Non-invasive Detection of Glycolytic Stress-induced Reactive Oxygen Species in Human Retinal Pigment Epithelium Cells using Autofluorescence Multispectral Imaging

Abstract

Purpose : To determine auto fluorescent multispectral imaging (AFMI) signatures in a human retinal pigment epithelium (RPE) cell culture system under normal conditions and under glycolytic stress.

Methods : Human RPE cells (h1RPE7 cell line) were treated with a glycolytic inhibitor, iodoacetic acid (IAA) at 1 µM, 5 µM, and 10 µM concentrations for 24 hours. Cell viability was assessed using a fluorescent live/dead assay and dihydroethidium (DHE) was used to quantify cellular reactive oxygen species (ROS). Imaging was initially performed with routine confocal microscopy. ROS detection was then performed in the absence of exogenous fluorescent probes using a novel autofluorescence multispectral imaging (AFMI) microscopy method. A total of 33 spectral channels spanning specific excitation (345-505 nm) and emission (414-675 nm) wavelength ranges were used.

Results : A dose-dependent cell death occurred after 24 hours of exposure to IAA, peaking at 80% cell death at the 10 µM concentration. ROS production was significantly increased in cells under glycolytic stress (p < 0.001). There were highly significant changes in the patterns of spectral signals between treated and control groups using the AFMI microscopy (p < 0.001).

Conclusions : These results show that ROS can be detected using non-invasive multispectral imaging in retinal cells. This imaging technique may be adapted to in vivo use and has potential for the detection and monitoring of clinically relevant retinal disease states such as macular degeneration and glaucoma.

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