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Application of label‐free 2‐photon fluorescence lifetime imaging microscopy to measure endogenous melanin profiles in human eye melanocytes, naevus and melanoma cells
Author(s) -
Sitiwin E.,
Madigan M.,
Jager M.,
Conway R.,
Cherepanoff S.,
Macmillan A.
Publication year - 2017
Publication title -
acta ophthalmologica
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.534
H-Index - 87
eISSN - 1755-3768
pISSN - 1755-375X
DOI - 10.1111/j.1755-3768.2017.03345
Subject(s) - melanin , melanoma , fluorescence , phasor , fluorescence lifetime imaging microscopy , fluorescence microscope , chemistry , biology , biophysics , optics , physics , genetics , power (physics) , electric power system , quantum mechanics
Purpose The progression of choroidal melanoma ( CM ) is complex, involving genetic and immune‐related factors. Melanin/pigmentation genes and forms ( eu melanin/dark and pheo melanin/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. Methods Sections 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. Results The measured sampled regions displayed distinct intracellular melanin ( eu melanin: pheo melanin) profiles with varying dominant melanin‐mapped clusters. The dominant ‘highest pixel fraction’ cluster measured in ‘light CM ’ mapped to long FL s, 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 FL s (high eu:pheo ratio). Conclusions Our 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.