Multiphoton laser tomography and fluorescence lifetime imaging of melanoma: morphologic features and quantitative data for sensitive and specific non-invasive diagnostics

Abstract

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.

Figure 1. Multiphoton laser tomography and fluorescence lifetime imaging of healthy skin, melanocytic lesions and basal cell carcinoma.

A and B. Images referring to the same epidermal area (healthy skin of a phototype 4 subject); in A, the multiphoton image, in B, the corresponding color coded image, enabling the recognition of green large keratinocytes (keratinocytes without pigment, asterisk), large orange melanin-containing keratinocytes (thick arrow) and small red melanocytes (thin arrow). C, D and E. Melanocytic nevus; C, red-orange cells corresponding to melanocytes, regular in size and shape in the upper epidermal layers; D, a melanocytic nest, consisting of aggregated orange melanin containing cells at the dermal-epidermal junction (the arrows indicate the papillae); E, a small melanocytic nest close to the tip of the papilla (arrow), where red collagen fibers are visible (asterisk). F, basal cell carcinoma; a nest of blue basaloid cells is clearly visible (thick arrow), infiltrating green normal keratinocytes (thin arrow); asterisks indicate blood vessels.

Figure 2. Multiphoton laser tomography and fluorescence lifetime imaging of melanoma.

A, MPT image of upper melanoma layers; B, FLIM image of the same spot displayed by a 0–2000 ps fluorescence lifetime range showing that all cells are melanocytic; C, 0–400 scale, employed to increase contrast for the distinction of cellular details, such as the cytoplasmic halo and the speckling. Dots indicate nuclei with undefined contours; arrows indicate polinucleated cells; asterisks indicate cytoplasmic halo, and triangles speckled melanin. FLIM values do not change, they are only displayed in a different way.

Figure 3. Fluorescence lifetime imaging of melanoma at different depth.

A, dermoscopic images and stacks of 3 melanomas; B, typical dermoscopic features; C, and D, upper melanoma layers, characterized by orange atypical large short-lifetime cells (ASLCs) irregularly distributed or forming aggregates. Cells are pleomorphic and show a nucleus with undefined contours and a non-homogeneous cytoplasm with speckled melanin. A peripheral cytoplasmic halo is observable in some cells (triangles), some of which are polinucleated (dots). E, F and G, deeper MM layers with ASLCs variable in size, shape and distribution forming aggregates and infiltrating papillae (thick arrows) and hair follicles (thin arrows).

Figure 4. Fluorescence lifetime imaging: different features of melanoma.

A, dendritic cells in upper melanoma layers (asterisks); B, melanoma cells infiltrating a non edged papilla (arrows); C, red collagen fibers surrounding ASLC nests (arrows).