparticles with unconventional shape is a very active field of research [112]. By intrusive modification of existing procedures for spherical nano-gold, a number of nonspherical gold nanopar- ticles have been demonstrated, including dielectric-core/metal-shell silica or gold-sulphide/gold nano-shells [113–115], complex hollow shells as gold nano- cages [116], or high aspect ratio gold nano-rods [106,117,118]. Tuning of size and shape of these nanoparticles allows for tuning of plasmon resonances in good agreement with theoretical calculations. In particular, absorption within the near-infrared range of interest is becoming a mature achievement. Near-infrared-light irradiation of biological media dispersed with nonspheri- cal nano-gold was proven to result in selective, controllable, and significant heating [119,120]. Photo-activated nonspherical nano-gold holds the promise of manifold ap- plications in the emerging field of nano-medicine. Proposals of extreme interest are e.g., in the treatment of tumors by selective ablation of individual malig- nant cells [120–122]. In our context, the potential of silica/gold nano-shells in the laser welding of tissues has recently been demonstrated in combination with an albumin solder [99]. Absorption of 820 nm diode laser radiation by a low concentration of nano-shells was shown to induce successfully the coagu- lation of albumin proteins and the ensuing soldering of muscles ex vivo and of skin in vivo (rat model). Preliminary results are very promising. However, much progress is still required. We estimate that future innovation will take special advantage of the adaptable functionalization of nano-gold. This may for example enable the selective targeting of individual biological structures, which may in turn result in the engineering of tissue power absorption pro- files with resolution in the nano-range. This is a completely novel and powerful perspective in the laser-welding of biological tissues. Currently, the replacement of traditional organic molecules with nanos- tructured chromophores is an inspiring possibility, which is yet far from the clinical application. As a very recent concept and technology, there exists at present basically no experimental evidence of the superiority of these new materials in the welding of tissues. Additional aspects of practical concern include their biocompatibility and overall sustainability (e.g., economical). In short, nano-medicine is a broad and thriving context, offering a wealth of 296 R. Pini et al. novel opportunities. This is true also in the laser-welding of tissues, which is yet a poorly explored frontier. We foresee exciting evolution in the very next future, possibly along the guidelines sketched in this section. References 1. K.M. McNally, in Biomedical Photonics Handbook, ed. by T. Vo-Dihn (CRC Press, Boca Raton, 2003), p. 1 2. L.S. Bass, M.R. Treat, Lasers Surg. Med. 17, 315 (1995) 3. G.E. Kopchok, R.A. White, G.H. White, et al., Lasers Surg. Med. 8, 584 (1988) 4. S.D. DeCoste, W. Farinelli, T. Flotte, et al., Lasers Surg. Med. 12, 25 (1992) 5. D.P. Poppas, D.S. 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The epidermis is supported underneath by the dermis, the portion of the skin that is responsible for the physical integrity of the skin, and gives it the capacity to undergo stretching and flexing and resist trauma [3–5]. Interspersed in this tough matrix is the skin’s vascular network and the nerves that permit the transmission of signals indicating pain or itch. Lastly, there are many adnexae situated in skin, such as the hair, nails, sebaceous, and sweat glands. Collectively, the key function of these structures is to keep toxic environmental influences out, and prevent loss of bodily fluids [1]. Some specific aspects of these functions are: • Temperature regulation; sweating and vasodilation for heat loss [6] • Physical integrity; dermal toughness and elasticity [4, 5] • Maintenance of water balance; providing an intact evaporative barrier [2] • Microorganism defense; skin has active innate and acquired immunity [7] • Appearance [8] • Use of light to synthesize Vitamin D [9] • Protection from hazardous UV radiation [10] The cell types present in the epidermis are keratinocytes, melanocytes, and langerhans cells. Keratinocytes constitute approximately 90–95% of the cells in epidermis, and are responsible for its structure [1]. Keratinocytes are pro- duced in the innermost layer of the epidermis adjacent to the dermis. They 302 A.P. Pentland Stratum Corneum Skin cell Types Stratum granulosum - Keratinocytes - Melanocytes Stratum Spinosum - Langerhans cells Stratum Basale - Fibroblasts - Vascular cells - Mast cells - Adnexal cells Fig. 16.1. Skin is a multilayered organ. Epidermis comprises the most superficial layer. The cells of the epidermis divide in the basal layer, then differentiate, moving upward to the skin surface to slough. The dermis underlies the epidermis, providing structural support for blood vessels and adnexae, as well as the epidermis then differentiate, flattening and progressing toward the surface of the skin as the process of differentiation proceeds, until they are no longer viable. Dur- ing this differentiation process, keratinocytes synthesize and secrete an array of lipids intercellularly in organelles termed lamellar bodies. As differentia- tion is completed, these organelles are secreted into the extracellular space where their lipid contents spread and disperse among the cells of the stratum corneum, creating a “brick and mortar” motif that is extraordinarily imper- meable [2,11]. The barrier is resistant to moisture, invasion of microorganisms, and even oxygen. The constant sloughing of fully differentiated epidermal cells also is an adaptive strategy that prevents microorganism invasion and pro- vides a constant new supply of skin as we wash and scrub the skin surface in the course of work and play. Keratinocytes are also capable of innate immune responses, secreting inflammatory cytokines and chemotactic mediators that help to repel invading organisms [7], and are responsible for the synthesis of the vitamin D precursor, Vitamin D3 [9, 12]. A second important cell type located in the epidermis is the melanocyte, which represents ∼4% of the cells in the epidermis [13]. It is responsible for our skin color because of its capacity to synthesize melanin granules and se- crete them to be taken up by keratinocytes [14,15]. The melanin produced by melanocytes is the chief photoprotective substance within the skin, as it is ca- pable of absorbing light throughout the visible and ultraviolet light spectrum. One melanocyte provides melanin for about 36 keratinocytes (Fig. 16.2). In addition to these two key cell types, the epidermis also contains ∼4% Langerhans cells, which are the resident immune cell of the epidermis. When microorganisms do successfully penetrate the epidermal stratum corneum Epidermis Dermis 16 Photobiology of the Skin 303 Fig. 16.2. The melanocyte is responsible for the synthesis of melanin. Melanocytes reside in the basal layer of the epidermis. They extend dendrites to many nearby ker- atinocytes. Melanin is delivered to keratinocytes by transfer of pigment-containing melanosomes to these nearby keratinocytes, where it is situated above the nucleus. Melanin is responsible for skin color barrier, it is the Langerhans cell that identifies and transports microorgan- ism proteins to regional lymph nodes so an adaptive, long-term, and specific immune response can be initiated. The dermis underlies the epidermis, creating its physical support structure. It contains fibroblasts, vascular cells, adnexal cells, mast cells, and dermal den- drocytes [4,5]. Fibroblasts are responsible for the synthesis of dermal proteins. The most abundant of these is type 1 collagen, which is present in the dermis as a highly cross-linked protein network. In addition to collagen, fibroblasts synthesize and secrete elastic fibrils, which impart resiliency and elasticity to skin and comprise ∼1–2% of skin protein. Mature elastic fibers in the dermis 304 A.P. Pentland are composed of an amorphous elastin core surrounded by microfibrils, also produced by fibroblasts. Interestingly, most synthesis of elastin occurs in the first trimester, so that as chronic UV exposure degrades this protein, our skin loses its stretchiness, causing wrinkles [8, 16]. In addition to fibroblasts, the dermis contains a vascular network that provides nourishment to the skin, and which can dilate and contract as needed to adjust the core temperature [6]. Mast cells are located at the intersections of the small vessels in the dermis where they can readily detect minor trauma and invading microorganisms. The mast cell is the key cell responsible for hives observed in allergic reactions [17]. The mast cell also contributes to the skin’s innate immune defenses. When stimulated physically or by proteins present on invading organisms, it releases granules that contain vasodilator substances and mediators that can activate the immune system. These substances “kick start” the host immune response. Interposed between the epidermis and the dermis is a thin layer of elegantly assembled attachment proteins, termed the basement membrane [18, 19]. There are approximately 15 different proteins present in the basement mem- brane zone, which work in concert to permit the rapidly proliferating epidermis to maintain its attachment to the underlying dermis. 16.2 Effects of Light Exposure on Skin As in any other system, light interacting with the epidermis is reflected, re- mitted, scattered, or absorbed. Only absorbed solar radiation has a biological effect on the skin. The most bioactive wavelengths in skin are those in the ultraviolet range, from 280 to 400 nm [10]. Although some work has been done to demonstrate that 670 nm light may improve wound repair [20, 21], most literature on the interaction of light with skin focuses on wavelengths in the ultraviolet range, because of their capacity to promote synthesis of vitamin D [9], cause photoaging and skin cancer [22], as well as their thera- peutic utility. Radiation in the ultraviolet range is divided into two types, on the basis of the associated biology. Wavelengths from 280 to 320 nm are termed UVB light, while those from 320 to 400 nm are termed UVA light. UVB light is primarily responsible for initiation and promotion of skin cancer, and vitamin D synthesis [10, 12]. UVA wavelengths also contribute to skin cancer, but are much less effective. Their primary impact is on photoaging. Both UVA and UVB wavelengths stimulate melanin synthesis by melanocytes to protect against the unwanted effects of light [23]. The biological effects of these two types of light are dictated by the cellular structures that absorb light. DNA absorbs light |
readily in the UVB range, and therefore UVB light is far more mutagenic than UVA light [24]. When skin is exposed to UV light, it produces both acute and chronic effects. In the acute response, observed 4–48 h after exposure to UV light, 16 Photobiology of the Skin 305 skin can develop redness due to vasodilation, swell, become painful, or even blister if the dose of light is sufficiently large. Somewhat after these changes occur, in 2–7 days, tanning and stratum corneum thickening occur. Those individuals who have very little capacity to tan will not produce much extra melanin after UV exposure, but will still produce thickening of the stratum corneum [9]. The time course of the acute changes resulting from UV exposure is also dependent on the dose of light. Doses of light that are in large excess of the photoprotective capacity of an individual’s skin will result in much more long-lasting and intense erythema [9]. Thus, the changes produced in skin are very dependent on the genetic capacity for melanin synthesis, which is termed the skin type [25]. Skin types are rated on a scale of 1 through 6 (Fig. 16.3). Individuals with type 1 skin always burn and never tan when exposed to UV light. Individuals with type 2 skin burn initially, but then can tan modestly after recurrent exposure to light. Those with type 3 skin may burn initially in the spring, but will reliably tan thereafter. Those with type 4 skin have significant coloration without sun exposure, and always tan readily. Those with type 5 skin have deep coloration without sun exposure, but tan- ning is evident after sun exposure. Lastly, individuals with type 6 skin have constant very deep pigmentation in which tanning is difficult to detect. Light- related skin cancer is prevalent primarily in skin types 1–3. Over the course of years, recurrent exposure of individuals to UV light produces chronic changes in susceptible, light skinned, individuals. Most evident is photoaging. Char- acterized by deep wrinkling, uneven pigmentation, vascular dilatation, and hyperkeratotic changes in epidermis, photoaging is what most people think Fig. 16.3. Skin types: capacity to tan, burn, and risk of skin cancer 306 A.P. Pentland of as aging because of the passage of time [8]. However, vigilant protection against exposure to UV light or a naturally dark complexion are highly ef- fective in preventing these skin changes [9]. For those individuals who are light skinned, chronic exposure to light can also result in immunosuppression and skin cancer formation [22]. Despite these negative consequences of UV light exposure, UV light can be useful in the treatment of skin disease. The response of skin to light is shaped by the presence of many chromophores in skin. These include aromatic amino acids, DNA, NADH, beta-carotene, porphyrins, and most importantly melanin, which has an absorptive range from 280 nm through the whole visible spectrum of light [22, 26]. Although absorption of light by melanin is generally harmless, absorption of light by DNA, amino acids, and membrane lipids produces immediate cellular dam- age. Therefore, intricate defensive mechanisms are in place to assess the extent of UV damage that has been produced by a particular exposure to UV light and to initiate appropriate cellular responses to it. Immediately after exposure to UV light, the cell division process is paused (Fig. 16.4). UVA wavelengths produce damaging free radical production, while both UVA and UVB can produce photoproducts in DNA [10,27]. The key pro- tein initiating this halt in cell cycling is p53, sometimes called “the guardian of the genome” because of its role in response to UV injury. At about the same time, a transcriptional activation occurs. This permits induction of DNA ex- cision/repair enzymes so that damaged DNA can be repaired. Efforts at DNA repair are focused mostly on actively transcribed genes. In addition, oxidized or denatured proteins are degraded, and the synthesis of their replacements is initiated. Similar mechanisms are carried out to repair damaged lipids. Synthesis of inflammatory cytokines is also initiated in response to light ex- posure, including TNFα, IL-1, INFγ and TGFα, which act to regulate repair responses and also are immunomodulators [24, 28, 29]. As new antigens and DNA fragments can be produced by exposure of the skin to light, these cy- tokines help to ensure that sun exposure does not result in sun allergy or autoimmune disease. About 72 h after sun exposure, melanin synthesis and increased differentiation of keratinocytes is prominent [30]. By 7 days after exposure to light, the repair of DNA and proteins is back to baseline values, and the photoprotective capacity of the exposed epidermis has been increased as needed. In some instances after UV exposure, the damage to cells is so severe, that repair is not a viable option. In this case, cellular apotosis is induced. Cells undergoing apoptosis after UV injury were initially described as sunburn cells. Apoptotic destruction of injured cells allows their orderly removal from injured tissue [31]. The process of repair that occurs after UV exposure is imperfect, mak- ing chronic UV exposure problematic. Mutations in DNA can escape repair processes, an error that is key for cancer initiation. Mutations may increase expression of genes that support proliferation or block genes that inhibit it. The capacity of UV light to both cause mutations and to stimulate a proliferative response, as described earlier, is the reason that UV light is 16 Photobiology of the Skin 307 Fig. 16.4. After exposure to UVB radiation, a cellular process to assess the response of epidermis to UV injury is activated. Cell division is halted, the extent of injury is assessed, and repair processes are initiated. Concurrently, those individuals capable of tanning induce melanin synthesis and pigment transfer to keratinocytes. Those cells that have been severely injured are removed by the process of apoptosis a complete carcinogen, both initiating and promoting cancerous growth in skin [24]. Clones of these initiated cells proliferate as recurrent UV exposure occurs over time, increasing the opportunity for a second mutation to occur in a cell, and thereby causing cancer [10]. The added twist that UV light is immunosuppressive further increases the capacity of UV light to cause cancer, as the immune surveillance capability of irradiated skin is reduced, permit- ting survival of more initiated cells. After UV exposure, Langerhans cells, 308 A.P. Pentland the resident immune cell in epidermis, are depleted [24]. Immunosuppressive cytokines are released, including IL-10, TNF, αMSH and PGE2. PGE2 also powerfully stimulates keratinocyte cell division [32]. Although this immuno- suppression is necessary to prevent sun allergy and autoimmune disease, it decreases the ability of individuals with poor native photoprotection to en- dure sun exposure over the course of their lifetime without developing skin tumors. The mutations in DNA induced by UV exposure have been well character- ized. Adjacent thymine residues in DNA form cyclobutane pyrimidine dimers. Also formed are pyrimidine–pyrimidone photoproducts, which can isomerize to a Dewar isomer. Should replication of DNA occur before repair of these photoproducts, base substitution and therefore mutation, can occur [10]. In addition to this type of DNA damage, UV light can also produce strand breaks. Additionally, UVA light is capable of producing an 8-oxo-guanine photoprod- uct in DNA. Once DNA photoproducts occur, they are removed over time. Those that are most likely to be mutagenic are removed more efficiently than those that are not. The removal of photoproducts is increased in areas of ac- tively transcribed DNA. It is also much quicker in newborns than in older individuals (Fig. 16.5). With so much evident harm resulting from exposure of the skin to light, it is not surprising that the skin is well endowed with defenses against UV injury. In addition to the repair pathways already described, and the broad UV absorbing capacity of melanin, the epidermis also has other substances in abundance that can absorb UV energy harmlessly. Glutathione is present in the cell cytosol in large amounts, as is urocanic acid, which isomerizes when absorbing energy in UV wavelengths. In addition, epidermis contains catalase and superoxide dismutase to detoxify UV-induced free radicals [9]. Fig. 16.5. UV light is a complete carcinogen, capable of initiating mutations in DNA that also decrease the capability of cells to detect these alterations. It also promotes mutated cells to divide, increasing the chance of a second mutation within a cell, and therefore increasing the chance of cancer progression 16 Photobiology of the Skin 309 One good activity that occurs when skin is exposed to UV light is the syn- thesis of Vitamin D [9,12]. Provitamin D (7-dehydrocholesterol) is synthesized in epidermis. When this compound is exposed to light wavelengths between 280 and 315 nm, it is altered to become previtamin D3. This compound is then isomerized by the warmth of the skin to become Vitamin D3 (cholecal- ciferol). Vitamin D3 is sufficiently soluble that it can then enter circulation to be metabolized to 25(OH)D3 termed calcediol, or 25-hydroxy vitamin D3. This vitamin is important in calcium deposition in bones and calcium home- ostasis in the kidney. UV exposure is not a requirement for adequate supplies of Vitamin D; however, it is readily obtained from dietary sources. 