Figure 3
2PM FLIM phasor fingerprints of fluorophore controls. FLIM phasor distribution profiles of various control fluorophores. (a) FLIM phasor clusters mapping to synthetic melanin and dark brown human hair cortex (melanin pigmentation: ‘dark’ to ‘very dark’; eumelanin-enriched) with short average fluorescence lifetimes. (b) FLIM phasor clusters mapping to red-brown bird feather (melanin pigmentation: ‘medium’; medium mixture of eumelanin and pheomelanin) with medium average fluorescence lifetimes. (c) FLIM phasor clusters mapping to red human hair cortex, amelanotic melanoma and lightly pigmented human fetal eye choroid (melanin pigmentation: ‘light’ to ‘very light’; pheomelanin-enriched) with long average fluorescence lifetimes. (d) FLIM phasor clusters mapping to choroidal ECM and light pigmented melanins in human choroid flatmount tissue. (e) FLIM intensity image of sampled choroid flatmount tissue. (f) Segmented FLIM image of light pigmented HCMs and ECM in choroidal tissue. (g) The collagen fibers (*) in the ECM surrounding the HCMs were imaged in SHG mode (excitation wavelength: 840 nm, emission range: 420/10 nm). (h) Brightfield image of the choroid tissue. M = Melanocytes, * = Collagen fibers, SHG = Second harmonic generation, G = X coordinate of phasor transform (‘real’ unitless phasor component), S = Y coordinate of phasor transform (‘imaginary’ unitless phasor component).
