Fluorescence lifetime imaging microscopy of pigmented cells in neovascular age-related macular degeneration

This study characterized fibrosis-associated pigment changes in neovascular age-related macular degeneration (nAMD) using fluorescence lifetime imaging microscopy (FLIM). Retinal cross sections from human donor eyes with nAMD (n = 5; mean age 91.0 ± 2.8 years) and control eyes without maculopathy (n = 5; 83.0 ± 1.9 years) were analyzed at λ_exc 488 nm and λ_exc 780 nm. In total, 116 regions of interest (50 μm width each) were assessed across the fovea, parafovea, and areas of subretinal fibrosis in nAMD eyes and compared with corresponding locations in controls. Fluorescence lifetimes (FLTs) were evaluated using a linear mixed-effects model across pigment in unremarkable retinal pigment epithelium (RPE), pigment in RPE above fibrosis, pigment within fibrosis, and pigment in the choroid. At λ_exc 488 nm, FLTs were significantly prolonged in RPE above fibrosis (0.54 ± 0.04 ns) compared with unremarkable RPE (0.49 ± 0.04 ns, linear mixed-effect model: p = 0.04). Pigment within fibrosis (0.51 ± 0.03 ns) showed similar lifetimes to unremarkable RPE (p = 0.58), while choroidal pigment exhibited markedly shorter lifetimes (0.23 ± 0.04 ns; p < 0.001). At λ_exc 780 nm, pigment within fibrosis displayed shorter lifetimes (0.27 ± 0.03 ns) than unremarkable RPE (0.34 ± 0.04 ns, p = 0.04), and choroidal pigment again showed the shortest values (0.11 ± 0.01 ns; p < 0.001). Dual-wavelength FLIM reveals distinct lifetime patterns at fibrotic locations consistent with an RPE origin of fibrosis-associated pigment in nAMD.