16.3 Sun Protection and Sunscreens Skin cancer is the most common form of cancer, with nearly a million new cases occurring every year. The most dangerous type of skin cancer, melanoma, is rapidly rising in incidence, with lifetime risk now approaching 1 in 50. All forms of skin cancer are linked to sun exposure [22, 33]. It is, therefore, quite important to be cautious about sun exposure, particularly for those with light complexions, and to adopt sun protection practices to minimize skin cancer risk and also to decrease the effects of photoaging. The simplest and most effective method for minimizing sun exposure is to avoid outdoor activities during the period of the day when UVB exposure is most intense, from 10am to 4pm. In addition, when outdoors, good sun protection can be achieved by wearing protective clothing, a hat with a 4′′ brim, and a sunscreen [9]. Some clothing is also now rated by its sun protection factor, so an indi- vidual can determine the degree of protection provided by a garment [34,35]. Some garments provide more protection than others. The style of the garment matters – it must cover the skin. Two layers are better than one, and newer garments and thicker garments are more UV resistant than older ones. Inter- estingly, even the type of fiber makes a difference. The UV blocking capacity of fibers is as follows: polyester > wool > silk > nylon> cotton and rayon. Of course wet, see-through fabric also does a poorer job filtering the light than dry fabric. As individuals vary so widely in their sunburn responses to UV light, the common method to rate protection is called the sun protection factor, or SPF. The SPF of a sun protection device or lotion is determined empirically. An initial exposure of a subject’s skin to graded doses of a UV light source is made to determine the minimum amount of light that can produce detectable erythema with a distinct border 24 h later. Clearly, the more melanin an in- dividual has in their skin, the larger the dose of light necessary to produce redness. Once the minimal erythema dose (MED) is determined for an in- dividual, the protective measure to be tested is applied to the area to be irradiated and the skin again exposed to light (Fig. 16.6). The capacity of the protective measure to block erythema production 24 h later is expressed as 310 A.P. Pentland Fig. 16.6. The sensitivity of an individual to damage from sun exposure is measured by determining their minimal erythema dose. The least amount of light capable of producing a clearly outlined spot of redness, (termed erythema) is called the minimal erythema dose for that individual. The amount of light required to produce erythema varies nearly 100-fold because of the differences in constitutive skin color the sun protection factor, or SPF. For instance, if a garment has an SPF of 15, then an individual who ordinarily develops erythema after 10 min of light exposure without protection will now be able to tolerate 150 min of exposure when wearing the garment [9]. Use of sunscreens as a method of protection from skin cancer risk |
has been actively debated in recent years. There is definitive evidence that their use protects animals from skin cancer in the laboratory. Similar trials have not been conducted in humans, but there is strong supportive evidence. Some debate has arisen about whether sunscreens may interfere with Vitamin D metabolism, but it is clear that the amount of Vitamin D needed can be readily obtained from dietary sources. In addition, very little light is needed to produce Vitamin D from endogenous sources. It would be the rare individual who could be sufficiently assiduous in their sun protective measures to produce Vitamin D deficiency. Most currently available sunscreens were initially formulated to protect against UVB wavelengths, from 280 to 320 nm. There are two major classes of these compounds: UVB absorbing and physical sunscreens [9]. With recent rapid advances in material science, many new sunscreen compounds are being created and tested. Some of these new sunscreens have already been tested for safety and met standards for European approval, while some aspects of safety for new sunscreen materials are still being evaluated. Physical sunscreens are effective across a broad range of wavelengths. They are made up of particulates that can scatter and reflect UV light. In general, they contain zinc oxide, titanium dioxide, magnesium oxide, talc, or kaolin (up to 25%). Sunscreens in this category provide the best block available in 16 Photobiology of the Skin 311 the UVA range (up to 380 nm), and are also very effective at blocking UVB. Their chief drawback is that when formulated to have a high sun protection factor, they are visible as a white substance when applied to skin. Much work has been conducted lately to alter their physical properties to decrease scattering such that visibility is decreased. Some of this alteration has been done by making nanoparticle formulations. Unfortunately, their capacity to protect against light in the UVA range is decreased when the particle size is decreased into the nanometer range. This problem is being addressed by grouping some nanoparticles in the sunscreen vehicle, and also by coating the particles with dimethicone or silica. A recent concern has been raised that the capacity of nanoparticles to penetrate the skin may be different from that of larger particles. Because of the character of the stratum corneum, it may be they cannot penetrate readily into viable tissue in significant quantities. Research is ongoing in this area [9]. UVB absorbing sunscreens are capable of harmlessly absorbing UV light, and are generally degraded over time by their absorption of light. Sunscreens of this type are wavelength selective, unlike the physical sunscreens. Therefore, it is necessary to combine several chemical sunscreens to gain optimal coverage across the UV spectrum. More consideration is also now being given to group- ing sunscreen ingredients so that their absorption of UV can be transferred to heat, thereby leaving the sunscreen ingredient intact and able to function in its absorbing capacity longer. UVB blockers include paraaminobenzoic acid (up to 15%), Padimate O, a PABA ester (up to 8%), Octocrylene (up to 10%), Cinnamates (such as octyl methoxycinnamate, up to 8% ), and Sali- cylates (up to 5%). Some chemical sunscreens are broader in their protective capacity, such as the benzophenones and oxybenzone (up to 6%), which are useful for UVB protection, but also protect against the shorter wavelengths in the UVA spectrum. Until recently, the only available absorbing sunscreen protective in most of the short UVA spectrum was Parsol 1789, which is utilized in concentrations up to 3%. New formulations of UV absorbing mole- cules are now becoming available, which will greatly expand the capacity of sunscreen manufacturers to make a product that protects broadly across UV wavelengths. 16.4 Phototherapy: Use of Light for Treatment for Skin Disease Despite the problems associated with exposure to UV light, it has been recog- nized for centuries that it is effective in the treatment of skin disease. Diseases such as psoriasis, eczema, and vitiligo have been managed with moderate ef- ficacy, using sun exposure for centuries [9]. With the advent of artificial light sources, it became practical to expand the use of light as a treatment option, as therapy could be administered throughout the year and in cold climates. 312 A.P. Pentland Three types of phototherapy are in common use today, on the basis of the light source in use for treatment. The type of lamp selected for use in treatment depends upon the patient’s diagnosis, the disease location and the patient’s skin type. All types of phototherapy are less effective in dark-skinned individuals because melanin blocks some of the therapeutic light transmission. The most widely available is UVB therapy, using lamps that emit light pri- marily in the wavelengths from 280 to 320 nm. Because of its similarity to solar UVB emission, treatment can be administered daily, taking advantage of the body’s adaptive responses to UVB light. In use since the early part of the last century, it is a well-characterized treatment method. Because the wavelengths of light emitted are the same wavelengths responsible for photocarcinogenesis, it is used sparingly. Studies examining whether UVB phototherapy produces increased cancer risk suggest that the increase is modest, due to physician supervision of light treatment and the fact that its use is generally intermit- tent for disease therapy. Tanning also occurs when patients are treated with this light source, which decreases the efficacy of light treatment. Disease lo- cated deep within the skin is difficult to treat because of absorption of UVB wavelengths primarily in the epidermis [9]. In addition to UVB phototherapy, two other light sources have been in- troduced in the more recent past for phototherapy treatment. The creation of UVA light sources (320–400 nm) triggered an investigation of whether this light source could be useful for disease located deep in the skin, which is re- sistant to UVB treatment because of poor skin penetrating capacity. To make UVA wavelengths efficacious as a treatment method, light exposure occurs after patients have taken the photosensitizing drug psoralen. The treatment method is, therefore, termed “PUVA,” meaning psoralen + UVA. When pso- ralen is present in the skin during UVA irradiation, psoralen becomes cross- linked to DNA. This initiates the repair pathways discussed earlier, and has a beneficial effect on a number of diseases. Because the treatment produces more inflammatory change than does UVB therapy, it is only given 2–3 times per week to allow ample time for repair. As this treatment also produces crosslinks in DNA, the risk of photocarcinogenesis is quite high; studies have demonstrated that the rate of squamous cell skin cancer occurring after PUVA increased eightfold and that melanoma rates are increased as well. Nonethe- less, PUVA treatment is significantly more efficacious than UVB treatment, and therefore in cases of refractory disease, it is used [9]. The lamps used in tanning parlors are the same as those used for PUVA treatment, but the public does not take a photosensitizing drug prior to their visit! To try and reduce the undesirable side effects of UVB and PUVA treat- ment, lamps that emit light most intensely at 312 nm have been brought to market, termed as narrow-band UV lamps. This light source avoids some of the cancer risk of broad-band UVB lamps, and can work well without pro- ducing intense tanning of patients. This form of treatment has been found to be nearly as effective as PUVA, and is becoming the standard of care in many treatment centers. The diseases most responsive to phototherapy using any 16 Photobiology of the Skin 313 of the light sources mentioned earlier are psoriasis, atopic dermatitis, vitiligo, refractory itching, some forms of lymphoma, and many more [9]. In summary, the skin is a complex organ with many functions. Although we are constantly reminded of its role in appearance, it has important utility in protecting us from the environment. There are many special adaptations of skin that are involved in protecting us from UV light, some of which we are able to exploit to treat disease. References 1. D. Chu, A. Haake, K. Holbrook, L. Loomis, The Structure and Development of Skin, 6th edn. (McGraw-Hill, New York, 2003) 2. R.R. Wickett, M.O. Visscher, Am. J. Infect. Control 34, S98 (2006) 3. J.M. Waller, H.I. Maibach, Skin Res. Technol. 12, 145 (2006) 4. J. Uitto, L. Pulkkinen, M. Chu, Collagen, 6th edn. (McGraw-Hill, New York, 2003) 5. J. Uitto, M. Chu, Elastic Fibers, 6th edn. (McGraw-Hill, New York, 2003) 6. B. Wenger, Thermoregulation, 6th edn. (McGraw-Hill, New York, 2003) 7. G. Stingl, D. Maurer, C. Hauser, K. Wolff, The Skin: An Immunologic Barrier, 6th edn. (McGraw-Hill, New York, 2003) 8. M. Yaar, B. Gilchrest, Aging of Skin, 6th edn. (McGraw-Hill, New York, 2003) 9. H. Lim, H. Honigsman, J. Hawk, Photodermatology (Informa Healthcare, New York, 2007) 10. D.I. Pattison, M.J. Davies, Experientia Supplementatum 96, 131 (2006) 11. P. Elias, K. Feingold, J. Fluhr, Skin as An Organ of Protection, 6th edn. (McGraw-Hill, New York, 2003) 12. R. Ebert, N. Schutze, J. Adamski, F. Jakob, Mol. Cell Endocrinol. 248, 149 (2006) 13. R. Halaban, D. Hebert, D. Fisher, Biology of Melanocytes, 6th edn. (McGraw- Hill, New York, 2003) 14. G. Scott, S. Leopardi, S. Printup, N. Malhi, M. Seiberg, R. Lapoint, J. Invest. Dermatol. 122, 1214 (2004) 15. Z. Abdel-Malek, M.C. Scott, I. Suzuki, A. Tada, S. Im, L. Lamoreux, S. Ito, G. Barsh, V.J. Hearing, Pigment Cell Res. 13(Suppl 8), 156 (2000) 16. A. Oba, C. Edwards, Skin Res. Technol. 12, 283 (2006) 17. F. Hseih, C. Bingham, K. Austen, The Molecular and Cellular Biology of the Mast Cell, 6th edn. (McGraw-Hill, New York, 2003) 18. T. Masunaga, Connect. Tissue Res. 47, 55 (2006) 19. L. Bruckner-Tuderman, Basement Membranes, 6th edn. (McGraw-Hill, New York, 2003) 20. M.T. Wong-Riley, X. Bai, E. Buchmann, H.T. Whelan, NeuroReport 12, 3033 (2001) 21. H.T. Whelan, R.L. Smits Jr., E.V. Buchman, N.T. Whelan, S.G. Turner, D.A. Margolis, V. Cevenini, H. Stinson, R. Ignatius, T. Martin, J. Cwiklinski, A.F. Philippi, W.R. Graf, B. Hodgson, L. Gould, M. Kane, G. Chen, J. Caviness, J. Clin. Laser Med. Surg. 19, 305 (2001) 22. R.N. Saladi, A.N. Persaud, Drugs Today 41, 37 (2005) 314 A.P. Pentland 23. W. Westerhof, O. Estevez-Uscanga, J. Meens, A. Kammeyer, M. Durocq, I. Cario, J. Invest. Dermatol. 94, 812 (1990) 24. M. Kripke, H. Ananthaswamy, Carcinogenesis: Ultravioloet Radiation, 6th edn. (McGraw-Hill, New York, 2003) 25. T.B. Fitzpatrick, Arch. Dermatol. 124, 869 (1988) 26. F.R. de Gruijl, H.J. van Kranen, L.H. Mullenders, J. Photochem. Photobiol. B 63, 19 (2001) 27. X. Chen, A. Gresham, A. Morrison, A.P. Pentland, Biochim. Biophys. Acta 1299, 23 (1996) 28. V.E. Reeve, Methods 28, 20 (2002) 29. T. Nakamura, I. Kurimoto, S. Itami, K. Yoshikawa, J.W. Streilein, J. Dermatol. Sci. 23(Suppl 1), S13 (2000) 30. G. Scott, A. Deng, C. Rodriguez-Burford, M. Seiberg, R. Han, L. Babiarz, W. Grizzle, W. Bell, A. Pentland, J. Invest. Dermatol. 117, 1412 (2001) 31. B.J. Nickoloff, J.Z. Qin, V. Chaturvedi, P. Bacon, J. Panella, M.F. Denning, J. Investig. Dermatol. Symp. Proc. 7, 27 (2002) 32. R.L. Konger, R. Malaviya, A.P. Pentland, Biochim. Biophys. Acta 1401, 221 (1998) 33. A.C. Geller, G.D. Annas, Semin. Oncol. Nurs. 19, 2 (2003) 34. V.E. Reeve, M. Bosnic, D. Domanski, Photochem. Photobiol. 74, 765 (2001) 35. D. Domanski, M. Bosnic, V.E. Reeve, Redox Rep. 4, 309 (1999) 17 Advanced Photodynamic Therapy B.C. Wilson 17.1 Introduction Photodynamic therapy (PDT) is the use of drugs (photosensitizers) that are activated by visible or near infrared light to produce specific biological effects in cells or tissues [1]. The basic steps in a PDT treatment are application of the photosensitizer (systemically or topically), a time interval to allow for photo- sensitizer accumulation in the target diseased tissue or cells, and illumination of the target area or volume with light of an appropriate wavelength to activate the sensitizer. PDT is a highly multidisciplinary topic, involving optical bio- physics and bioengineering, synthetic chemistry, pharmacology, photophysics and photochemistry, photobiology, and different clinical specialties. The main emphasis in this chapter is on those aspects of greatest interest to specialists in biophotonics. The term “photodynamic” was first coined a century ago with the ob- servation that light and an acridine dye in the presence of oxygen could kill microorganisms [2]. At around the same time, light therapy was being used in |
