Identification, distribution and intracellular melanin profiling of melanocytes and melanocytic lesions in the human choroid

Choroidal melanocytes are the melanin-producing cells in the vascular uveal tract 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 from skin melanocytes. Melanocytes (and melanins) are important in normal human eye physiology, with roles including photoprotection, regulation of oxidative damage and potentially immune regulation. Our current understanding of the regional distribution, spatial melanin composition and melanosome distribution in choroidal melanocytes (and melanocytic lesions) is primarily informed by immunolabelled eye tissues, or by melanin quantification following tissue-destructive techniques. To address these knowledge gaps, the thesis initially quantified the regional distribution of human choroidal melanocytes (HCMs) in histology eye sections. The distribution of HCMs was statistically preserved in central (sub-foveal and near the optic disc), sub-perifoveal and far peripheral (near the ora serrata) choroidal regions. The quest for understanding eye melanocytes continued next with the visualisation of pigmented HCMs using specific melanocyte-related target proteins, fluorescent immunohistochemistry (IHC) and melanin bleaching in post-mortem tissue. A combined fluorescent-IHC and 5% H2O2 melanin bleaching protocol provided a balance between fluorescence signal detection from target proteins of dark pigmented HCMs and tissue integrity including HCM cytomorphology. However, melanin bleaching did incur loss of the fluorescence emission spectral and lifetime signatures of the cytoplasmic melanins. As such, melanins (eumelanin and pheomelanin) and melanosomes in label-free cultured and whole tissue HCMs were ultimately profiled and spatially visualised using non-invasive 2-photon microscopy (2PM) fluorescence spectral and lifetime acquisition and phasor plot segmentation. This approach successfully and consistently confirmed intracellular melanins as endogenous fluorophores, and as quantifiable and reliable biomarkers for variably pigmented HCMs. As a proof-of-concept, the 2PM FLIM acquisition and phasor plot analysis methods were used to define the melanin profiles in a small series of human choroidal melanocytic lesions