Recent advances in multiphoton microscopy for clinical skin imaging

. This presentation will discuss recent advances in multiphoton microscopy (MPM) as a tool for in-vivo imaging of human skin to evaluate MPM’s potential to enhance the diagnostic accuracy of skin diseases and guide effective treatment. There is an important clinical need to enhance the diagnostic accuracy of skin diseases, to understand dynamic cellular and molecular processes during treatment procedures and to guide effective treatment. Technologies based on optical spectroscopy and imaging can provide non-invasive solutions to these longstanding clinical challenges. Among these, MPM imaging is unique in that it provides sub-micron resolution images with contrast that closely resembles the histological sections dermatopathologists use for diagnosis. MPM contrast in skin is derived from second harmonic generation (SHG) of collagen and two-photon excited fluorescence (TPEF) of tissue components such as the co-factors NADH and FAD+, elastin, keratin, and melanin. This presentation discusses recent advances in clinical skin imaging by using MPM for assessing the potential of this technology to enhance the accuracy of early melanoma detection and guide therapy of pigmentary skin disorders such as vitiligo (white patches on skin due to loss of melanocytes). For the melanoma study

Abstract.This presentation will discuss recent advances in multiphoton microscopy (MPM) as a tool for in-vivo imaging of human skin to evaluate MPM's potential to enhance the diagnostic accuracy of skin diseases and guide effective treatment.
There is an important clinical need to enhance the diagnostic accuracy of skin diseases, to understand dynamic cellular and molecular processes during treatment procedures and to guide effective treatment.Technologies based on optical spectroscopy and imaging can provide non-invasive solutions to these longstanding clinical challenges.Among these, MPM imaging is unique in that it provides sub-micron resolution images with contrast that closely resembles the histological sections dermatopathologists use for diagnosis.MPM contrast in skin is derived from second harmonic generation (SHG) of collagen and two-photon excited fluorescence (TPEF) of tissue components such as the co-factors NADH and FAD+, elastin, keratin, and melanin.This presentation discusses recent advances in clinical skin imaging by using MPM for assessing the potential of this technology to enhance the accuracy of early melanoma detection and guide therapy of pigmentary skin disorders such as vitiligo (white patches on skin due to loss of melanocytes).
For the melanoma study, we imaged melanocytic lesions at three different stages: common nevi, dysplastic nevi and melanoma in 75 patients (25 in each group).In order to account for inter-observer variability, common nevi, when not biopsied, were diagnosed by three independent dermatologists.Pigmented lesions considered suspicious of dysplasia and melanoma were biopsied after imaging and diagnosed by three independent dermatopathologists.We analyzed the data both qualitatively and quantitatively.For the qualitative analysis we identified and correlated the histological and MPM morphological features.The quantitative analysis is based on an algorithm we have previously introduced and tested on a limited number of pigmented lesions (1).We defined metrics to quantify morphological changes in endogenous biomarkers such as melanocytes and collagen fibers within a certain volume across the dermo-epidermal junction.
We use these metrics to develop a melanoma multiphoton index (MMI) that distinguishes among the three groups of common, dysplastic nevi and melanoma.
For the vitiligo imaging study, we evaluate the MPM potential to image in vivo, non-invasively the kinetics of vitiligo re-pigmentation of human skin.We image the re-pigmentation process during two treatment approaches, UVB light therapy and micro-grafting procedure, which also provides the opportunity for monitoring the wound re-epithelialization process.We imaged migration of melanocytes at different time points in 10 patients who underwent UVB light therapy and in 2 patients who underwent vitiligo micro-transplantation followed by UVB light therapy.Besides monitoring melanocyte migration, the ability to monitor metabolic changes in cell populations involved in re-epithelialization is of great interest.For this purpose, we also employ an imaging metric derived from an innovative mitochondrial clustering analysis (2).It is a biomarker of cellular mitochondrial organization based on mitochondrial clustering measured through detection of the endogenous TPEF contrast of NADH.We tested this metric successfully on vitiligo vs normal skin and currently apply it to images acquired from treated vitiligo areas.This metric is expected to reveal important cellular metabolic information without having to acquire optical redox data from multiwavelength excitation and/or fluorescence lifetimes.
These results demonstrate the potential of the MPM technology to be used as a non-invasive label-free imaging tool for early detection of melanoma and for understanding dynamic cellular and molecular processes during vitiligo treatment.