patients but without any administered photosensitizing agent. The first reported clinical use of PDT was in 1904 when eosin was applied locally to a tumor on the lip and exposed to light. There was limited follow up to this early work, probably due to the lack of potent photosensitizers and of suitable light generation/delivery technologies. The modern era of PDT started with the discovery of hematoporphyrin derivative (HpD) in the 1950s/1960s, and this was first used in patients in the 1970s to treat bladder cancer. Substan- tial preclinical and clinical studies were started in the late 1970s and the first government approval for PDT was in 1993, with a purified version of HpD (Photofrin©R ). In addition to being further developed since as a treatment for solid tumors and precancerous conditions, PDT is currently approved in various countries for nononcological applications [3,4]. These include actinic keratosis (sun-damaged skin) and age-related macular degeneration, in which abnormal blood vessel growth in the retina causes central vision loss, particularly in the 316 B.C. Wilson elderly. Recently, there has also been a large interest in PDT to treat localized infection [5], in part driven by the rise of bacteria and other microorganisms that are multidrug resistant. For example, PDT using the agent methylene blue was recently approved for treating gum infection (periodontitis) and is also approved for treating acne using the agent ALA (see later): in both cases the sensitizer is applied topically. These applications exploit the different biological mechanisms that can be selectively activated, depending on the PDT treatment parameters: cell killing, blood vessel shut down, and possibly immunological effects [6, 7]. The invention of the laser and optical fibers in the 1960s was also an important driver for the development of PDT, since these enable light of adequate intensity to be delivered to almost anywhere in the body and so make the treatment generically applicable. It should be emphasized, however, that PDT remains largely a localized treatment, since the penetration of light in tissues is limited, so that it is not generally useful for systemic diseases. 17.2 Basic Principles and Features of “Standard PDT” Before discussing the potential novel approaches to PDT, we will summarize the “standard” PDT technique that is characterized by the following: – High doses of photosensitizer and light, given as a single treatment (even if this is subsequently repeated to achieve complete clinical response). – Single-photon activation (see Fig. 17.1 and discussion), using continuous- wave (CW) or quasi-CW light sources, and exploiting the Type II pho- toreaction. – Photosensitizers that are always activatable. – Photosensitizers that comprise organic molecules that are directly acti- vated by the light. Figure 17.1 shows the Jablonski, or energy-level, diagram for PDT. The ground state (S0) of the photosensitizer absorbs the photon energy and is raised to an electronic excited (S1) state (lifetime approximately nanosec- ond). This can de-excite either nonradiatively by fluorescence emission to S0 or by intersystem crossing to a triplet state (T1). The T1 →S0 transition is quantum-mechanically forbidden, so that T1 is long-lived (approximately microsecond) and can exchange energy with ground-state molecular oxygen (3O2) to generate singlet-state oxygen, 1O2, which is highly reactive and leads to oxidative damage to nearby biomolecules (within 10 s of nanometer). Most often these are cell membrane components because of the photosensitizer mi- crolocalization. Following this photoreaction, S0 is regenerated, so that the photosensitizer essentially acts as a catalyst. It may eventually be destroyed (photobleached), either directly by the interaction or indirectly through dam- age due to the 1O2. In addition to the properties common to any therapeutic drug, photosensitizers should have high 1O2 quantum yield, high absorption 17 Advanced Photodynamic Therapy 317 Fig. 17.1. Jablonski diagram for singlet oxygen generation by PDT (Type II pho- toreaction) at red/near-infrared wavelength to get good light penetration, high target-to- nontarget uptake ratio in tissues or cells, and low concentration in the skin and eyes to avoid complications from accidental light exposure. Photosensi- tizers that are not water soluble require formulation into liposomes or lipid emulsions in order to achieve efficient delivery in vivo. The absorption spectra of some PDT photosensitizers are shown in Fig. 17.2, together with a plot showing the general dependence of light penetration in tissues. For example, HPD has its highest absorption in the UVA/blue region and only a very small red absorption (at about 630 nm). This contrasts with most second-generation photosensitizers where the molecule is designed to have the largest absorption peak above 630 nm where the hemoglobin absorption falls rapidly, but below about 800 nm in order to maintain high 1O2 quantum yield and avoid the NIR water absorption bands. One agent that is used widely is aminolevulinic acid (ALA). While not itself a photosensitizer, administering ALA to cells leads to synthesis of the photosensitizer protoporphyrin IX (PpIX) [8]. The advantage is that there is a degree of intrinsic tissue selectivity that does not just rely on enhanced photosensitizer uptake. ALA-PDT is used in treating tumors, particularly early-stage, and other indications mentioned above. Its fluorescence has also made it useful for early cancer/precancer imaging, either for tumor detection or in guiding tumor surgery, particularly in the brain [9]. As well as selection of good photosensitizer properties (and enough oxygen), successful PDT requires that enough light must be delivered to the target tissue to generate biologically effective levels of 1O2 (>108 molecules per cell). This can be technologically challenging, since the amount of light required is quite high [10], typically >100 J cm−2 of red/NIR light incident on the tissue surface. Since this amount of light has to be delivered in at most tens of minutes for clinical practicality 318 B.C. Wilson efficiency Photofrin mTHPC AIPcS4 400 500 600 700 Wavelength (nm) Useful range for most (non-superficial) applications 10 1 m Monte Carlo 5 s(1-g) = 1 mm 104 0 1000 W cm2 100 z [mm]1 per W incident10 1 power 1 2 0.1 −1 0 1 400 600 800 1000 r [mm] Wavelength (nm) Fig. 17.2. Absorption spectra of some typical photosensitizers (top) and the de- pendence of light penetration in tissues (bottom), showing the range of effective penetration (1/e) depth spectra in typical tissues (left) and a representative Monte Carlo calculation of the distribution of light fluence in tissue of given optical ab- sorption and scattering properties (right) (but see mPDT below), the light sources require typically a few Watts of output power at the right wavelength for the photosensitizer. Suitable light sources are diode lasers, wavelength-filtered lamps, and light emitting diode arrays (LEDs) [3, 11]. Each has its own advantages and limitations. Overall, lasers are preferred when delivery via single optical fibers is required, for example, in endoscopy or when treating a larger tissue volume by multiple interstitial fibers; LED arrays are particularly useful for irradiation of easily accessible tissue surfaces. Lamps have a role when treating very large surface areas and have the advantage of easy (and low cost) change of wavelength to match different photosensitizers. Different “modes” of target illumination by the light are used: surface, in- terstitial, or intravascular; external, endoscopic, or intraoperative. In all cases, making the total system (light source, power supply, light delivery devices, etc.) ergonomic and economic for specific clinical applications is critical. For the highest efficacy, the spatial distribution of the light in the tissue must be “matched” as far as possible to the volume and shape or the target tis- sue. This can be very difficult, given (a) the limited penetration of light in effective penetration depth (mm) Absorption 17 Advanced Photodynamic Therapy 319 tissue (the 1/e depth of red/NIR light in tissues is typically only a few mil- limeter), (b) the high variability optical scattering and absorption properties of tissues, and (c) the limited degree of “tailoring” of the applied light dis- tribution that can be achieved, especially in body locations where access is restricted. A variety of light-delivery devices has been developed for different applications [3, 10]. For example, optical fibers can be modified to produce a cylindrical irradiation volume along the last several centimeter at the tip, and recently it has become possible to make the output distribution deliberately nonuniform to match the target volume profile. Likewise, various balloon de- vices have been developed to irradiate within body cavities. With multiple fiber delivery, efficient splitting of the output of the light source is needed, and recent developments have included instruments in which nonequal distri- bution among sources can be achieved, again to match the target geometry in individual patients. These different technologies are still evolving as new clinical applications are investigated. A major challenge in some applications is to make the light source and delivery much cheaper and simpler to use than the established systems where, typically, the source cost is ∼$10 K per Watt and single-use delivery devices are hundreds of dollars. 17.3 Novel PDT Concepts In this main section we will present several new concepts that are still under development but which, if eventually adopted into clinical practice, would represent “paradigm sifts” in the way that PDT is performed, either in terms of the underlying principles used and/or in the technologies involved. 17.3.1 Two-Photon PDT The photophysical interaction that has been used in essentially all PDT appli- cations to date is that shown in Fig. 17.1 (although, in some cases, there may be Type 1 contributions to the photobiological effect). Thus, the S0 state of each molecule is activated by absorbing a single photon. Two-photon (2γ) ac- tivation, representing a fundamental alternative to this scheme, is illustrated in Fig. 17.3, in two different implementations both involving the absorption of two photons of light for each activation cycle. Consider Case A (referred to here simply as 2γ activation), where a wavelength is used at which the photosensitizer has a nonzero 2γ cross-section, σ. The probability of simulta- neous absorption of two photons is then proportional to σI2, where I is the instantaneous light intensity. Hence, to avoid requiring too much total energy (which could cause thermal damage to the tissue) an ultrashort laser pulse is needed, typically ∼100 fs. Since the energy required for the S0 →S1 transition is essentially the same as for 1γ activation, the wavelength of the pulsed laser can typically be pushed further into the NIR range, thereby increasing the penetration depth in tissue. This is one of the putative advantages of this ap- proach, although the situation is complex, since it involves a balance between 320 B.C. Wilson Tn S1 S1 T1 1O T 2 1 S 3 0 O2 S0 Fig. 17.3. Jablonski diagrams for two different types of 2γ activation involving either (a) simultaneous absorption of two photons by S0 (left) or (b) sequential absorption of two separate photons by S0 and then T1 (right) the reduced tissue attenuation and the quadratic dependence on the activa- tion intensity, which falls off faster with depth than the linearly-dependent 1γ intensity. The second advantage is that, because of the I2 dependence, focusing the laser beam activates the photosensitizer only within a very small (∼femtoliter) tissue volume. This is the same effect as used in 2γ confocal mi- croscopy, where it offers the analogous advantage of reduced photobleaching of the sample above and below the focal plane. A potential application of this concept is to treat AMD, which is a major application for (1γ) PDT, involving targeting of the abnormal blood vessel growth in the retina. 2γ PDT may be able to achieve vessel closure but min- imize any collateral damage caused by activation of photosensitizer outside the blood vessel layer. Recent research indicates that this is possible, in prin- ciple [12], but that there are several substantial technical challenges to be overcome, due primarily to the fact that absorption of two photons by S0 is a very low probability event [13]. As a result, since activation is proportional to σI2, either σ and/or I must be very high. The former requires the de- velopment of new molecules that have σ values several orders of magnitude higher than conventional PDT photosensitizers: in turn, this may also require the use of a “designer” delivery vehicle, since the pharmacological properties may then be difficult to control (e.g., limited water solubility). In principle, the intensity, I, may be arbitrarily high. However, in practice, this is limited by several factors: (a) the need |
to keep the total energy within sub-thermal range, and thus limiting the pulse energy, which means shorter pulses, (b) the cost of such laser sources, (c) nonlinear processes in the propagation of the laser pulses in the delivery system and the tissue itself, and (d) the onset of photomechanical damage to the tissue even in the absence of photosensitizer, which loses the potential selective advantage of photosensitizer localization. The practical implementation envisaged for 2γ PDT is to image the region of abnormal blood vessels, or “feeder” vessels, using a confocal scanning laser ophthalmoscope (cslo), as illustrated in Fig. 17.4. This instrument operates on the same principle as confocal microscopy, using a scanning laser beam focused at a specific plane in the object (in this case the neovascular layer). 17 Advanced Photodynamic Therapy 321 Fig. 17.4. Confocal scanning laser ophthalmoscope under development for image- guided 2γ PDT of age related macular degeneration, showing the optical design (top), the system in use, and a typical high-resolution retinal image (normal) The operator would then define the “target” and the laser beam would be switched to “treatment mode,” using the high peak-power fs laser. Apart from the fs light source itself, there are several optics challenges in implementing 2γ PDT for AMD, in particular, the following: – Efficient coupling of the fs laser source into the cslo is complicated by ef- fects such as pulse dispersion that take place as the laser beam propagates through the optical elements – Achieving diffraction-limited focusing at the back of the eye is restricted by the low numerical aperture of the eye and possibly by scattering (e.g., by cataract) and wavefront distortion (e.g., from astigmatism). To tackle the second problem, adaptive optics approaches will likely be needed and, in addition, automatic eye tracking will be required to keep the focal spot at the correct location. Both technologies are under development. Case B, so-called two-photon/two-color activation (2γ/2λ)-PDT, uses two laser pulses of different wavelength and separated in time. The first pulse 322 B.C. Wilson generates T1 via the S1 state. A second pulse, at a wavelength that is strongly absorbed by T1, then generates higher-order triplet states, Tn. The biological effects are then mediated by the Tn states interacting with nearby biomole- cules. The key point is that this process does not require molecular oxygen, and so may avoid the limitations of Type II photosensitizers, which do require adequate free oxygen in the target tissue. Oxygen-independent cell killing by 2γ/2λ PDT has been demonstrated in cells, although using a photosensitizer that is poorly suited to clinical use. However, there are major challenges in moving this concept to the clinic: (a) the photosensitizer must have high ab- sorption in both the S0 and T1 states and these must be at useful wavelengths for tissue penetration and (b) a 2γ/2λ laser source is required, preferably with independent control over the intensity and wavelengths of each pulse and pulse–pulse time delay to match the sensitizer photokinetics. Work is in progress to address both challenges. For example, Mir et al. [14] are devel- oping phthalocyanine with specific structural modifications to yield optimum absorption spectra in the ground and excited states, while rapid developments in pulsed laser sources, such as fiber lasers, are likely to yield practical laser technologies in the next few years. (Note that, in principle, this pathway could be activated to operate in CW mode with both wavelengths present simulta- neously, although the photoefficiency would be very low.) 17.3.2 Metronomic PDT At the other end of the light intensity scale is the concept of metronomic pho- todynamic therapy (mPDT). This is based on the observation that it may be possible to increase the target-specific cytotoxicity by delivering the photosen- sitizer and the light at very low dose rates over an extended period (hours or days) rather than applying a single, high dose-rate treatment. An example of such specificity is seen in treating brain tumors [15]. In this case, the clinical challenge is that the tumor cells around the edges of a solid tumor infiltrate the normal brain tissue. With standard, high dose-rate treatment, it may not be possible to kill the tumor cells without also destroying normal brain tis- sue. However, tumor cell-specific programmed cell death (apoptosis) can be achieved by mPDT. This has been demonstrated for ALA. It is not known if the mPDT effect is unique to ALA or applies also to other photosensitizers, or whether the mPDT principle can be applied also to other tumor sites or diseases. From the biophotonics perspective, a challenge is to develop light sources that could deliver light over a long period and still be practicable, either for clinical use or in preclinical animal models. Figure 17.5 shows an example based on high-efficiency coupling of LEDs into optical fibers, with battery and driver electronics incorporated into a lightweight “backpack.” For clinical use, the technology challenges will include (a) how to distribute the light, e.g., in the brain or other site after surgery to remove the bulk tumor tissue, (b) how to integrate the power supply, light source (diode laser or LED), and the 17 Advanced Photodynamic Therapy 323 Fig. 17.5. Fiber-coupled LED sources developed for mPDT use in rodent brain, and a source in use to treat brain tumor in a rat model, with the battery and electronics in a wearable backpack light delivery components, (c) how to maintain sterility and biocompatibility of such an implant and (d) how to provide power, for which one option is to have an external power supply and remote coupling to the source, as is used in some other implantable devices. The fundamental biological issue with mPDT is whether or not it is pos- sible to kill the target cells faster than their proliferation, while avoiding non- apoptotic killing (to avoid inducing an inflammatory response that could cause secondary damage to normal tissues) and avoiding direct PDT damage to nor- mal tissues. 