Near-Infrared Fluorescence Lifetime Imaging Microscopy Reveals Loss of RPE Cell Body Polarity in Age-Related Macular Degeneration

Purpose: Retinal pigment epithelium (RPE) cells exhibit a markedly polarized distribution of organelles and membrane specializations. We aimed to determine whether fluorescence lifetime imaging microscopy (FLIM) using different excitation wavelengths reveals altered polarity in age-related macular degeneration (AMD). Methods: Retinal cross-sections from human donors (11 with AMD; 87.9 ± 4.5 years, and 9 healthy controls; 83.4 ± 2.7 years) were imaged at the fovea, perifovea, near-, mid-periphery, and ora serrata using FLIM (excitation = 488, 780 nm; 5 regions of interest [ROI; 50 µm width] per location). At each location, individual RPE cells were classified as healthy, non-uniform, or pathological. Cell bodies were divided into four equal apical-to-basal compartments to determine the fluorescence lifetime (FLT) gradient. Post-processing included multiexponential decay modeling and phasor analysis. Ordinal logistic regression (LR) and linear mixed-effects models (LMMs) compared healthy and diseased cells. Results: In 477 ROIs (3-5 cells/ROI), healthy RPE at 780 nm displayed a distinct intracellular FLT gradient. In 52 pathological ROIs, this was significantly reduced (regression coefficient = -0.029, P = 0.007). Ordinal LR confirmed an association between polarity loss and dysmorphia (log cumulative odds ratio = -6.50, P = 0.033). Mean FLT was shorter in non-uniform (-0.009 nanoseconds [ns], P = 0.03) and pathological RPE (-0.036 ns, P < 0.001) than in healthy RPE. At 488 nm, pathological RPE exhibited prolonged FLT without an apical/basal gradient. Conclusions: At 780 nm, FLIM detects a loss of RPE cell polarity that likely reflects intracellular reorganization and metabolic change. Disruption in NIR-autofluorescence lifetimes may provide an early biomarker for pathological RPE alterations in AMD.