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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 \u2018fit-free\u2019 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\u2013625\u2009nm) and pheomelanin-enriched lighter pigmented HCM regions mapped to phasors with longer lifetimes and shorter spectral emission (550\u2013585\u2009nm). Overall, we demonstrated that these methods can identify and quantitatively profile the heterogeneous eumelanins\/pheomelanins within<jats:italic>in situ<\/jats:italic>HCMs, and visualize melanosome spatial distributions, not previously reported for these cells.<\/jats:p>","DOI":"10.1038\/s41598-019-54871-y","type":"journal-article","created":{"date-parts":[[2019,12,9]],"date-time":"2019-12-09T11:03:02Z","timestamp":1575889382000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":26,"title":["Shedding light on melanins within in situ human eye melanocytes using 2-photon microscopy profiling techniques"],"prefix":"10.1038","volume":"9","author":[{"given":"Ephrem","family":"Sitiwin","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Michele C.","family":"Madigan","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Enrico","family":"Gratton","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Svetlana","family":"Cherepanoff","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Robert Max","family":"Conway","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Renee","family":"Whan","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Alexander","family":"Macmillan","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2019,12,9]]},"reference":[{"key":"54871_CR1","doi-asserted-by":"publisher","first-page":"144","DOI":"10.1016\/j.preteyeres.2009.12.002","volume":"29","author":"DL Nickla","year":"2010","unstructured":"Nickla, D. 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