17.3.3 PDT Molecular Beacons Molecular beacons were introduced several years ago as a way to enhance the target specificity of molecular imaging agents [16]. In that case the con- cept was to link a fluorescent reporter molecule (fluorophore) with a molecule 324 B.C. Wilson Triplet energy transfer hνν 1O hνν 2 1O2 O2 Q O2 PSQ PS Enzyme Enzyme Activation AS-ON 6.E+04 Tumor Specific mRNA Loop mRNA 5.E+04 G A AS-ON G C G G -C 4.E+04 A -T Linker Stem G -C C -G 5’3G’ -C PPSS 3.E+04 Linker Linker O O2 2 2.E+04 hνν PS Q hνν 1O2 1.E+04 Triplet energy transfer 1O2 0.E+00 Buffer P30 P30 P30C P30C P30C Alone +RNA +RNA+30min Fig. 17.6. PDT molecular beacons. showing the concept with enzyme cleavage of the linker (top), and using an antisense loop (bottom), with an example of the low 1O2 luminescence signal (third bar) compared to controls (first and second bars) that increases with unquenching (fourth bar) and then decreases with inhibition of the unquenching (fifth bar) (quencher) that suppresses the fluorescence when the two are in close prox- imity. The quenching efficiency has a very strong (∼1/r6) dependence on the quencher-fluorophore distance, r. The linker molecule could then be broken by, for example, interaction with an enzyme that is found in high concentration in the target tissues or cells. This concept has recently been transferred into PDT using a photosensi- tizer instead of the fluorophore (Fig. 17.6). Proof-of-principle studies of this concept have been reported [17], showing, for example, that 1O2 can be gen- erated by light exposure at much higher levels after unquenching than before. Selective toxicity has also been shown, both in vitro and in vivo, where only cells/tumors that express the specific enzyme used in the beacon are sensitive to PDT. There are several possible variations on this idea, in terms of the linker structures. One of the most interesting, but challenging in its synthetic chem- istry, is to use a so-called antisense loop, in which the quencher and photo- sensitizer are held in close proximity when the “hairpin” structure is closed, but then the linker opens up and unquenches the photosensitizer when it hybridizes with a specific nucleotide sequence in the target cells. Antisense beacons have been demonstrated to date in solution. This could have tremen- dous disease specificity, since it is based on highly defined genetic biomarker characteristics of, for example, tumor cells. Total 1O2 Luminescence + + 17 Advanced Photodynamic Therapy 325 17.3.4 Nanoparticle-Based PDT There has been great excitement recently in the potential of nanoparticles (NPs) for a wide range of applications, including medical diagnostics and therapeutics [18]. This disruptive technology is based on the special optical, electrical, mechanical, or chemical properties that emerge when materials are formulated at the nanoscale (typically, 1–100 nm) compared to the bulk prop- erties. Optical NPs include the following: – Metal NPs with light absorption and scattering spectra that are highly dependent on the size, shape, and form (e.g., solids vs. shells, spheres vs. rods) – Quantum dots (Qdots), semiconductor NPs that are fluorescent in the visible and/or near-infrared, which are very bright, have very low photo- bleaching, have narrow emission spectra with the peak emission increasing with the Qdot diameter, and have a wide excitation spectrum so that a single light source can excite many different sizes of Qdots – NPs that are themselves not optically active but that can carry a “pay- load” of optically active molecules, such as fluorescent dyes or photosen- sitizers In addition to these specific novel properties, NPs have the advantage that they can be “decorated” with molecules, such as antibodies or peptide sequences, to target them to specific cells or tissues. The potential of NPs for cancer diagnostics (particularly imaging) and therapeutics has been recently reviewed [19]. An important subset of these techniques is photonics-based, including fluorescence imaging and photothermal and photodynamic treat- ments. For PDT there are several potential and distinct ways to utilize NP char- acteristics, as illustrated in Fig. 17.7, namely (a) as “carriers” of PDT photo- sensitizers, (b) as photosensitizers themselves, or (c) as “energy transducers.” Fig. 17.7. Schematic of three different uses of nanoparticles for PDT: (a) as targeted “carriers” of the photosensitizer, (b) as direct 1O2 generators, and (c) as energy transducers 326 B.C. Wilson 1O2 luminescence peak in suspension 9000000 8000000 7000000 6000000 5000000 4000000 3000000 2000000 1000000 0 1200 1220 1240 1260 1280 1300 1320 1340 Wavelength (nm) Fig. 17.8. 1O2 signal from porous silicon NPs in water (relative to background): insert shows the ultrastructure of these NPs For Case A, a variety of NPs can be used that either encapsulate many pho- tosensitizer molecules per NP or have many attached to the surface in order to deliver a high payload of photosensitizer to the target cells/tissues. There are several demonstrations in the literature of this approach. One advantage that PDT has over, say chemotherapeutic drugs, is that generally the toxicity of most photosensitizers is very low in the absence of light activation, so that there is a higher tolerance for nontarget “leakage” in the delivery system. For Case B, Fig. 17.8 shows an example where singlet oxygen is generated upon light irradiation of porous silicon NPs, without any added molecular photosensitizer. This is believed to be due to direct energy transfer to the molecular oxygen in the liquid that permeates the NPs, which have an ex- tremely large surface-to-mass ratio. It has not yet been shown that this can actually be exploited to produce PDT cell killing: these studies are in progress. Potential barriers are getting the NPs into the cells and having the 1O2 dif- fuse from the (inner) NP surface to the biological target. (Note that in the case of NPs as photosensitizer delivery vehicles, it may not be necessary for the NPs themselves to penetrate the cell wall if the payload can be released within the target tissue, such as in the interstitial space, from which it would then be taken up by the cells.) In the third scenario, Case C, Qdots are used as the primary light absorber. The energy is then transferred to a photosen- sitizer molecule that is conjugated to the Qdot, activating it to the S1 state, as in standard PDT. The generation of 1O2 via Förster Resonant Energy Transfer has been demonstrated in solution and should work in cells/tissues. Photon counts 17 |
Advanced Photodynamic Therapy 327 Again, the advantage in principle would be the ability to target to Qdots to the desired tissues/cells without compromising the optimum photosensitizer properties. A variation of this approach would be to use it for 2γ PDT, since Qdots are known to have very high σ values (∼104–105 GM units compared with ∼10 GM units for conventional 1γ drugs and ∼103 GM units for the best “designer” 2γ molecules to date). 17.4 PDT Dosimetry Using Photonic Techniques PDT dosimetry, i.e., the measurement of the “dose” that determines the effi- cacy of the treatment, is complex, due to the following: (a) The multiple factors involved (photosensitizer, light, oxygen) (b) The heterogeneity of the local photosensitizer concentration, light fluence rate, and oxygen level in the tissue, which may result in nonuniform de- position of PDT dose throughout the target volume, and (c) The interdependence and dynamic behavior of the parameters, e.g., 1O2 destroys the photosensitizer molecules (photobleaching), reducing the amount available for activation; the photosensitizer may increase the at- tenuation of light in the tissue, further limiting the penetration depth; if the photosensitizer concentration and the light intensity (fluence rate) are high enough, this can deplete the ground-state oxygen faster than the blood supply can replenish it. Three main dosimetry approaches have been used to address this complexity, termed explicit, implicit, and direct [20]. In the first, the attempt is to measure the three basic parameters of light fluence rate, photosensitizer concentration, and molecular oxygen concentration, which are then combined into a predic- tive estimate of the effective PDT dose. There are many technical details in each of these three measurements [3, 10]. Figure 17.9 illustrates light dosime- try in PDT to destroy prostate cancer by delivering light through multiple diffusing optical fibers to the whole prostate volume. To optimize the distribution of light throughout the prostate treatment planning is used through a series of steps: 3D MRI is done to determine the volume and shape of the prostate; source fibers are placed at selected posi- tions to cover the prostate volume; the spatial light fluence-rate distribution resulting from delivering selected powers to each of the source fibers is cal- culated; this is compared with the desired light distribution; and the steps are iterated to optimize the number, length, position, and power from each source. This is analogous to treatment planning in radiation therapy (e.g., brachytherapy using implanted radioactive wires or seeds). For photosensi- tizer dosimetry, the main technique has been to measure the fluorescent by point spectroscopy or imaging. However, while it is straightforward to make relative measurements, it is more difficult to derive absolute photosensitizer concentration values, since the measured signal depends also on the tissue 328 B.C. Wilson Fig. 17.9. Example of light dosimetry during PDT of prostate cancer, showing the patient set-up, the placement of fibers and detector fibers through a template, the multichannel light dosimeter measurements of the local light fluence rate as a function of time during treatment at three different locations the graph shows: (a) in the prostate, (b) the urethra and (c) the rectum optical properties. One solution is to measure these properties separately and apply a light propagation model to correct for the attenuation. Alternatively, fiberoptic probes have been developed to reduce this attenuation dependence, for example, by sampling only a very small tissue volume [22]. The measurement of oxygen levels in tissue is well-established, for example, using interstitial microelectrodes or fiberoptic probes. Noninvasive measure- ments are less well developed and do not directly measure the pO2, e.g., diffuse reflectance spectroscopy can detect changes in the relative concentration of 17 Advanced Photodynamic Therapy 329 Fig. 17.10. Example of treatment planning for PDT of prostate [21], showing the process for calculating the 3D light distribution in the tissue (top, two-source ex- ample) and the diffusion-theory equation that is solved on a nonuniform mesh by finite-element analysis (bottom, six-source example) hemoglobin and oxyhemoglobin, from which the oxygen saturation, SO2, can be derived. (This is also used in studies of brain and muscle physiology [23].) In implicit dosimetry a surrogate measure of the integrated interplay of light, drug, and oxygen is employed, most commonly the photosensi- tizer photobleaching. Referring to Fig. 17.1, if the 1O2 can react with and disrupt the photosensitizer molecules such that they become nonphotoac- tive/nonfluorescent, then this is related to the 1O2 production. This method is technically fairly simple and can be applied at multiple locations in the tissues. 330 B.C. Wilson However, interpreting the data rigorously is not simple, since the photobleach- ing may or may not be 1O2-dependent and there may be non-1O2 dependent cytotoxic pathways if the tissue pO2 is low. Nevertheless, under well-controlled conditions, photobleaching can be a very useful PDT dose metric [24]. Lastly, in direct dosimetry, 1O2 itself is quantified by detecting the 1,270 nm luminescence emission from the 1O2 →3O2 transition. The signal is very weak, since 1O2 is so reactive in biological media, with only about 1 in 108 molecules decaying by luminescence. However, this has been demon- strated recently both in vitro and in vivo and is highly correlated to the PDT response [25–27]. The challenge now is to simplify and reduce the cost and complexity of the technology. 17.5 Biophotonic Techniques for Monitoring Response to PDT Partly in response to the continuing challenge of PDT dosimetry, but also to gain more direct understanding of the biological effects of PDT treatment, there has been ongoing interest in assessing directly the tissue responses fol- lowing or even during PDT. The last, in particular, is also motivated by the possibility to use this information for feedback control of the treatment while it is in progress. A first example that is used for preclinical studies is bioluminescence imag- ing (BLI). For this the cells of interest are transfected with the luciferase (“firefly”) gene, so that, when luciferin is supplied the cells emit visible light through chemiluminescent. Although weak, this can be imaged noninvasively using a high sensitivity photodetector, such as a cooled CCD array. BLI has been used to quantify changes in implanted tumors [28] or the survival of bac- teria [5] treated with PDT in animal models. The method can be extended by having the luciferase gene only “turned on” when another gene of interest (e.g., a stress-response gene) is up-regulated by the PDT treatment, so that BLI gives direct in vivo imaging of the response of the target cells at the specific molecular level. A second example is optical coherence tomography (OCT), the analogue of high-frequency ultrasound imaging, based on interferometry (Fig. 17.11) (explained in detail in the chapter by S. Boppart). Depth scans (A-scans) are produced by detecting the interference signal between the reflected light from a given depth in the tissue and a reference beam: high spatial resolution is obtained by using a light source with a very short coherence length. A 2D image (B-scan) is then generated by scanning the beam across the tissue sur- face. OCT is approved in ophthalmology and is being widely investigated for a range of other applications, including endoscopic and intravascular imag- ing using fiberoptic implementation. As with ultrasound, OCT can operate in Doppler mode to image blood flow (by measuring the small shift in phase be- tween successive A-scans caused by movement of the red blood cells or other tissue components). 17 Advanced Photodynamic Therapy 331 reference structure blood flow mirror coherence length beam splitter low-coherence CW optical source tissue detector Change in microvascular blood flow index during and post PDT 1 ------DURING---- 0 3 5 7 20 PRE 1 3 6 12 24h POST Hours post PDT treatment Fig. 17.11. Doppler OCT for monitoring vascular response of tumor tissue to PDT treatment. (Top) Principle of OCT and example of cross-sectional imaging in vivo. (Bottom) Structural and DOCT images at different time points following PDT treat- ment showing blood vessel shut down (left) and plot of volume-averaged measure of blood flow in the tissue during and following Photofrin-PDT treatment of melanoma For monitoring the changes to tissue from PDT, one can look at either the structural or the Doppler images. The latter have proved to be more useful to date, since the changes in tissue structure are rather subtle. Figure 17.11 also shows examples of the changes in blood flow in individual microvessels due to PDT. Such changes can be seen in some cases even during the light treatment [29], so that there is the possibility to use the DOCT information to modify the treatment “on line.” A limitation with OCT is that the depth of imaging is small (∼1–2 mm). Recently, we reported an OCT probe within a small-diameter needle that can be placed interstitially [30], e.g., at the tumor base to ensure that adequate PDT dose is delivered. 17.6 Biophotonic Challenges and Opportunities in Clinical PDT We end with a brief overview of some of the major outstanding limitations of PDT and how these might be overcome, at least in part by applying biopho- tonic science and technologies. Certainly PDT can be both safe and effective. However, there can be sig- nificant variations in the response. One of the obvious problems is that there is little or no “tailoring” of the treatment parameters used to the individual 332 B.C. Wilson patient or lesion. The various dosimetry methods outlined above could im- prove this situation, but they are rarely used in routine clinical practice at this time, in part because they are still rather complicated to use and inter- pret. A major technical challenge is to devise the next generation of dosimetry devices that will be simple and fast to use, minimally invasive, inexpensive, and reliable. Until this happens, PDT will continue to evolve largely as an empirical procedure with optimization based on dose ranging in clinical tri- als. A second issue is the continuing high cost of light sources. Using existing technologies it is not clear how this can be significantly reduced. Certainly, part of the cost comes from dealing with medical devices where significant investments are required to ensure safety and reliability. It is becoming clear that, for some potentially important applications, it is the cost of the light source that is limiting, not the cost of the photosensitizer. Hence, there are significant opportunities for optical engineers and physicists to devise new devices. A further major limitation is that the specificity of photosensitizer target- ing of disease has not been high enough to use unlimited light safely. Attempts have been made to improve this, for example, by linking the photosensitizer to antibodies to target tumors cells, but these have not been particularly suc- cessful to date. PDT beacons could change this significantly. However, it will be challenging to get these into clinical trials and then through regulatory approvals, so that the preclinical results in vivo will have to be outstanding to make the effort worthwhile. With respect to tissue response monitoring, there are many novel biopho- tonic imaging and spectroscopic techniques being developed for other appli- cations that could also be valuable in PDT: fluorescence imaging is a prime example, while others include Raman spectroscopy (to report biochemical changes) and intravital second harmonic generation imaging (changes in tis- sue architecture). PDT monitoring will not drive the development of these techniques but will benefit from them. Three other major aspects of PDT are worth mentioning: – The parallel development of “photodynamic diagnostics,” particularly for tumor detection and localization [23] – The combination of PDT with other therapeutic modalities, e.g., (fluo- rescence guided) tumor resection followed by PDT to eliminate residual minimal disease – The extension of PDT into new applications, particularly to modify cells or tissue function rather than destroying them: recent examples are for modifying bone growth and for targeting epilepsy. In addition to these clinical applications, the principle of light-activated drugs may also be useful for basic life sciences research. One example is the use of 2γ PDT to uncage growth factors in order to direct the growth of neurons in tissue engineering [31]. 17 Advanced Photodynamic Therapy 333 17.7 Conclusions Although PDT is over a century old, it is far from a mature science. Address- ing some of the challenges in PDT, particularly in the optical technologies and dosimetry, has been a major driving force in biophotonics as a whole, with substantial spin-off into other |
