Super-Resolution Near-Infrared Autofluorescence Microscopy Depicts Melanin Containing Organelles of the Retinal Pigment Epithelium in Human Donor Eyes.

Purpose: In the human retinal pigment epithelium (RPE), melanin localizes to melanosomes as well as melanolipofuscin. Changes in the organelles' volumetric density may have different clinical implications, especially in autofluorescence (AF) imaging. Here, short wavelength (SW)-AF and near-infrared-AF (NIR-AF) of RPE cells including apical processes was evaluated regarding the histological source of the NIR-AF signal. Methods: Retinal cross-sections from seven human donors (age = 80-89 years) were imaged at the fovea and near periphery using a confocal microscope with different laser excitation wavelengths (λexc; 488, 514, 642, 705, 750, and 785 nm). To further characterize AF RPE granules, the RPE cells were also imaged by structured illumination microscopy (SIM, λexc 488 nm and 785 nm). Results: The RPE cell body is the main source of AF-emission in both SW (λexc 488 nm and 514 nm) and NIR excitation (λexc 750 nm and 785 nm). Melanosomes within apical processes exhibit a bright NIR-AF signal and lack SW-AF signal. Quantitative signal analysis revealed SW-AF intensity is highest in the basolateral RPE, corresponding to the localization of lipofuscin granules, and decreases towards apical processes. The RPE cell body containing melanolipofuscin granules generate the highest NIR-AF intensity observed - surpassing even that of melanosomes in the apical processes. In the basal quartile of the cell body, the NIR-AF signal sharply decreases toward the basolateral end. Conclusions: The combined evidence from high-resolution confocal and super-resolution NIR-AF microscopy reveals that melanin-containing organelles of the RPE in human donor eyes aged 80 to 89 years are specifically detected at λexc 750 nm and 785 nm, but not at 705 nm. These histological findings will inform future clinical NIR-AF imaging studies.