Multiphoton laser tomography (MPT) combined with fluorescence lifetime imaging (FLIM) is a non-invasive imaging technique, based on the study of fluorescence decay times of naturally occurring fluorescent molecules, enabling a non-invasive investigation of the skin with subcellular resolution. The aim of this retrospective observational ex vivo study, was to characterize melanoma both from a morphologic and a quantitative point of view, attaining an improvement in the diagnostic accuracy with respect to dermoscopy. In the training phase, thirty parameters, comprising both cytological descriptors and architectural aspects, were identified. The training set included 6 melanomas with a mean Breslow thickness±S.D. of 0.89±0.48 mm. In the test phase, these parameters were blindly evaluated on a test data set consisting of 25 melanomas, 50 nevi and 50 basal cell carcinomas. Melanomas in the test phase comprised 8 in situ lesions and had a mean thickness±S.D. of 0.77±1.2 mm. Moreover, quantitative FLIM data were calculated for special areas of interest. Melanoma was characterized by the presence of atypical short lifetime cells and architectural disorder, in contrast to nevi presenting typical cells and a regular histoarchitecture. Sensitivity and specificity values for melanoma diagnosis were 100% and 98%, respectively, whereas dermoscopy achieved the same sensitivity, but a lower specificity (82%). Mean fluorescence lifetime values of melanocytic cells did not vary between melanomas and nevi, but significantly differed from those referring to basal cell carcinoma enabling a differential diagnosis based on quantitative data. Data from prospective preoperative trials are needed to confirm if MPT/FLIM could increase diagnostic specificity and thus reduce unnecessary surgical excisions.

Multiphoton laser microscopy is a new, non-invasive technique providing access to the skin at a cellular and subcellular level, which is based both on autofluorescence and fluorescence lifetime imaging. Whereas the former considers fluorescence intensity emitted by epidermal and dermal fluorophores and by the extra-cellular matrix, fluorescence lifetime imaging (FLIM), is generated by the fluorescence decay rate. This innovative technique can be applied to the study of living skin, cell cultures and ex vivo samples. Although still limited to the clinical research field, the development of multiphoton laser microscopy is thought to become suitable for a practical application in the next few years: in this paper, we performed an accurate review of the studies published so far, considering the possible fields of application of this imaging method and providing high quality images acquired in the Department of Dermatology of the University of Modena.

This study describes new morphological descriptors of BCC enabling its characterization and its distinction from healthy skin and other skin lesions in ex vivo samples, and demonstrates for the first time that MPT represents a sensitive and specific technique for the diagnosis of BCC.

Multiphoton laser tomography (MPT) combined with fluorescence lifetime imaging (FLIM) is a non‐invasive imaging technique, which gives access to the cellular and extracellular morphology of the skin. The aim of our study was to assess the sensitivity and specificity of MPT/FLIM descriptors for basal cell carcinoma (BCC), to improve BCC diagnosis and the identification of tumor margins. In the preliminary study, FLIM images referring to 35 BCCs and 35 healthy skin samples were evaluated for the identification of morphologic descriptors characteristic of BCC. In the main study, the selected parameters were blindly evaluated on a test set comprising 63 BCCs, 63 healthy skin samples and 66 skin lesions. Moreover, FLIM values inside a region of interest were calculated on 98 healthy skin and 98 BCC samples. In the preliminary study, three epidermal descriptors and 7 BCC descriptors were identified. The specificity of the diagnostic criteria versus ‘other lesions’ was extremely high, indicating that the presence of at least one BCC descriptor makes the diagnosis of ‘other lesion’ extremely unlikely. FLIM values referring to BCC cells significantly differed from those of healthy skin. In this study, we identified morphological and numerical descriptors enabling the differentiation of BCC from other skin disorders and its distinction from healthy skin in ex vivo samples. In future, MPT/FLIM may be applied to skin lesions to provide direct clinical guidance before biopsy and histological examination and for the identification of tumor margins allowing a complete surgical removal.

Confocal laser scanning microscopy (CLSM) has been introduced in clinical settings as a tool enabling a quasi-histologic view of a given tissue, without performing a biopsy. It has been applied to many fields of medicine mainly to the skin and to the analysis of skin cancers for both in vivo and ex vivo CLSM. In vivo CLSM involves reflectance mode, which is based on refractive index of cell structures serving as endogenous chromophores, reaching a depth of exploration of 200 μm. It has been proven to increase the diagnostic accuracy of skin cancers, both melanoma and non-melanoma. While histopathologic examination is the gold standard for diagnosis, in vivo CLSM alone and in addition to dermoscopy, contributes to the reduction of the number of excised lesions to exclude a melanoma, and to improve margin recognition in lentigo maligna, enabling tissue sparing for excisions. Ex vivo CLSM can be performed in reflectance and fluorescent mode. Fluorescence confocal microscopy is applied for “real-time” pathological examination of freshly excised specimens for diagnostic purposes and for the evaluation of margin clearance after excision in Mohs surgery. Further prospective interventional studies using CLSM might contribute to increase the knowledge about its application, reproducing real-life settings.