applications. For example, much of the early work in tissue optics was motivated by the need to understand and control the distribution of light in PDT of solid tumors. In turn, this laid the foundation for diffuse optical tomography and other therapeutic and diagnostic tech- niques. In the same way, studies of photosensitizer fluorescence led directly to imaging based on tissue autofluorescence: subsequently, attempts to improve the image contrast stimulated work on targeted fluorophores, including most recently quantum dots. It is reasonable to expect that PDT will continue, both academically and commercially, especially with the extension into a diverse range of new ap- plications and using some of the new concepts outlined here. There is still certainly much to be done that will require the efforts of many different dis- ciplines. Acknowledgments The author thanks the following agencies for support of work illustrated here: the National Cancer Institute of Canada (1O2 dosimetry and DOCT monitoring), the National Institutes of Heath, US (prostate PDT and mPDT [(CA-43892)], beacons), the Canadian Institute for Photonic Innovations (2-photon PDT), and Photonics Research Ontario (PDT technology devel- opment). Also, thanks to many colleagues and students in the PDT program at the Ontario Cancer Institute and to our clinical and industry collaborators. References 1. T. Patrice (ed.), Photodynamic Therapy, (Royal Society of Chemistry, UK, 2003) 2. R. Ackroyd et al., Photochem. Photobiol. 74, 656 (2001) 3. B.C. Wilson, S.G. Bown, in Handbook of Lasers and Applications, ed. by C. Webb, J. Jones (IOP Publ, UK, 2004), p. 2019 4. T.J. Dougherty, J. Clin. Laser Med. Surg. 20, 3 (2002) 5. T.N. Demidova, M.R. Hamblin, Int. J. Immunopathol. Pharmacol. 17, 245 (2004) 6. B.W. Henderson, T.J. Dougherty, Photochem. Photobiol. 55, 145 (1992) 7. N.L. Oleinick, H.H. Evans, Radiat. Res. 150, S146 (1998) 8. Q. Peng et al., Photochem. Photobiol. 65, 235 (1997) 9. W. Stummer et al., Acta Neurochir. Suppl. 88, 9 (2003) 10. M.S. Patterson, B.C. Wilson, in Modern Technology of Radiation Oncology, ed. by J. Van Dyke (Medical Physics Publishing, Madison, WI, USA, 1999), p. 941 334 B.C. Wilson 11. L. Brancaleon, H. Moseley, Lasers Med. Sci. 17, 173 (2002) 12. K.S. Samkoe, D.T. Cramb, J. Biomed. Opt. 8, 410 (2003) 13. A. Karotki et al., Photochem. Photobiol. 82, 443 (2006) 14. Y. Mir et al., Photochem. Photobiol. Sci. 5, 1024 (2006) 15. S.K. Bisland et al., Photochem. Photobiol. 80, 2 (2004) 16. U. Mahmood, R. Weissleder, Mol. Cancer Ther. 2, 489 (2003) 17. J. Chen et al., J. Am. Chem. Soc. 126, 11450 (2004) 18. M. Ferrari, Nat. Rev. Cancer 5, 161 (2005) 19. B.C. Wilson, in Photon-Based NanoScience and Technology, ed. by J. Dubowski, S. Tanev (Springer, Netherlands, 2006), p. 121 20. B.C. Wilson et al., Lasers Med. Sci. 12, 182 (1997) 21. R.A. Weersink et al., J. Photochem. Photobiol. 79, 211 (2005) 22. B.W. Pogue, G. Burke, Appl. Opt. 37, 7429 (1998) 23. B.C. Wilson, in Handbook of Lasers and Applications, ed. by C. Webb, J. Jones (IOP, UK, 2004), p. 2087 24. J.S. Dysart, M.S. Patterson, Photochem. Photobiol. Sci. 5, 73 (2006) 25. M. Niedre et al., Photochem. Photobiol. 75, 382 (2002); 81, 941 (2005) 26. M. Niedre et al., Cancer Res. 63, 7986 (2003) 27. M. Niedre et al., Br. J. Cancer 92, 298 (2005) 28. E. Moriyama et al., Photochem. Photobiol. 80, 242 (2004) 29. H. Li et al., Lasers Surg. Med. 38, 754 (2006) 30. V.X. Yang et al., Opt. Lett. 30, 1791 (2005) 31. Y. Luo, M.S. Stoichet, Nat. Mater. 3, 249 (2004) Index 1D photonic crystal, 103 Genome, 177 2D photonic crystal, 103 Genomics, 219 Gram bacteria, 114 Active pixel sensor, 239 Greenhouse effect, 15 Avidin, 200 Hydrogenases, 19 Biosensor, 107, 199 BSA, 120 IgG, 117 Carotenoids, 3, 31, 42 Keratinocytes, 301 CARS:Coherent anti-Stokes Raman, 48 Label-free, 88 CCD, 239 Label-free optical biosensing, 109 Chlorophylls, 3, 29 Laser tissue welding, 275 Chloroplasts, 2 Light cone, 104 DHM, 164 Light harvesting complexes, 6 DHM Life Cell Imaging, 171 Mach–Zehnder interferometer, 128 DHM numerical focus, 170 Michelson-type interferometer, 128 DHM resolution, 170 Microcavity, 94, 105, 110, 111, 121 Digital Holographic Microscopy, 164 Microscopic Speckle Interferometry, 161 Digital holographic reconstruction, 166 Multi-photon microscopy, 47 Distal endoscopic ESPI, 158 Mutant Type protein, 195 DNA, 114 Double exposure subtraction ESPI, 152 NDRM, 166 Non diffractive reconstruction, 166 Effective dielectric constant, 110 Electronic Speckle Pattern Interferome- Ophthalmology, 127 try, 152 Optical tweezers, 249 ESPI, 152 Optrodes, 204, 207 Fluorescence, 49 Phosphorescennce, 49 Fluorescent dye molecule, 87 Photonic bandgap, 105 Functionalization, 111 Photonic crystals, 101 Photosynthesis, 1, 17 Genetic engineering, 202 Plasmid, 194 336 Index Porous silicon, 107 Singlet oxygen, 30 Proteomeics, 219 Spatial phase shifting, 153 Proteomics, 221 SPS, 153 Proximal endoscopic ESPI, 156 Stable transfection, 194 Streptavidin, 115 Quantum dots, 178 Second harmonic generation, 48 Thylakoids, 2, 6 Sensitivity vector, 153 Transfection, 194 Setups for DHM, 165 Transient transfection, 194 Silanization, 114 Two-photon cross-section, 58 Single photon avalanche diode, SPAD, 239 Vector, 194 |
Encyclopaedia of MEDICAL PHYSICS SECOND EDITION Encyclopaedia of MEDICAL PHYSICS SECOND EDITION Edited by Slavik Tabakov Franco Milano Magdalena S. Stoeva Perry Sprawls Sameer Tipnis Tracy Underwood Second edition published 2021 by CRC Press 6000 Broken Sound Parkway NW, Suite 300, Boca Raton, FL 33487-2742 and by CRC Press 2 Park Square, Milton Park, Abingdon, Oxon, OX14 4RN © 2021 Slavik Tabakov, Franco Milano, Magdalena S. Stoeva, Perry Sprawls, Sameer Tipnis & Tracy Underwood First edition published by Taylor & Francis 2012 CRC Press is an imprint of Taylor & Francis Group, LLC The right of Slavik Tabakov, Franco Milano, Magdalena S. Stoeva, Perry Sprawls, Sameer Tipnis & Tracy Underwood to be identified as the authors of the editorial material, and of the authors for their individual chapters, has been asserted in accordance with sections 77 and 78 of the Copyright, Designs and Patents Act 1988. This book contains information obtained from authentic and highly regarded sources. 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ISBN: 978-0-367-63251-9 (pbk) ISBN: 978-1-138-59214-8 (hbk) ISBN: 978-0-429-48996-9 (ebk) Typeset in Times Deanta Global Publishing Services, Chennai, India Contents Preface: The Road Which Led to This Encyclopaedia . vii How to Use EMITEL Website (www .emitel2 .eu) . xi Editorial Board of the Encyclopaedia of Medical Physics (2nd Edition) .xv Contributors . xvii VOLUME I A–K Numerals .3 A .7 B .77 C .135 D .233 E .305 F .353 G . 405 H .433 I .459 J.521 K .523 VOLUME II L–Z L .533 M .573 N .639 O .661 P .677 Q .755 R .765 S .825 T .917 U .985 V . 997 W.1013 X .1027 Y .1031 Z .1035 Index .1037 v Preface: The Road Which Led to This Encyclopaedia Almost 25 years have passed from the birth of the idea to this Our next project EMERALD – INTERNET ISSUE second updated edition of the Encyclopaedia of Medical Physics. (EMERALD II, 1999–2000) developed the first educational During this period of time, the medical physics profession has had website in medical physics (www .emerald2 .net currently www incredible growth – from about 12,000 medical physicists globally .emerald2 .eu), which continues to have thousands of users each in 1995 (starting from 6,000 in 1965) to about 30,000 globally in month. This activity assured us that the idea of an Encyclopaedia 2020. The most substantial growth has been in low- and middle- with Dictionary would have to be developed initially as an open- income countries. It is without doubt that a significant part of this access e-Encyclopaedia (at that time Wikipedia did not yet exist). growth was underpinned by the educational projects carried out Naturally, this was an extremely complex endeavour and it by various teams, including our projects and the Encyclopaedia required careful planning in several phases. and Dictionary team. This focus on education will undoubtedly continue in order to reach over 60,000 medical physicists in 2035 (as predicted in various publications). THESAURUS AND MULTILINGUAL One of the key aspects of developing the education materials SCIENTIFIC DICTIONARY was the ease of access for university students and hospital medical physicists; this is especially relevant for professional development The first phase of the large Encyclopaedia with Dictionary proj- in low- and middle-income countries. Thus the numerous con- ect was to develop a Thesaurus – a bank of necessary scientific tributors to our projects over the years have striven to make these terms. This task, plus the Multilingual Scientific Dictionary of materials free where possible. In a world where medical care is Medical Physics Terms, was included in our next project EMIT of increasing importance and visibility, this generous approach (2001–2003), which widened the scope of the training materials and collegial attitude is one of the truest forms of international created in project EMERALD. EMIT added Ultrasound Imaging collaboration. and MR Imaging training modules, as well as the development This preface gives a short description of the projects and of a Dictionary – initially in five European languages: English, activities which led to the development of the Encyclopaedia of French, German, Italian and Swedish. The projects mentioned so Medical Physics and the Scientific Dictionary of Medical Physics far were supported financially by the EC program Leonardo and Terms. included a core of partners from universities and hospitals from the UK, Sweden, Italy and ICTP, plus partners from Portugal, France, Ireland, Czech Republic, Bulgaria, EFOMP and col- THE NEED FOR AN ENCYCLOPAEDIA leagues from other countries, specially mentioning P. Sprawls WITH DICTIONARY (with his educational website Sprawls Educational Resources), colleagues from several IAEA activities and the ICTP College The idea for the Scientific Dictionary and Encyclopaedia of on Medical Physics. All our projects are described in the free Medical Physics was triggered by our first e-learning project downloadable e-book The Pioneering of e-Learning in Medical EMERALD (1995–1998), which developed training materials Physics (www .emerald2 .eu /mep _15 .html). (e-books and image databases) to address the initial training of During the second phase, we created an original system young medical physicists. These were the first e-learning materi- which uses identification numbers (IDs) for each term from the als in the profession and included specific training tasks covering Thesaurus. This was necessary for the cross-translation between the physics of X-Ray Diagnostic Radiology, Nuclear Medicine and any of the languages in the Multilingual Scientific Dictionary. In Radiotherapy. These materials (written in English) included the this system, all translations to various languages were based on first e-books with educational image databases in medical phys- the IDs of the English terms from the Thesaurus. ics – the carrier of these was the second in the world CD-ROM Groups of translators were formed for each language, usually with ISBN number. including specialists in the main fields of the profession (Physics The first tests of these e-learning materials were in 1996 at the of: X-Ray Diagnostic Radiology, Nuclear Medicine, Radiotherapy, College of Medical Physics (ICTP, Trieste, Italy) and in 1997 at Ultrasound Imaging, Magnetic Resonance Imaging and Radiation the new MSc in Medical Physics at Plovdiv, Bulgaria (part of our Protection). General terms were covered by all translators (mainly international project ERM). These events revealed the need for terms related to relevant frequently used terminology from phys- a Scientific Dictionary of Medical Physics Terms for the inter- ics, mathematics, medicine, etc.). national medical physics community, since most medical phys- After several updates and consolidation, the Thesaurus ics publications were in English. The same was evident from reached 3500 terms by 2003. The Thesaurus and first-edit the First International Conference on Medical Physics Training Scientific Dictionary were engraved on a mini-CD and distrib- (1998 at ICTP, Trieste), which we organised to test the usability of uted for free at the World Congress on Medical Physics and EMERALD materials. The representatives of many universities Biomedical Engineering in Sydney, Australia (2003). The need from various countries at this Conference underlined the require- for a Dictionary triggered another wave of demands for additional ment of use of national language for education. This led to the translations, while some Asian countries even used our Dictionary idea that the profession needed an Encyclopaedia with Scientific CD as a catalyst to create their own national Dictionaries to/from Dictionary to support the first steps of the newly formed medical English. The First International Conference on e-Learning in physics courses around the world. Medical Physics, which we organised in November 2003 in ICTP, vii viii Preface: The Road Which Led to This Encyclopaedia Trieste, further supported the value of this Multilingual Scientific our students of the MSc Programme Medical Engineering and Dictionary. Physics at King’s College London, UK. In December 2004 our EMIT project received the inaugu- The fourth phase, created in parallel to the third, was the devel- ral EU Award for vocational training – the Leonardo Da Vinci opment of the web database and the website to host the e-Encyclo- Award. Our unique Scientific Dictionary of Medical Physics paedia. The website was created and maintained by our IT partner Terms played a significant role in selecting the project among AM Studio (M. Stoeva and A. Cvetkov). The website was made almost 500 applications. The award was presented at the high-level with a user-friendly and flexible structure, and crucially with our Conference ‘Strengthening European Co-operation in Vocational own design, not based on external templates. All our educational Education and Training’, held in Maastricht, the Netherlands (part websites were purpose-built without external software and have of the summit of all European Ministers of Education). This was been running non-stop since their creation. It is important to note an exceptional recognition for the project and all its partners and that the longevity of any such product is affected by updates of translators, and it added greatly to the visibility of our profession third-party software. The original website built by our project has – medical physics. flawlessly served the profession for over 10 years. The website was built with its own Content Management System (CMS) to allow easy future updates. ENCYCLOPAEDIA OF MEDICAL PHYSICS The main validation of the Encyclopaedia at the International Once the Thesaurus was compiled, we were able to proceed to the Conference (ICTP, Trieste, 2008) included 21 present and past third phase of the project – the development of the entries/articles Presidents of Medical Physics Societies. This was also made at for the Encyclopaedia of Medical Physics. This was made as a a Topical Workshop at the World Congress on Medical Physics consecutive EU project, EMITEL (2005–2009), supported by the and Biomedical Engineering in Munich, Germany (2009). All core of the previous partners and also colleagues from all over participants had online access to the Encyclopaedia and assessed Europe and other countries associated with the IOMP. It was also highly the usefulness of the project and gave recommenda- necessary to create an original methodology for the development tions. Their feedback was implemented in the final editing of of an Encyclopaedia. |
