Application of label‐free 2‐photon fluorescence lifetime imaging microscopy to measure endogenous melanin profiles in human eye melanocytes, naevus and melanoma cells

Abstract

PurposeThe progression of choroidal melanoma (CM) is complex, involving genetic and immune‐related factors. Melanin/pigmentation genes and forms (eumelanin/dark and pheomelanin/light) can also impact on CM progression. In this study, an optimised 2‐photon fluorescence lifetime imaging microscopy (FLIM), with phasor analysis, was used to identify melanin fluorescence lifetime (FL) profiles in formalin‐fixed, label‐free paraffin sections of human melanoma and naevus, with surrounding heterogeneously pigmented melanocytes.MethodsSections of ‘light CM’ (n = 3) with ‘mixed surrounding melanocytes’ and ‘dark naevi’ (n = 3) with ‘dark surrounding melanocytes’ were imaged with FLIM (3 sampled regions). Fluorescence lifetimes were measured at every pixel of captured FLIM images, Fourier transformed and presented in a ‘fit‐free’ phasor plot. These plots were segmented by 7 phasor clusters of linearly increasing fluorescence lifetimes mapped to intracellular melanin in melanocytes, naevus and melanoma cells. The mean fraction of FLIM image pixels linked to each melanin‐mapped cluster was obtained for all sampled regions to form melanin FL profiles.ResultsThe measured sampled regions displayed distinct intracellular melanin (eumelanin:pheomelanin) profiles with varying dominant melanin‐mapped clusters. The dominant ‘highest pixel fraction’ cluster measured in ‘light CM’ mapped to long FLs, implying a low eu:pheo ratio. The ‘mixed pigmented melanocytes around CM’ showed a mixed eu:pheo content based on the dominant mid‐valued FL cluster. The ‘dark naevi and surrounding melanocytes’ were mapped to mostly short FLs (high eu:pheo ratio).ConclusionsOur FLIM‐phasor method provides a fast ‘model‐free’ way to unmix melanin fluorescence lifetimes in melanocytes, naevi and CM, and provides a basis for exploring the role of melanin forms in eye melanoma pathogenesis.