Microscopy

PURPOSE. Retinal pigment epithelium (RPE) cells show strong autofluorescence (AF). Here, we characterize the AF spectra of individual RPE cells in healthy eyes and those affected by age-related macular degeneration (AMD) and investigate associations between AF spectral response and the number of intracellular AF granules per cell. METHODS. RPE–Bruch’s membrane flatmounts of 22 human donor eyes, including seven AMD-affected eyes (early AMD, three; geographic atrophy, one; neovascular, three) and 15 unaffected macula (80 years, seven), were imaged at the fovea, perifovea, and near-periphery using confocal AF microscopy (excitation 488 nm), and emission spectra were recorded (500–710 nm). RPE cells were manually segmented with computer assistance and stratified by disease status, and emission spectra were analyzed using cubic spline transforms. Intracellular granules were manually counted and classified. Linear mixed models were used to investigate associations between spectra and the number of intracellular granules. RESULTS. Spectra of 5549 RPE cells were recorded. The spectra of RPE cells in healthy eyes showed similar emission curves that peaked at 580 nm for fovea and perifovea and at 575 and 580 nm for near-periphery. RPE spectral curves in AMD eyes differed significantly, being blue shifted by 10 nm toward shorter wavelengths. No significant association coefficients were found between wavelengths and granule counts. CONCLUSIONS. This large series of RPE cell emission spectra at precisely predefined retinal locations showed a hypsochromic spectral shift in AMD. Combining different microscopy techniques, our work has identified cellular RPE spectral AF and subcellular granule properties that will inform future in vivo investigations using single-cell imaging.