the materials made in early 2010. With this, the EU project was It was important to establish what type of Encyclopaedia was completed. to be created. A number of specialist Encyclopaedias include a In parallel to all this, the second phase (translations of the relatively small number of large articles plus an extensive index Scientific Dictionary terms) continued with full speed and by of terms, mentioned in the articles. Other Encyclopaedias consist 2009 the number of languages had increased to 27 (in nine of a large number of small articles – these are easier to search alphabets): Arabic, Bengal, Bulgarian, Chinese, Croatian, and update, however such Encyclopaedias are more difficult to Czech, English, Estonian, French, German, Greek, Hungarian, organise as they include many authors, many entries and many Italian, Japanese, Latvian, Lithuanian, Malaysian, Persian, reviewers. A well-known general knowledge Encyclopaedia with Polish, Portuguese, Romanian, Russian, Slovenian, Spanish, a large number of small articles is Larousse. Swedish, Thai and Turkish. Many of these were translations As we already had the Multilingual Scientific Dictionary, made by former attendees to the ICTP College on Medical based on our Medical Physics Thesaurus, it was logical to accept Physics. the second design (with a large number of small articles). This The huge parallel coordination of so many simultaneous con- was also suitable as a Reference in the dynamic profession of tributions to the Encyclopaedia and to the Dictionary by various Medical Physics, where updates would be necessary quite often. workgroups from many countries required the Project Manager By that time Wikipedia was gaining popularity and it also used and Coordinator (S. Tabakov) to devote all his spare time to this concept. achieve the harmonious development of the project. This activity It was agreed that the educational level of the Encyclopaedic was the largest international project in the profession, its results entries should be at Master level and above (MSc, or equivalent, being used by thousands of colleagues each month. which is usually the case for most medical physics university In order to combine the Encyclopaedia entries (in English) courses around the world). Being linked with the previous educa- with the Multilingual Dictionary, the website (www .emitel2 .eu tional projects, EMITEL included a number of images from their and also www .emitel2 .net) was extended and was equipped with image databases. The Encyclopaedia was developed with around two search engines – one multilingual (for the Dictionary) and 2800 full articles/entries. All entries were written in English, but one in English (to search inside the text of the entries). The fact the Encyclopaedia was developed to work online together with that the Internet browsers at that time were already support- the Dictionary. ing various alphabets was very important for the work of the The coordination of such a huge project was extremely com- Dictionary. plex, as the articles/entries were developed by seven groups working in parallel. These were groups in Medical Physics of: FURTHER DEVELOPMENT OF X-Ray Diagnostic Radiology, Nuclear Medicine, Radiotherapy, THE ENCYCLOPAEDIA Ultrasound Imaging, Magnetic Resonance Imaging, Radiation Protection and General terms. The fifth phase of the Encyclopaedia (2010–2013) was a self- A special system was developed to organise the large number funded independent project. Its aim was to support and update of entries, each with its own text, image files and other data. For the Encyclopaedia, including preparations for a paper print of the ease of reference, we kept the ID numbers of the entries identical encyclopaedic entries by CRC Press. to those in the Dictionary and each entry file had to go through The main CRC paper print phase was carried out by the several stages of reviewing. The internal process of reviewing coordination office of the Encyclopaedia and the first editors – included not only the authors and reviewers, but also the ultimate S. Tabakov, F. Milano, S.-E. Strand, C. Lewis, P. Sprawls and users of our materials – the students. Useful feedback came from Editorial Assistant V. Tabakova (all of them members of the Preface: The Road Which Led to This Encyclopaedia ix previous projects). This activity underwent another editing of the some have retired and some are no longer with us. All these col- content (together with CRC Press). leagues made free contributions to this huge project (mostly in The CRC paper print was ready by 2014 and a number of uni- their spare time) in order to support the global development of versity libraries included it in their catalogues. Alongside this, the medical physics. open-access web site continued to be used by colleagues from all As I developed and coordinated the described projects/phases, over the world with thousands of users per month, especially from and invited personally most of these colleagues, I am truly grate- low- and middle-income countries. ful to each one. Given the numerous and frequent use of our results Over the next few years, the support for the Encyclopaedia during the past 25 years, I am also sure that most young medical update and website was mainly through its coordination office. physicists around the world appreciate highly and are grateful Another update of the Thesaurus and entries was made during for this generous collegial contribution. In this book, and in the this period and the Dictionary was enriched with five more lan- website (www .emitel2 .eu), we have listed all colleagues who con- guages – Finnish, Korean, Georgian, Ukrainian and Vietnamese. tributed to the project for Encyclopaedia of Medical Physics and Currently the Multilingual Scientific Dictionary of Medical Multilingual Scientific Dictionary of Medical Physics Terms. The Physics Terms cross-translates in 32 languages (11 alphabets). entire network of these colleagues from over 50 countries is a real In 2016 this activity entered the current phase (sixth phase) example of international collaboration. – also a self-funded independent project – preparing an update We gratefully acknowledge the contribution of all colleagues and second paper print of the e-Encyclopaedia by CRC Press. to this huge endeavour, the initial financial support from EU to This phase was facilitated by a group of the active members of the the listed projects and the support from various institutions and initial stages of EMITEL. A new team of contributors was gath- organisations. We are also grateful to the teams of CRC Press ered to revise the existing material and to add new medical phys- and Deanta (in particular Kirsten Barr, Rebecca Hodges-Davies ics terms, as well as create new encyclopaedic entries for these. and Lillian Woodall) with whom we collaborated during the final This project was guided by the Encyclopaedia Editorial Board – S. stages of the preparation of this book. Tabakov (Chair), F. Milano, M. Stoeva, P. Sprawls, S. Tipnis and T. Medical physics is a dynamic profession – in the past decades Underwood – and was supported by many colleagues from various it has changed dramatically and will continue to grow and countries and by alumni from the MSc at King’s College London. develop. The constant introduction of new methods and equip- This project phase also added new fields to the Encyclopaedia ment will require constant update of medical physics education. and Dictionary – Non-Ionising Radiation Safety and Medical This will undoubtedly be reflected in the future updates of the Equipment Management. This resulted in over 650 new terms Encyclopaedia with Dictionary, as one of the main reference related to the new developments of medical physics. The current resources of the profession, supporting our educational activities, phase was completed in early 2020. and also as evidence of the important contribution of medical After this update, the current Encyclopaedia of Medical physics to healthcare. Physics, Second Edition includes over 3300 cross-referenced full entries related to medical physics and associated technologies. Prof. Slavik D. Tabakov, PhD, Dr h.c., The materials are supported by over 1300 figures and diagrams. FIPEM, FHEA, FIOMP, FIUPESM The Encyclopaedia also includes over 600 synonyms, abbrevia- Developer & Coordinator of the Dictionary tions and other linked entries. and Encyclopaedia Projects IUPESM Vice-President (2018–2022) CONCLUSION AND ACKNOWLEDGEMENTS IOMP President (2015–2018) Around 150 contributors from 30 countries took part in the devel- opment of the Encyclopaedia of Medical Physics through its vari- ANNEX: MULTILINGUAL SCIENTIFIC ous stages (these colleagues are in the List of Contributors to the DICTIONARY OF MEDICAL PHYSICS TERMS Encyclopaedia). Additionally, over 200 colleagues took part in the transla- Dictionary Lead Coordinator: Slavik Tabakov tions of the Multilingual Scientific Dictionary of Medical Physics Dictionary Contributors per Language (All Translation Terms into various languages (these colleagues are listed in the Coordinators are underlined): Annex below). The huge number of professionals who contributed to the Arabic: Farida Bentayeb; Rachida El Meliani; Nagi Hussein; Dictionary and Encyclopaedia projects includes many promi- Ibrahim Elyasseery; Salem Sassi; Youssef Bouzekraoiu nent medical physicists – Past and Present Presidents and Senior Bengal: Hasin Azhari Anupama; Golam Abu Zakaria; Md Officers of all Medical Physics and Engineering Professional Akhtaruzzaman; Safayet Zaman; Mohammad Ullah International Organisations and of 41 National Medical Physics Shemanto Societies/Associations: IUPESM, IFMBE, IOMP and its Bulgarian: Jenia Vassileva; Venceslav Todorov; Petar Federations (AFOMP, SEAFOMP, MEFOMP, FAMPO, EFOMP, Trindev; Slavik Tabakov; Borislav Konstantinov; ALFIM), Australia, Austria, Bangladesh, Brazil, Bulgaria, Anastas Litchev; Magdalena Stoeva; Valentin Turiyski; Canada, PR China, Croatia, Cyprus, Czech Republic, Denmark, Ivan Kolev; Nikolay Nedelev; Vasil Andreev; Plamen Estonia, Finland, France, Georgia, Germany, Greece, Hungary, Mihailov; Vasil Dinkov India, Iran, Ireland, Japan, Latvia, Lithuania, Malaysia, Malta, Chinese: Andy Zhu; Wu Wenkai; Bao Shanglian; Dai Liyan; Morocco, Poland, Portugal, Romania, Russian Federation, S. Geng Jianhua; Dai Xiangkun; Fu Guishan; Geng Hui; Korea, Slovenia, Spain, Sweden, Thailand, Turkey, Ukraine, Wang Jianhua; Wang Yunlai; He Zhengzhong; Xu Xiao; United Kingdom, United States of America, Vietnam. Xu Zhiyong; Yin Yong; Zhang Jiutang; Zhang Yue; Hu Most of these colleagues continue to be among the leading Yimin; Jianrong Dai; Fugen Zhou; Jianhu Geng; Yong figures in the national and international medical physics fields, Yin; Kuo Men x Preface: The Road Which Led to This Encyclopaedia Croatian: Mario Medvedec; Jurica Bibic; Ana Buinac; Malay: David Bradley; Suhairul Hashim; Hrvoje Hrsak; Sandra Kos; Nenad Kovacevic; Srecko Persian: Azam Niroomand-Rad; E. Ishmael Parsai; Ali Loncaric; Tomislav Bokulic; Iva Mrcela; Zeljka Knezevic; Mahmoud Pashazadeh; Behrouz Rasuli; Alireza Binesh; Saveta Miljanic; Dario Faj; Slaven Jurkovic; Deni Ali Asghar Mowlavi Smilovic Radojcic; Igor Lackovic; Branko Breyer; Milica Polish: Marta Wasilewska-Radwanska; Zenon Matuszak; Mihaljevic; Ticijana Ban; Marija Majer; Nadica Maltar Katarzyna Matusiak; Aleksandra Jung Strmečki; Katarina Ružić; Marija Surić Mihić; Gordana Portuguese: Ana Pascoal; Nuno Teixeira; Paulo Ferreira; Žauhar Nuno Machado; Paolo R. Costa; Simone Kodlulovich Czech: Ivana Horakova; Anna Kindlova; Simona Renha; Elisabeth M. Yoshimura; Alessandra Tomal; Lídia Trampotova; Daniela Kotalova; Vaclav Husak; Jaroslav Vasconcellos de Sá; Denise Y. Nersissian Ptacek; Josef Pacholik; Pavel Dvorak; Libor Judas; Romanian: Loredana Marcu; Aurel Popescu; Octavian Jaroslav Tintera; Irena Koniarova; Simona Borovickova; Duliu; Daniela Andrei; Cristina Petroiu; Raducu Popa; Daniela Skibova; Dana Prchalova; Leos Novak; Vladimir Constantin Milu Dufek Russian: Valery Kostylev; Nina Lutova; Boris Narkevich; Estonian: Kalle Kepler; Sigrid Kivimae; Kalju Meigas; Juri Tatiana Ratner; Marina Kislyakova; N. N. Blokhin; Vedru; Eduard Gerskevitch; Mait Nigul; Markus Vardja; Alexey Moiseev Ando Aasa; Joosep Kepler; Kristjan Pilt Slovenian: Ervin B. Podgoršak; Božidar Casar; Viljem Finnish: Hannu Eskola; Paivi Laarne Kovač; Damijan Škrk; Petra Tomše; Urban Simončič; French: Alain Noel; Jean-Yves Giraud; Helene Bouscayrol; Urban Zdešar; Boris Sekeres; Igor Serša Louis Blache; Adam Jean-François; Bulin Anne-Laure; Spanish: Ana Millan; Ignacio Hernando; Alejandro García Charvet Anne-Marie; Elleaume Hélène; Sancey Lucie Romero; Luis Roso; Felix Navarro German: Markus Buchgeister; Gunther Helms; Stefan Swedish: Inger-Lena Lamm; Monica Almquist; Ronnie Delorme; Dimos Baltas; Inge Beckers; Martin Fiebich; Wirestam; Sven-Erik Strand; Bo-Anders Jonsson; Michael Jens Heufelder; Mark Ladd; Bernhard Sattler; Ulrich Wolf Ljungberg; Freddy Stahlberg; Thomas Jansson; Lena Georgian: Giorgi Archuadze; Giorgi Kukhalashvili; Beka Jonsson; Linda Knutsson; Crister Ceberg; Sofie Ceberg Poniava; Nino Budagashvili; Nino Khitarishvili; Giorgi Thai: Anchali Krisanachinda; Sivalee Suriyapee; Tanawat Kiladze Sontrapornpol; Panya Pasawang; Chotika Jumpangern; Greek: Stelios Christofides; Prodromos Kaplanis; Taweap Sanghangthum; Isra Na Ayuthaya; Sorn-jarod George Christodoulides; Charalambos Yiannakkaras; Oonsiri; Kitiwat Khamwan; Titipong Kaewlek; Patsuree Nicolaos Papadopoulos; Demetrios Kaolis; Georgiana Cheebsumon; Nuntawat Udee; Pachuen Potup; Supawitoo Kokona; Georgios Menikou; Christos Papaefstathiou; Sookpeng; Thanyawee Pengpan; Prathan Wongtala; Yiannis Gerogiannis; Demetra Constantinou; Spyros Arunee Hematulin; Sumalee Yabsantia; Thunyarat Spyrou; Andreas Mikelides; Anastasia Sissou; Christodoulos Chusin; Kingkarn Aphiwatthanasumet; Ausanai Prapan; Christodoulou; Irene Polycarpou; George Kagadis Chitsanupong Butdee; Chanyatip Suwannasing; Bouripat Hungarian: Pal Zarand; Istvan Polgar; Tamas Porubszky; Kadman; Somsak Wanwilairat; |
Nisa Chawapun; Janos Martos; Geza Safrany; Tamas Daboczi; Jozsef Narongchai Autsavapromporn; Wannapha Nobnop; Varga; Richárd Elek; András Barta; László Ballay; Zoltán Anirut Watcharawipha; Malulee Tantawiroon; Pachee Harkanyi; József Bakos Chaudhaketrin; Thannapong Tongprapan; Chumpot Italian: Franco Milano Kakanaporn; Utumporn Puangragsa; Chanida Japanese: Koichi Ogawa; Hidetaka Arimura; Katsuyuki Sathithanawirot; Tanwiwat Jaikuna; Amporn Funsian; Nishimura; Kanae Nishizawa; Suoh Sakata; Hidetoshi Nipha Chumsuwan; Chitchaya Suwanraksa; Wasinee Saitoh; Keiko Imamura; Kiyonari Inamura; Hiraku Fuse; Thiangsook; Suphalak Khamruang Marshall; Natee Jun Takatsu; Kana Motegi; Masaya Tamura; Shigeto Ina; Saowapark Poosiri; Chatsuda Songsang; Jongwat Kabuki; Tatsuya Fujisaki; Hajime Monzen; Hidekazu Cheewakul Nanbu; Hidetoshi Saitoh; Keiichi Akahane Turkish: Perihan Unak; Turgay Karali; Serap Teksoz; Korean: Chang-Hoon Choi; Wee-Saing Kang; Tae Suk Suh; Zumrut Biber Muftuler; Fatma Yurt Lambrecht; Elçin Sangwon Kang; Kyeon-Hyeon Kim Ekdal Karali; Ayfer Yurt Kilcar; Çiğdem İchedef Latvian: Yuri Dekhtyar; Alexei Katashev; Marite Ukrainian: Natalka Suchowerska; Ruslan Zelinskyi; Chaikovska; Emzinsh Dzintars; Sergei Popov; Lada Volodymyr Nahirnyak; Olena Chygryn; Anastasiia Bumbure; Juris Rauzins; Plaude Sandija; Aldis Balodis; Myronenko Raimonds Dreimanis; Katrina Caikovska; Laura Antra Vietnamese: Anh-Tung Hoang; Nhu-Tuyen Pham; Thanh- Grikke; Agnese Katlapa; Sanda Kronberga; Toms Kusins; Luong Dang Kristaps Palskis; Indra Surkova Lithuanian: Diana Adliene; Arunas Lukosevicius; Algidas The website www .emitel2 .eu (and www .emitel2 .net) includes Basevicius; Dovile Serenaite; Jurgita Laurikaitienė; Reda both the Multilingual Scientific Dictionary of Medical Physics Čerapaitė-Trušinskienė Terms and the e-Encyclopaedia of Medical Physics. How to Use EMITEL Website (www .emitel2 .eu) HOW TO USE THE ONLINE SCIENTIFIC The Encyclopaedia and Dictionary website is served by two DICTIONARY OF MEDICAL PHYSICS dedicated Search Engines. The first Search Engine serves the Encyclopaedic articles The online Scientific Dictionary and Encyclopaedia of Medical (entries in English), this way allowing search for words/terms Physics now contains about 3300 articles/entries with many illus- within the text of the articles. This Search Engine can also search trations, diagrams and tables. Over 350 medical physics special- within articles specific to one area of the Encyclopaedia (see the ists from around the world have voluntarily contributed to this example in Figure 1 where various Diagnostic Radiology articles project. It is published online as an open resource, at its dedicated including the term ‘Artefact’ have been listed, and one of these is web site: www .emitel2 .eu (also www .emerald2 .net). displayed). This reference resource is designed to support educational pro- The second Search Engine serves the Multilingual Dictionary grammes. Its online Dictionary provides immediate translation of (it can work with various alphabets, as per the Internet browser terminology among 32 different languages, making it of value in settings of the user). This allows the user to make a search in most countries of the world. An example from the server statistics any of the 32 languages, which will display the term translation shows that in a randomly selected month last year the web site was (and its text in English) – Figure 2 shows translations of the term used as follows: 3957 users from Asia; 2705 users from Europe; ‘Artefact’ in Spanish (plus a list of several others in the 30 avail- 1296 users from North America; 771 users from South America; able languages). 