The stepwise two‐photon excited melanin fluorescence is a unique diagnostic tool for the detection of malignant transformation in melanocytes

Abstract

Malignant transformation of melanocytes is associated with changes in melanogenesis. Therefore, fluorescence of melanin may be an informative indicator of this process. But the conventionally excited autofluorescence of melanin in skin tissue is ultra-weak and its main part in the visible spectral region is hidden by the much stronger fluorescence from other endogenous fluorophores. Here, using a new mode of stepwise two-photon excitation, melanin-dominated fluorescence spectra of pigmented skin lesions are reported. From these, pure melanin fluorescence spectra of normal pigmented skin, melanocytic nevi and malignant pigmented melanoma were analyzed. They show distinctly different spectral shapes: melanoma gave a characteristic fingerprint with a fluorescence band peaking at 640 nm, independent of the melanoma subtype. The melanin fluorescence spectra peaked at 590 nm for all types of common melanocytic nevi. These differences in the fluorescence spectra are probably based on different contents of eumelanin and pheomelanin. In a series of 167 cases with melanocytic nevi and melanomas, the sensitivity of this new method to diagnose melanoma was 93.5%, the specificity 80.0% and the diagnostic accuracy 82.6%. The two-photon excitation fluorescence method is a new diagnostic tool which may in future supplement conventional dermatohistopathology.

Advances and challenges in label-free nonlinear optical imaging using two-photon excitation fluorescence and second harmonic generation for cancer research

“…In the work of Giorgi et al [88] multi-spectral imaging could discriminate BCC from normal skin. Later Leupold et al [89] was able to spectrally characterize and distinguish clearly between malignant melanoma, common nevi, oculocutaneous albinism and vitiligo.…”

Two-photon excited fluorescence lifetime imaging and spectroscopy of melaninsin vitroandin vivo

“…The first results for bulk melanin emission spectral properties in human skin were reported for simultaneous 14 and stepwise [15][16][17] two-photon excitation using 810-nm light. In this work we extend these previous studies by characterizing naturally occurring eumelanin and pheomelanin using two-photon excited fluorescence spectra and lifetimes.…”

Application of multiphoton microscopy in dermatological studies: A mini-review

“…157 This was pursued further, incorporating SHG imaging, and imaging the wound with greater time resolution and over a longer period, in order to yield more insight into the wound-healing process. 93 Later, Luo et al . used SHG and image analysis to investigate wound healing in KunMing mice over a period of 14 days.…”

“…Further studies have been performed using mouse models, 90 as well as a study in humans that attempted to image melanogenesis on excised tissue samples. 91 Fluorescence lifetime has also been used to separate melanoma from nevi, 92 and attempts have also been made to use ordinary two-photon fluorescence 93,94 along with a demonstration of the use of two-spectral-channel lifetime imaging to classify nevi and basal cell carcinoma. 51 Today, while the identification of melanoma based on the spectral differences between pheomelanin and eumelanin is far from proven, this approach does show enough promise to justify further investigation.…”