Abstract
Purpose :
Histologically, three major granule types with distinct autofluorescence (AF) properties are found within the cell bodies of human RPE: lipofuscin (L), melanolipofuscin (ML), and melanosomes (M). In contrast to L and ML, Ms block short wavelength light but may exhibit AF in the NIR range. Clinically, NIR FAF is believed to reflect M abundance, but histological data are sparse. This study reports the subcellular origin of NIR AF within human RPE cells using super-resolution microscopy.
Methods :
RPE flatmounts and retinal cross sections from human donors were microscopically imaged using a custom built NIR SIM device (excitation: 785 nm, emission >800 nm; plan-apochromatic 63X 1.46 oil immersion objective), a commercially available SIM device (Elyra S1, Zeiss; excitation: 488 nm, 633 nm; plan-apochromatic 63x 1.4 oil immersion objective), and a light microscope. Tissues were imaged at the fovea, perifovea, and near-periphery. The NIR SIM signals were further magnified using a 1.6 magnification changer (Optovar, Zeiss) and detected using an Orca Flash4.0 V3 camera (Hamatsu). The SIM data was reconstructed using custom written algorithms (NIR SIM) or the device's internal software (Elyra S1). For signal co-localization, SIM images were superimposed and the light microscope images confirmed the melanin within the pigmented granules.
Results :
Based on AF phenotype, SIM imaging enables clear distinction between intracellular RPE granule types. 488 nm SIM shows bright signals from L and the L part within ML. The same signal pattern, though less intense, is detectable at 633 nm excitation. At 785 nm excitation, AF pattern changes and AF arises from the pigmented part of ML. M are only sporadically visible due to preparation artefacts that remove or damage apical processes, and do not contribute substantially to the AF signal in cell bodies.
Conclusions :
ML seems to be the major source in NIR imaging of RPE cell bodies. This is in line with recent histological findings showing ML to be the leading granule type at the fovea. Therefore, clinical NIR AF imaging of the posterior pole with its high AF signal arising from the fovea might be a good indicator for intracellular RPE ML abundance.
This is a 2020 ARVO Annual Meeting abstract.