561 users from Africa. More information about user statistics in previous years is available in the free downloadable e-book The Pioneering of e-Learning in Medical Physics (www .emerald2 .eu HOW TO USE THE ONLINE /mep _15 .html). ENCYCLOPAEDIA OF MEDICAL PHYSICS The articles in the online Encyclopaedia are grouped into eight areas of Medical Physics: X-Ray Diagnostic Radiology, The website has been created to allow effortless use. Here are the Radiotherapy, Nuclear Medicine, Radiation Protection, Non- three types of website use: Ionising Radiation Safety, Magnetic Resonance Imaging, Select Encyclopaedia > select Search in ‘Title’ > type the Ultrasound Imaging, and General terms. The Dictionary and searched term/word in the window (in English) > click Enter (or Encyclopaedia also include over 600 synonyms and abbreviations Search). A list with terms is displayed – against each one is a blue hyperlinked to the respective entries/articles. hyperlink related to the area of the term > click the hyperlink to FIGURE 1 Encyclopaedia mode: search has been performed for the term ‘Artefact’ inside all articles related to Diagnostic Radiology. After clicking on the field hyperlink (to the right of the term), the term text (in English) is displayed. xi xii How to Use EMITEL Website (www.emitel2.eu) FIGURE 2 Dictionary mode: each article with the word ‘Artefact’ in its name is translated to Spanish (some of the other available languages are also displayed). access the full text of the article/entry. The website can also search supports the Input language and the desired Output languages). within the text of the articles. To do this, select Search in ‘Full Against each term is its translation into the Output language. This Text’; after this specify the area (DR, RT, NM, etc., or ALL) and mode translates only the titles of the entries/articles. See example proceed as above. In case of UK or American English differences in Figure 2. (i.e. colour>color; optimise>optimize) try both spellings or search Select Combined > to use both the Encyclopaedia and only part of the term (e.g. colo, optim). See example in Figure 1. Dictionary > choose the Input and Output languages (normally Select Dictionary > choose the Input and Output languages > the Input language is your own language) > type the term in your type the searched term in the window > click Enter (or Translate). language in the window > click Enter (or Translate). The term A list of terms in the Input language is displayed, where the terms in the Input language is displayed (plus translation in the chosen are found either alone, or in combination with other words (the Output language) > click the field hyperlink to read the article in e-Dictionary assumes that the user’s Internet browser already English. See example in Figure 3. How to Use EMITEL Website (www.emitel2.eu) xiii FIGURE 3 Combined mode: the term ‘Aliasing (Wraparound artefact)’ has been included in the local language (Chinese in the example) and the term entry is displayed in English. Additionally the term name can be translated to any Dictionary language (in the box: second example with translation in Spanish). The term text is displayed in English. Editorial Board of the Encyclopaedia of Medical Physics (2nd Edition) Slavik Tabakov, PhD, Dr.h.c., FIUPESM, medical physics are recognized by many awards including the FIOMP, FIPEM, FHEA, is IUPESM Harold Johns Medal by the IOMP and the Edith Quimby Medal Vice-President (2018–2022) and IOMP by the AAPM. President (2015–2018). He has been an active contributor in the international Magdalena Stoeva, PhD, FIOMP, development of medical physics for 25 FIUPESM is Professor in Diagnostic years. He has developed/coordinated 7 Imaging at Medical University of Plovdiv, international projects, which have cre- Bulgaria, and an elected member of the ated the first e-learning and first educa- governing bodies of the International tional website in the profession, the first Union for Physical and Engineering Medical Physics Scientific Dictionary (in 31 languages) and Sciences in Medicine (IUPESM) and the the first e-Encyclopaedia of Medical Physics (www.emitel2. International Organization for Medical eu). He is Founding Co-Editor of the IOMP Journal Medical Physics (IOMP). Dr. Stoeva has exper- Physics International, Co-Director of the International College tise in medical physics, engineering and on Medical Physics at the International Centre for Theoretical computer systems at academic and clinical level. She is Editor- Physic (ICTP), Trieste, Italy, Emeritus Founding Director of MSc in-Chief of the Health and Technology journal, Springer Nature- Clinical Sciences at King’s College London, UK, and Visiting IUPESM-WHO. Dr. Stoeva’s work has been recognized at various Professor at Medical University Plovdiv, Bulgaria, and Rajasthan levels, including the inaugural EU Leonardo da Vinci Award, the University, India. Among his awards are the IOMP Harold Johns IUPAP Young Scientist Award for Medical Physics, the Award of Medal and the EU Leonardo da Vinci Award. the Mayor of Plovdiv, Bulgaria, and the IOMP & IUPESM Fellow awards. Franco Milano, Dr.h.c. graduated in Physics at the University of Florence, Sameer Tipnis, PhD, DABR is cur- Italy, and a post-graduate training rently Professor of Radiology and in Medical Physics at University Chief, Division of Medical Physics of Pisa, Italy. He was appointed in the Department of Radiology and Professor at the University di Catania Radiological Science at the Medical and University of Florence, Italy. University of South Carolina (MUSC). He During his academic career he has is board certified in Diagnostic Medical been very active in cooperation with Physics and Nuclear Medical Physics. At many foreign universities and par- MUSC he provides guidance on optimiz- ticipated as a partner or manager in various European and NATO ing imaging protocols in CT, Nuclear science programs dedicated to the training of medical physi- Medicine and also teaches radiology residents and medical stu- cists. He is Co-Director of College on Medical Physics at ICTP, dents about the physics of clinical imaging. Dr. Tipnis serves as Trieste, Italy, and Visiting Professor at Chulalongkorn University, an examiner for the American Board of Radiology’s Diagnostic Thailand, Zhytomyr State Politeknik University, Ukraine, and and Nuclear Medicine certifying exams and is a member AAPM Riga Technical University, Latvia. He was expert of the IAEA, sub-committees for radiation protection and radiology resident Vienna, from 1995 to 2012 and member of the Audit Team of education. Dr. Tipnis has published over 40 research articles QUATRO IAEA project from 2008 to 2012. His awards include related to clinical imaging. the EU Leonardo da Vinci Award. Tracy Underwood, DPhil is currently Perry Sprawls, PhD, FAAPM, FACR, a Dean’s Prize Research Fellow in FIOMP, is Distinguished Emeritus Radiotherapy Physics at the University of Professor at Emory University, Atlanta, Manchester, UK. Throughout her career and Co-Director of College on Medical so far, she has worked to improve radio- Physics at the ICTP, Trieste, Italy and therapy at some of the most innovative also Founding Co-Editor of the IOMP clinics and academic departments world- Journal Medical Physics International wide, including Massachusetts General (www.mpijournal.org). His specialisa- Hospital/Harvard Medical School in tion is the clinical application of phys- Boston, IUCT Oncopole in Toulouse, ics in diagnostic radiology supported by France, and the University of Oxford, innovations in education. This experi- UK. She won the 2015 IMechE JRI: Best Medical Engineering ence and resulting educational resources are shared through his PhD Prize and the 2017 Early Career Academic Prize from the textbooks and scientific publications available from the Sprawls UK Institute of Physics and Engineering in Medicine. She is the Educational Foundation: www.sprawls.org. His contributions to proud mum of Jen and Alex. xv Contributors ENCYCLOPAEDIA Jacques Bittoun1 Stelios Christofides1, EDITORIAL BOARD CIERM-Hôpital de Bicêtre, France Nicosia General Hospital, Nicosia, Cyprus Karin Bloch1 Gillian Clarke1 Slavik Tabakov Lund University, Lund, Sweden (Chair), King’s College London, UK King’s College Hospital NHS Foundation Kirsty Blythe1 Trust, London, UK Franco Milano King’s College Hospital NHS Foundation James Clinch1 University of Florence, Florence, Italy Trust, London, UK King’s College Hospital NHS Foundation Magdalena Stoeva Cari Borras1 Trust, London, UK Medical University Plovdiv, Bulgaria University of Pernambuco, Brazil Patrick Conaghan1 Perry Sprawls Chloe Bowen2 King’s College Hospital NHS Foundation Sprawls Educational Foundation, Imperial College Healthcare NHS Trust, Trust, London, UK NC, USA London, UK Asen Cvetkov1,2 Sameer Tipnis Gerard Boyle1 Medical University South Carolina, AM Studio Ltd, Plovdiv, Bulgaria St James’s Hospital, Dublin, Ireland SC, USA Thomas Davies2 David Bradley1 Tracy Underwood Brighton and Sussex University Hospitals University of Surrey, Guildford, UK University of Manchester, UK NHS Trust, Brighton, UK Paola Bregant1,2 Colin Deane1,2 Azienda Sanitaria Universitaria Giuliano ENCYCLOPAEDIA Isontina, Trieste, Italy King’s College Hospital NHS Foundation CONTRIBUTORS1,2 Trust, London, UK Sara Brockstedt1 Mario De Denaro1,2 Barry Allen1 Lund University, Lund, Sweden St George Hospital NSW, Australia Azienda Sanitaria Universitaria Giuliano Luca Brombal2 Isontina, Trieste, Italy Monica Almqvist1 University of Trieste, Trieste, Italy Lund University Hospital, Lund, Sweden Antonio De Stefano 2 Francesco Brun2 Queen Alexandra Hospital, Amany Amin2 University of Trieste, Trieste, Italy Portsmouth, UK Oxford University Hospitals, Oxford, UK Markus Buchgeister1 Charles Deehan1,2 Beuth University of Applied Sciences, King’s College London, London, UK Virginia Marin Anaya2 Berlin, Germany University College London Hospitals Harry Delis2 NHS Foundation Trust, London, UK Justine Calvert1 King’s College Hospital NHS Foundation University of Patras, Patras, Greece Cameron Anderson2 Trust London, London, UK Navneet Dulai1 Imperial College Healthcare NHS Trust, London, UK Carmel |
J. Caruana2 King’s College Hospital NHS Foundation University of Malta, Malta Trust, London, UK Philip Batchelor1 Alex Dunlop1 King’s College London, London, UK Elizabeth Chaloner1,2 King’s College Hospital NHS Foundation King’s College Hospital NHS Foundation Stefano Bergamasco2 Trust London, London, UK Trust, London, UK MedTech Projects Srl, Udine, Italy Kin-yin Cheung1 Hannu Eskola1 Anna Benini1 Hong Kong Sanatorium and Hospital, Tampere Technical University, Tampere, Ringshospitalet, Copenhagen, Denmark Hong-Kong, PR China Finland Eva Bezak2 Joan Chick1 Anthony Evans1 University of South Australia, Adelaide, King’s College London, London, UK University of Leeds, Leeds UK Australia Arun Chougule2 Phil Evans1 Adnan Bibic1 SMS Medical College & Hospital, Jaipur, Institute of Cancer Research and Royal Lund University, Lund, Sweden India Marsden Hospital, Sutton, UK 1 Encyclopaedia First Edition; 2 Encyclopaedia Second Edition xvii xviii Contributors Fiammetta Fedele2 Emily Joel1 Brendan McClean1 Guy’s and St Thomas’s NHS Foundation King’s College Hospital NHS Foundation St Luke’s Hospital, Dublin, Ireland Trust, London, UK Trust, London, UK Ruth McLauchlan1 Michelle Footman1 Bo-Anders Jonsson1 Charing Cross Hospital, London, UK King’s College Hospital NHS Foundation Lund University, Lund, Sweden Siddharth Mehta2 Trust, London, UK Lena Jonsson1 Mount Vernon Cancer Hospital, East George D. Frey1 Lund University, Lund, Sweden and North Hertfordshire Trust, Medical University of South Carolina, London, UK Stephen Keevil1 SC, USA King’s College London, London, UK Franco Milano1,2 Callum Gillies2 Kalle Kepler1 University of Florence, Florence, Italy University College London Hospitals University of Tartu, Tartu, Estonia Ana Millan1 NHS Foundation Trust, UK Linda Knutsson1 Radiophysics Techniques Inc, Zaragoza, Jean-Yves Giraud1 Lund University, Lund, Sweden Spain Grenoble University Hospital, Grenoble, France Anchali Krisanachinda1 Angelo Filippo Monti2 Chulalongkorn University, Bangkok, ASST GOM Niguarda, Milano, Italy David Goss1 Thailand Elizabeth Morris1 King’s College Hospital NHS Foundation Inger-Lena Lamm1 King’s College London, UK Trust, London, UK Lund University Hospital, Lund, Sweden Ewald Moser1 Mark Grattan1 Jimmy Latt1 Medical University of Vienna, Vienna, Northern Ireland Cancer Centre, Belfast Lund University, Lund, Sweden Austria City Hospital Trust, Belfast, UK Martin Leach1 Ben Newman2 Kristina Hakansson1 Institute of Cancer Research and Royal Guy’s and St Thomas’ NHS Foundation King’s College Hospital NHS Foundation Marsden Hospital, London, UK Trust, London, UK Trust, London, UK Lorenzo Leogrande2 Kwan Hoong Ng1,2 Nicola Harris1 Fondazione IRCCS Policlinico A. University of Malaya, Kuala Lumpur, King’s College Hospital NHS Foundation Gemelli di Roma, Rome, Italy Malaysia Trust, London, UK Cornelius Lewis1 Mattias Nickel1 Glafkos Havariyoun2 King’s College Hospital NHS Foundation Lund University, Lund, Sweden King’s College Hospital NHS Foundation Trust, London, UK Markus Nillson1 Trust, London, UK Maria Lewis1 Lund University, Lund, Sweden St George’s Hospital, London, UK Gunter Helms1 Anders Nilsson1 University Clinic Göttingen, Germany Kang Ping Lin2 Lund University, Lund, Sweden Chung Yuan Christian University, Taipei William Hendee1 Jonathan Noble1 Medical College of Wisconsin, Lefteris Livieratos2 King’s College Hospital NHS Foundation Milwaukee, WI, USA Guy’s and St Thomas’s NHS Foundation Trust, London, UK Trust, London, UK Ignacio Hernando1 Alain Noel1 University Hospital Río Hortega, Michael Ljungberg1 Centre Alexis Vautrin, Vandeouvre-les- Valladolid, Spain Lund University, Lund, Sweden Nancy, Nancy, France Naomi Hogg1 Renata Longo2 King’s College Hospital NHS Foundation University of Trieste, Trieste, Italy Emil Nordh (Lindholm)1 Lund University, Lund, Sweden Trust, London, UK Ratko Magjarevic1 Fridtjof Nuesslin1 University of Zagreb, Zagreb, Croatia Ivana Horakova1 Technical University Münich, Munich, National Radiation Protection Institute, Peter Mannfolk1 Germany Prague, Czech Republic Lund University, Lund, Sweden Crispian Oates1 Ernesto Iadanza2 Paul Marsden1 Newcastle Hospitals NHS Foundation University of Florence, Florence, Italy King’s College London, London, UK Trust, Newcastle-upon-Tyne, UK Samuel Ingram2 Kosuke Matsubara2 Kjeld Olsen1 University of Manchester, Kanazawa University, Kanazawa, Japan University Hospital Herlev, Manchester, UK Denmark George Mawko1 Tomas Jansson1 Queen Elizabeth II Health Sciences Johan Olsrud1 Lund University Hospital, Lund, Sweden Centre, Halifax, Canada Lund University, Lund, Sweden Contributors xix John Oshinski2 Fernando Schlindwein1 Graeme Taylor1 Emory University, Atlanta, USA University of Leicester, Leicester UK Guy’s and St Thomas’s NHS Foundation Trust, London, UK Renato Padovani2 Mario Secca1 ICTP, Trieste, Italy New University of Lisbon, Lisbon, Heikki Terio1 Portugal Karolinska University Hospital, Nikolas Pallikarakis1 Stockholm, Sweden University of Patras, Patras, Greece Christopher Sibley-Allen1 King’s College Hospital NHS Foundation Jim Thurston1,2 Silvia Pani2 Trust, London, UK Royal Marsden Hospital NHS Foundation University of Surrey, Guildford, UK Trust, London UK Leandro Pecchia2 Jonathan Siikanen1 University of Warwick, Coventry, UK Lund University, Lund, Sweden Sameer Tipnis2 Medical University of South Carolina, Mikael Peterson1 Andy Simmons1 Charleston, SC, USA Lund University, Lund, Sweden King’s College London, London, UK Tracy Underwood1,2 Jonathan Phillips1 Edward A. K. Smith2 University of Manchester, Manchester, King’s College Hospital NHS Foundation University of Manchester, UK Trust, London, UK Manchester, UK Emil Valcinov1 Davide Piaggio2 Peter Smith1 University of Patras, Patras, Greece University of Warwick, Coventry, UK Northern Ireland Regional Medical Physics Agency, Belfast, UK Bruce Walmsley1 David Platten1 Guy’s and St Thomas’ NHS Foundation Northampton General Hospital, Perry Sprawls1,2 Trust, London, UK Northampton, UK Emory University, Atlanta, GA, USA Mark Wanklyn2 Ervin Podgorsak1 Freddy Stahlberg1 GenesisCare, Sydney, Australia McGill University, Montreal, Canada Lund University, Lund, Sweden Stephen Wastling1 Marta Radwanska1 Magdalena Stoeva1,2 King’s College, London, London, UK AGH University of Science and Medical University Plovdiv, Plovdiv, Technology, Krakow, Poland Bulgaria Carla Winterhalter2 University of Manchester, Manchester, Hamish Richardson1 Sven-Erik Strand1 UK King’s College Hospital NHS Foundation Lund University, Lund, Sweden Trust, London, UK Ronnie Wirestam1 Slavik Tabakov1,2 Lund University, Lund, Sweden Luigi Rigon2 King’s College London, London, UK University of Trieste, Trieste, Italy Xia Yunzhou2 Vassilka Tabakova1,2 University of Manchester, Manchester, Anna Rydhog1 King’s College London, London, UK UK Lund University, Lund, Sweden Jan Taprogge2 Paul Zarand1 Tobias Schaeffter1 Royal Marsden Hospital NHS Foundation Uzsoki Hospital Medical Physics Lab. King’s College London, London, UK Trust, Sutton, UK Budapest, Hungary xx Contributors WORKING GROUP WORKING GROUP COORDINATORS COORDINATORS ENCYCLOPAEDIA ENCYCLOPAEDIA SECOND EDITION FIRST EDITION Diagnostic Radiology (X-ray): Slavik Diagnostic Radiology (X-ray): Slavik Tabakov, Perry Sprawls, Paola Bregant Tabakov, Perry Sprawls, Maria Lewis Magnetic Resonance: John Oshinski, Magnetic Resonance: Andrew Simmons, Renata Longo, Antonio de Stefano Stephen Keevil, Freddy Stahlberg Non-Ionising Radiation Safety: Nuclear Medicine: Sven-Erik Strand, Fiammetta Fedele, Elizabeth Chaloner Bo-Anders Jonson, Mikael Peterson Nuclear Medicine: Sameer Tipnis Radiation Protection: Cornelius Lewis, Peter Smith, Jim Thurston Radiation Protection: Magdalena Stoeva, Jim Thurston Radiotherapy: Franco Milano, Inger- Lena Lamm, Charles Deehan, Joan Chick Radiotherapy: Tracy Underwood, Franco Milano, Eva Bezak Ultrasound: David Goss, Tomas Janson Ultrasound: Sameer Tipnis, Kwan Ng General terms: Graeme Taylor, William Hendee General terms: Slavik Tabakov, Magdalena Stoeva, Franco Milano, Encyclopaedia Editorial Assistant: Ernesto Iadanza Vassilka Tabakova Encyclopaedia Web Software: AM Studio Ltd. – Magdalena Stoeva, Asen Cvetkov Volume I A–K Numerals # 1.5 D array 3D printing (Ultrasound) A 1.5 D array is a form of transducer where instead (General) 3D printing is a process to create three-dimensional of a single line of transducer elements, there are 5 to 6 lines objects under computer control. The process is also known as stacked atop one another (see figure). Due to this, these arrays additive manufacturing as the building material is deposited layer are able to focus and steer the ultrasound beam in the elevational by layer until the final 3D object is formed. direction as well as in the slice plane. The main advantage of such 3D Printers classification: an array is improved elevational resolution, due to the extra sensor elements in the elevational direction. • Stereolithography (SLA) • Selective Laser Sintering (SLS) 3D printing has numerous applications in various industries, as well as education and research. 