Adaptive optics fluorescence lifetime imaging ophthalmoscopy of the human RPE mosaic

Abstract

Purpose : In the retinal pigment epithelium (RPE), the fluorescence lifetime (FL) of lipofuscin granules, related to their composition, may be a useful biomarker of retinal health. Widefield fluorescence lifetime imaging ophthalmoscopy (FLIO) has been used to investigate RPE changes with age, eccentricity and disease but is limited by low sampling density and artifacts such as lens and macular pigment fluorescence. We improve the resolution and confocality by applying adaptive optics FLIO (AOFLIO), for the first time, to image the RPE mosaic in healthy people.

Methods : To specifically target lipofuscin, a custom-built adaptive optics scanning light ophthalmoscope was used for reflectance and FL imaging in 3 subjects (up to 12 locations across the macula; 30-38 yrs). RPE autofluorescence was excited with 532Δ10 nm (50 ps pulse width, 80 MHz repetition rate, 15 μW average power) and collected 575-725 nm (1.4-1.7° square field of view, 25 Hz frame rate, ≤60 s exposure). FL data was analyzed using custom and commercial software (Becker & Hickl GmbH). A two-component exponential function α₁e^-t/τ₁+α₂e^-t/τ₂ was fit to the decay curve at each binned pixel. Mean FL was calculated by τ_m=α₁τ₁+α₂τ₂ where τ_n is the FL and α_n is the relative contribution. τ_n and α_n were averaged across each AOFLIO image and compared using Student's t-test.

Results : Across all images, τ_m is 247.3±27.8 ps (ranging from ~200–270 ps, with an outlier at 330 ps); within an image, the average standard deviation of τ_m is 2.8±0.3 ps. α₁ was also consistent with mean 88.7±0.4% (ranging 88.1–89.4%). τ₁ and τ₂ ranged 120–230 and 830–1170 ps, respectively. The measured τ_m is within the range expected for lipofuscin (widefield FLIO long spectral channel attributed to lipofuscin: Dysli et al., 2017; ex vivo: Docchio et al., 1991; Schweitzer et al., 2007; Feldman et al., 2018). AOFLIO measurements taken 8 days apart in one subject showed no significant differences in τ_n and α_n (p ≥ 0.12). Between two subjects, no significant differences were found in the FLs (p ≥ 0.058), but α₁ showed a small but significant difference (s1: 88.6±1.7%, s2: 88.5±0.3%, p < 0.001).

Conclusions : These are the first measurements using AOFLIO to image the human RPE mosaic. Our results suggest AOFLIO will be repeatable within and between young healthy subjects. We expect larger variations with age and disease related to composition and function of the RPE mosaic.

This is a 2021 ARVO Annual Meeting abstract.