Organelles of the human retinal pigment epithelium (RPE): morphological subtypes, regional differences, and total 488nm autofluorescence (AF)

Purpose : Hundreds of lipofuscin (LF), melanolipofuscin (ML), and melanosomes (M) organelles are found within human RPE cells, as reported in histological (PMID 6698741) and 3D serial block electron microscopy studies (PMID 29066281). Information on the number and AF characteristics of these organelles would help inform models of disease pathogenesis involving RPE and interpretation of clinical imaging. Using super-resolution structured illumination microscopy (SIM), this study catalogues and reports number of AF intracellular RPE organelles. Methods : 15 human RPE flatmounts (8≤ 51 yrs, 7>80 yrs; no macular pathologies) were imaged at three predefined locations (fovea, perifovea, near periphery) using SIM (Zeiss Elyra.S1; 488 nm). Z-stacks were acquired from apical to basal through RPE cell bodies (step size 100 nm) for 10 adjacent cells in each region. Custom written FIJI plugin enabled manual marking and reporting x,y,z coordinates for each organelle. Based on their AF properties, organelles were further sub-classified. In addition, cell area and total AF intensity/cell expressed as a planimetric density were reported. Results : Within 450 cells, >193.000 organelles were analyzed and subdivided into nine different AF phenotypes (M1, M2; L1, L2, ML1-ML5). Total number of granules/cell is lowest at the fovea (322±114), compared to perifovea (508±197) and near periphery (456±243). Number of L/cell is lowest at the fovea (89±71), Number of ML/cell showed no differences between locations. A newly described ML sub-type (2x as large as common ML) is found preferentially at the perifovea/near-periphery. Only few M (<30/cell body) were found at each location. With age, L and ML increase while M decrease. RPE cell area is smallest at the fovea. AF/cell is highest at the perifovea (high L/ML ratio) and lowest at the fovea (low L/ML ratio). Conclusions : This is the largest sample of human RPE organelles analyzed to date. The different AF phenotypes may represent different stages of organelle lifecycle. The low number of L at the fovea might reflect cone photoreceptor-RPE specific features, while the overall low number of M might be related to preparation artefacts (loss of apical processes) or naturally occurring remodeling of M to ML. The intracellular organelle ratio is related to the total AF/cell, which serves as a basis for interpreting clinical AF imaging. This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.