3D printing has specific applica- tions in healthcare – personalized medicine, medical devices and Related Articles: Elevational resolution, Linear array instruments, simulation and planning purposes, assistive technol- transducer ogy and bioprinting/tissue engineering. Further Reading: Bushberg, Seibert, Leidholdt and Boone. 2012. The Essential Physics of Medical Imaging, 3rd edn., 3D reconstruction Lippincott Williams and Wilkins, 3rd Edition, 2012. (Diagnostic Radiology) 3D reconstruction in projection recon- struction techniques, e.g. x-ray computed tomography (CT), 2D arrays occurs when 2D imaging detectors are adopted. In the simplest (Ultrasound) 2D arrays are transducers in which there are mul- case of parallel beam geometry, 3D image reconstruction is more tiple rows of transducer elements in order to provide improved often decomposed into a series of slice-by-slice 2D image recon- control of beam width in the elevation plane or to provide beam structions. In this scenario, the projection rays can be divided into steering in the elevation plane. In the latter case, 2D arrays allow groups, where each group contains only those rays that are con- volume scanning of tissue without the need for moving parts, fined within a transaxial slice. However, in other cases – the most and these are available commercially for cardiac and abdominal significant one being the cone beam geometry – the projection imaging. The different geometries and nomenclatures are out- rays run through transaxial slices and therefore a slice-by-slice lined in the article on Matrix array. 2D reconstruction approach cannot be applied. Specific 3D meth- Related Article: Matrix arrays ods are required for cone-beam geometry and they can be adapted to non-circular (e.g. spiral or helical) trajectories. 3D (three-dimensional) An exact method for object reconstruction with cone-beam (General) 3D is an abbreviation for three-dimensional. This refers geometry based on complete set of Radon data and initially to a dimensional space built up by three arbitrary dimensions, arbitrary source trajectories was proposed by Grangeat (1990). e.g. length, width and depth. The position of any point in a 3D Grangeat’s method first tries to convert cone-beam ray-sums to space can be described using three coordinates in the Cartesian plane integrals by calculating the line-integrals on the cone-beam coordinate system. detector. Grangeat’s algorithm is not a filtered backprojection Related Article: Cartesian coordinate system algorithm. It requires data rebinning, which can introduce large interpolation errors. 3D imaging Complete Radon transforms can only be acquired if certain (Nuclear Medicine) This is the process of acquiring data repre- requirements with regard to the ray source trajectory are met. senting the three-dimensional radioactive source distribution in In the frequently used circular ray source orbit, one major prob- an object. A common approach used in nuclear medicine imag- lem is that there are so-called shadow zones in which no Radon ing is to acquire many planar images from different angles from data are available. However, approximation methods that can which to produce a 3D image dataset. This technique is called also deal with shadow zones are available. The most frequently tomographic imaging. The 3D distribution is reconstructed from used method was proposed by Feldkamp, Davis and Kress (FDK) the planar images in a post-processing procedure. Tomographic (1984). As long as the cone angle aperture of the ray beam is small, imaging techniques in nuclear medicine include single photon this method yields acceptable results. If this assumption does not emission computed tomography (SPECT) and positron emission hold, artefacts can appear especially at locations away from the tomography (PET). orbit plane. Feldkamp’s cone-beam algorithm is dedicated to the Related Articles: Positron emission tomography (PET), circular focal point trajectory. It consists of the following steps: (i) Single photon emission computed tomography (SPECT), Image pre-scale the projections by a cosine function cos α (α being the reconstruction cone angle aperture of the beam); (ii) row-by-row ramp filter the 3 4D computed tomography (CT) data 4 180° pulse pre-scaled data; (iii) cone-beam backproject the filtered data with as based on the 4DCT informed treatment plan. However, this # a weighting function of the distance from the reconstruction point involves a more technologically complex delivery, which is to the focal point. necessarily reliant on the collection and accurate real-time 3D methods like the ones previously introduced can be processing of reliable and reproducible respiration data during adapted to spiral or helical trajectories. An interesting method is each treatment fraction. Katsevich’s cone-beam algorithm (Katsevich, 2002). It was ini- Related Articles: Computed tomography, CT reconstruction tially developed for the helical orbit cone-beam geometry and it Further Readings: Bortfeld et al. 2006. Image-Guided was later extended to more general orbits. Katsevich’s method IMRT. Springer-Verlag, Berlin; Cole, A. J. et al. 2014. Motion solves the so-called long object problem. management for radical radiotherapy in non-small cell lung Related Articles: Filtered back projection, Cone beam CT cancer. Clin. Oncol. 26:67–80. Further Readings: Feldkamp, L. A., L. C. Davis, and J. W. Kress. 1984. Practical cone-beam algorithm. J. Opt. Soc. Am. 4D dose calculation A 1(6):612–619; Grangeat, P. 1990. Mathematical framework |
of (Radiotherapy) In radiotherapy treatment planning, four- cone-beam 3D reconstruction via the first derivative of the Radon dimensional dose calculations are performed on 4DCT datasets, transform. In: Mathematical Methods in Tomography, eds., G. T. which resolve a patient’s motion over time. 4D calculations are Herman, A. K. Louis, and F. Natterer, Springer, Berlin, pp. 66–97; commonly performed on either an average intensity projection Katsevich, A. 2002. Theoretically exact filtered backprojection- reconstruction of the dataset, or else through the (ideally) type inversion algorithm for spiral CT. SIAM J. Appl. Math. automated reproduction of a plan produced on a single 3D phase 62(6):2012–2026. of the dataset, across all other breathing phases. Related Article: 4D computed tomography (CT) data 4D computed tomography (CT) data (Radiotherapy) Four-dimensional computed tomography (4DCT) 4D imaging is an imaging technique that allows for the reconstruction of tem- (Ultrasound) The most common mode of clinical ultrasound poral as well as spatial patient CT data. Such data is most com- imaging is the 2D, ‘B-mode’. Here data are acquired in two monly utilised for the monitoring and modelling of the effect of a dimensions of the field of view, in the plane of the frame. Using patient’s breathing cycle on tumour position (as is particularly rel- 1.5 dimensional arrays, it is possible to also acquire data in the evant in the case of lung tumours), such as to allow for treatment third plane (orthogonal to the frame), thus allowing the rendering margin reductions, gated radiation delivery or to inform real-time of a 3D or volumetric image. When such 3D data are acquired at tracking of the tumour during treatment. several different time points, it is possible to view the resulting Standard 3D scanning effectively provides only a single data in a video format, which is commonly referred to as 4D snapshot in time of a patient’s anatomy, which will be arbitrary ultrasound imaging. with respect to internal anatomical motion. Planning and treating Related Articles: B-mode ultrasound, 1.5D array, Three- on such an image might then mean that the tumour falls outside dimensional ultrasound imaging of the PTV for much of the treatment. The only way to limit Further Reading: Bushberg, Seibert, Leidholdt and Boone. the effect of such movement on tumour control probability is to 2012. The Essential Physics of Medical Imaging, 3rd edn., utilise a very large margin for the PTV, but this will then lead to Lippincott Williams and Wilkins. additional irradiation of normal healthy tissues. 4DCT scans can be acquired either prospectively or 90° pulse retrospectively. In the former technique, the CT data is acquired (Magnetic Resonance) In MRI pulse sequences an arbitrary RF only when the patient is in a specific phase of their respiratory pulse can be applied to flip the magnetisation vector through some cycle (ascertained through some external metric such as arbitrary angle. 90° RF pulses are implemented very frequently movement of an infrared marker on the chest, or spirometry), in pulse sequence designs, primarily as excitations pulse in spin such that treatment delivery can then be gated to the same phase. echo type sequences. If B1 homogeneity is sufficient, 90° RF The downside is that acquisition and treatment both become much pulses provide the maximum MR signal from a particular sample longer. or region selected (Figure 1.1). In retrospective 4DCT, CT images are oversampled (through Related Articles: 180° pulse multiple tube rotations) at each position of the couch, in order to obtain separate full 3D scans at each phase of the breathing cycle. 180° pulse Once the multitude of individual CT slices have been obtained (Magnetic Resonance) In MRI pulse sequences an arbitrary RF and post-processed, they must be accurately binned (sorted) into pulse can be applied to flip the bulk magnetisation vector through the separate 3D datasets for each discretised breathing phase. As for prospective scans, this is conducted on the basis of breathing signals monitored while the scan is taking place, specifically z´ z´ according either to signal phase (i.e. the subdivided time window between each exhalation and inhalation peak) or amplitude. Once binned into the relevant 3D datasets, an internal target p volume (ITV) might be delineated according to the combined y´ y´ GTV across all datasets (or similarly on a maximum intensity projection amalgamation), or potentially from a time-averaged x´ x´ dataset (which stretches and blurs the GTV out along its total range of motion). Final treatment volumes may be reduced even further using FIGURE 1.1 90° pulse showing flipping of longitudinal magnetisation gating or real-time tracking of the tumour during treatment, onto the transverse plane. 180° pulse 5 180° pulse z´ z´ z´ z´ # 180° flip y´ 180° flip y´ y´ y´ x´ x´ x´ x´ Dephasing Rephasing Mz = Mz z Mz = – Mz z spins spins FIGURE 1.2 180° inverting pulse. FIGURE 1.3 Refocusing using a 180° pulse. some angle. 180° RF pulses are implemented very frequently in pulses are generically referred to as ‘inversion recovery type pulse sequence designs, utilised both as inverting pulses and refo- sequences’. cusing pulses. 180° pulses are also implemented in spin echo type sequences As an inverting pulse, the function of a 180° pulse is to to ‘refocus’ magnetisation and generate a spin echo. After an invert longitudinal magnetisation (Figure 1.2). Subsequent to the excitation pulse, magnetisation is flipped onto the transverse inverting pulse the longitudinal magnetisation starts to recover to (xy) plane. Individual spin transverse components dephase its equilibrium state through T1 relaxation. After a delay, tissues (Figure 1.3) in the transverse plane due to local variations in pre- with different T1 values will have recovered to different degrees. cession frequencies determined by T2* mechanisms. The result STIR and FLAIR type sequences exploit the longitudinal mag- is a ‘fan’ of spin components, moving apart from one another, or netisation differences that emerge after an inverting pulse to null dephasing. The application of a 180° pulse (Figure 1.3) moves the signal from fat and fluid, respectively. Equally, a preparatory slow, trailing edge of the fan of spins ahead in phase relative to the 180° inversion pulse can be utilised to enhance T1 weighting in fast, leading edge. As the fan now closes, the spin rephase and the a ‘host’ imaging sequence. Sequences utilising preparatory 180° signal builds up generating a spin echo. A A A number Further Readings: Baltas, D., L. Sakelliou and N. Zamboglou. (Nuclear Medicine) See Mass number 2007. The Physics of Modern Brachytherapy for Oncology, Taylor & Francis Group, Boca Raton, FL, pp. 220, 437–445; Nath, AAPM TG43 formalism R. et al. 1995. Dosimetry of interstitial brachytherapy sources: (Radiotherapy, Brachytherapy) The source models recommended Recommendations of the AAPM Radiation Therapy Committee, today for brachytherapy treatment planning systems are those Task Group No 43. Med. Phys. 22:209–234; Rivard, M. J. et al. based on the AAPM TG43 formalism. 2004. Update of AAPM Task Group No. 43 Report: A revised The TG43 formalism has water as the reference dosimetry AAPM protocol for brachytherapy dose calculation. Med. Phys. medium. Reference data for commercially available sources are 33:633–674; Rivard, M. J. et al. 2007. Supplement to the 2004 compiled from Monte Carlo calculations and experimental data. update of the AAPM Task Group No. 43 Report. Med. Phys. (Reference data are available from AAPM and ESTRO.) 34:2187–2205; Venselaar, J. and J. Pérez-Calatayud. eds. 2004. A Only cylindrical sources are considered, and cylindrical sym- Practical Guide to Quality Control of Brachytherapy Equipment, metry is assumed. A reference point is defined 1 cm from the ESTRO Booklet No. 8, ESTRO, Belgium, Germany. source centre on the transverse bisector of the source, (r0, θ0) with r0 = 1 cm and θ0 = π/2 (polar co-ordinates). Abdominal imaging According to the AAPM TG43 formalism, the dose rate to (Diagnostic Radiology) The physical parameters of anatomical water in water at point P(r, θ) is given by Equation A.1: structures have specific effects on the use of various imaging modalities. Abdominal imaging is difficult, as the anatomy of the region includes solid and hollow structures which complicate ( ) G (r, q) D r, q = SKL ( ( . ) 0 ) g (r )F (r, q) G r , q A 1 detection of lesions (compared with chest imaging). Imaging of 0 these structures requires the use of specific contrast media. The most commonly used intravenous contrast agents (x-ray S imaging) are iodine based. Barium-based contrast agents (x-ray K is the air kerma strength in units U, 1 U = 1 μGy m2/h = 1 cGy cm2/h. imaging) are usually used orally (e.g. barium meal to diagnose Λ is the dose rate constant in water, the dose rate to water the hollow gastro-intestinal tract). The high subject contrast in in water at the reference point per unit air kerma strength. The these cases requires the use of higher energies (kVp). The most -constant Λ must be determined for all source types and designs commonly used modalities for abdominal imaging are computed (unit cGy/h/U). tomography (CT) and magnetic resonance imaging (MRI), while G(r, θ) is the geometry function describing the distribution of ultrasound is very useful in the rare cases of imaging during the radioactive material in the source (proportional to 1/r2 for a pregnancy. point source). Related Article: Chest radiography g(r) is the radial dose function, describing the dependence of absorption and scatter of the photons in water along the transver- Abdominal imaging sal bisector (θ0 = π/2), also including the effect of interactions in (Nuclear Medicine) Abdominal imaging is a generic term for the source material and encapsulation. The function g(r) is dimen- medical imaging of abdominal disorders, including the alimen- sionless and normalised at 1 cm, g(r0) = 1. tary tract and the genitourinary system. It may include diag- F(r, θ) is the anisotropy function describing the anisotropy of nostic ultrasound imaging (US), CT, single photon emission the dose distribution around the source ‘in relation to the trans- tomography (SPECT), positron emission tomography (PET), versal bisector plane’, the absorption and scatter of photons in the and MRI. source itself, in the encapsulation and in the water. The function There are many nuclear medicine studies involving abdomi- F(r, θ) is dimensionless and normalised along the transversal nal imaging. A few examples of these are Tc-99 m colloid for bisector, F(r, θ liver imaging, Tc-99 m Iminodiacetates for biliary function stud- 0) = 1. Care must be taken when dose rate tables are used. The tables ies, Tc-99 m labelled red cells for GI bleeding, In-111 labelled to be used, e.g. as data in a treatment planning system must be white cells for infection and Ga-67 and I-123 MIBG for tumour derived using the same formalism/definitions as the formalism imaging. used in that specific system. For a detailed description of the TG43 formalism, please refer Abdominal imaging to the references given in the following. (Ultrasound) Abdominal imaging in ultrasound includes assess- Abbreviations: AAPM = American Association of Physicists ment of the liver, gall bladder, kidneys and renal tract, pancreas, in Medicine, ESTRO = European Society for Therapeutic spleen, retroperitoneum and gastrointestinal tract and surround- Radiology and Oncology. ing structures. B-mode, colour flow and spectral Doppler are all Related Articles: Treatment planning systems – Brachytherapy, used in abdominal imaging, usually with curvilinear transducers Source models in the range 1–6 MHz for adults. 7 Aberration 8 Absorbed dose conversion factor Aberration Cardiac ablation works by scarring or destroying tissue. In this A (Ultrasound) In optics, aberration refers to the failure of light rays case, ablation is used to treat atrial fibrillation by using the scar to converge at a single focus. The analogous problem in ultrasound formed to isolate the pulmonary vein from the left atrium of the imaging is that a focused beam will not produce a sharp focus, heart. and conversely, that a point reflector will appear blurred in the image. Ablation The first basic prerequisite for ultrasound imaging to work (Non-Ionising Radiation) See Photoablative effects at all is that echoes are produced, which occurs when there is a transition in acoustic impedance (the product of sound speed and density) between two types of tissues. Second, there should ideally Absolute risk be no difference in sound speed along the path of interrogation, as (Radiation Protection) The total risk of developing a particular the distance to the interface is given by the |