Shedding light on melanins within in situ human eye melanocytes using 2-photon microscopy profiling techniques
Autor: | Enrico Gratton, Michele C. Madigan, Ephrem I. Sitiwin, Renee Whan, Robert Max Conway, Svetlana Cherepanoff, Alexander Macmillan |
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Rok vydání: | 2019 |
Předmět: |
Male
0301 basic medicine In situ Cytoplasm Skin Neoplasms lcsh:Medicine 01 natural sciences Multiphoton microscopy Two-photon excitation microscopy lcsh:Science Melanoma Microscopy Melanosomes Multidisciplinary Pigmentation Chemistry Middle Aged Fluorescence medicine.anatomical_structure Melanocytes Female medicine.medical_specialty Fundus Oculi Article Eye cancer 010309 optics 03 medical and health sciences Ciliary body 0103 physical sciences medicine Humans Eye Disease and Disorders of Vision Aged Melanosome Melanins Photons Choroid lcsh:R Uvea NAD Spectral imaging HEK293 Cells 030104 developmental biology Biophysics Cancer imaging lcsh:Q Human eye sense organs Biological fluorescence |
Zdroj: | Scientific Reports, Vol 9, Iss 1, Pp 1-18 (2019) Scientific reports, vol 9, iss 1 Scientific Reports |
ISSN: | 2045-2322 |
Popis: | Choroidal melanocytes (HCMs) are melanin-producing cells in the vascular uvea of the human eye (iris, ciliary body and choroid). These cranial neural crest-derived cells migrate to populate a mesodermal microenvironment, and display cellular functions and extracellular interactions that are biologically distinct to skin melanocytes. HCMs (and melanins) are important in normal human eye physiology with roles including photoprotection, regulation of oxidative damage and immune responses. To extend knowledge of cytoplasmic melanins and melanosomes in label-free HCMs, a non-invasive ‘fit-free’ approach, combining 2-photon excitation fluorescence lifetimes and emission spectral imaging with phasor plot segmentation was applied. Intracellular melanin-mapped FLIM phasors showed a linear distribution indicating that HCM melanins are a ratio of two fluorophores, eumelanin and pheomelanin. A quantitative histogram of HCM melanins was generated by identifying the image pixel fraction contributed by phasor clusters mapped to varying eumelanin/pheomelanin ratio. Eumelanin-enriched dark HCM regions mapped to phasors with shorter lifetimes and longer spectral emission (580–625 nm) and pheomelanin-enriched lighter pigmented HCM regions mapped to phasors with longer lifetimes and shorter spectral emission (550–585 nm). Overall, we demonstrated that these methods can identify and quantitatively profile the heterogeneous eumelanins/pheomelanins within in situ HCMs, and visualize melanosome spatial distributions, not previously reported for these cells. |
Databáze: | OpenAIRE |
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