Spectral Analysis of Human Retinal Pigment Epithelium Cells in Healthy and AMD Affected Eyes

Abstract

Purpose : Individual retinal pigment epithelium (RPE) cells host hundreds of granules with AF properties (PMID: 32433758). Here, we characterized RPE cells in healthy and age-related macular degeneration (AMD) affected eyes by their AF spectra and correlate individual wavelengths with the amount of autofluorescent granules (lipofuscin, melanolipofuscin) per RPE cell body (RPECB).

Methods : Twenty-two RPE flat mounts of human donor eyes (7 AMD-affected (3 early AMD, 1 geographic atrophy, 3 neovascular) and 15 w/ unaffected macula (8<51 yrs; 7>80 yrs)) were imaged at three locations (fovea, perifovea, near periphery) using en face confocal AF (exc. 488 nm) and high-resolution structured illumination microscopy. Up to 110 cells/location/eye were marked manually and emission spectra were extracted (490-695 nm; 8.9 nm spectral channel width) from a projection image of each cell and stratified by disease status and location. Granules/cell were manually quantified and classified with computer-assistance. Mixed linear models were used to correlate spectra with the number of intracellular granules.

Results : Spectra of 5.549 RPE cells (<51yrs: 2.353, >80yrs: 2.053, AMD: 1.143) were included. Spectra of healthy cells showed similar emission spectral curves that peaked at 580 nm for the fovea and perifovea and between 575 and 580 nm for the near periphery. Compared to healthy, AMD spectral curves showed a 10 nm-shift towards shorter wavelengths (further statistical analysis in progress). The effect was strongest for the fovea and perifovea. Spectral curves at the perifovea appeared to be flattened at nAMD stage. Mixed linear models showed no statistically significant differences between the coefficients for wavelengths and granule load.

Conclusions : With the help of different microscopy techniques, identification of cellular (spectral AF) and subcellular (granules) properties of RPE cells allow to distinguish healthy from diseased RPE, especially at fovea and perifovea, areas highly impacted by AMD related sub-RPE deposits A spectral shift in AMD in this large series of RPE cells confirms earlier microscopy findings (PMID: 12091448) from tissue cross sections. Future studies will examine the impact of individual granule spectra on the total RPE spectrum. Overall, our results will help to interpret the RPE's AF in studies using in vivo single cell imaging.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.