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. 2019 Dec 9;9:18585. doi: 10.1038/s41598-019-54871-y

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© The Author(s) 2019

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

PMC Copyright notice

2PM FLIM/spectral phasor distribution mapping to HCM-localized melanins and surrounding human choroidal flatmount tissue. The image segmented FLIM/spectral phasor clusters mapping to (a) ‘very dark’ pigmented HCMs displayed short average fluorescence lifetimes/emission spectra: 580 to 625 nm. FLIM/spectral phasor clusters mapping to (b) ‘medium’ pigmented HCMs had longer fluorescence lifetimes/emission spectra: 565 to 610 nm. FLIM/spectral phasor clusters mapping to (c) ‘very light’ HCMs showed the longest fluorescence lifetimes/emission spectra: 550 to 585 nm. The unmixed FLIM phasor clusters mapped to fluorophores contained in ECM, RBCs and Hoechst 33342 nucleic DNA binding stain were distinct from the HCM-matched clusters but image segmented spectral phasor clusters showed overlapping emission spectra. M = HCM, ECM = Extracellular matrix, H = Hoechst 33342, RBC = Red blood cell, G = X coordinate of phasor transform (‘real’ unitless phasor component), S = Y coordinate of phasor transform (‘imaginary’ unitless phasor component), Scale bar = 10 μm.