Near-infrared adaptive optics fluorescence lifetime ophthalmoscopy of human retinal pigment epithelium reveals variations with eccentricity and disease

Abstract

Purpose : Infrared autofluorescence (IRAF) originates from oxidized melanin and its byproducts in retinal pigment epithelium (RPE). Variations in relative fluorophore composition may be a potential biomarker of cell health. Integrating an adaptive optics scanning light ophthalmoscope (AOSLO) with fluorescence lifetime (FL) measures allows visualization of the RPE mosaic and fluorophore changes with age and disease. Here, we present the first IR-AOFL imaging ophthalmoscopy (IR-AOFLIO) measurements of in vivo human RPE.

Methods : A custom AOSLO (1.4° x 1.4° FOV, 25 Hz frame rate, 120 s exposure) was used for IR reflectance and IRAF imaging (λ_ex 765/30 nm, 50 ps pulse width, 80 MHz rep rate, 300 µW; λ_em 800-900 nm). We performed IR-AOFLIO in 7 young (23-35 YO) and 3 older healthy subjects (49-64 YO) at 7 locations (fovea, 2°, 8°, and 12°; temporal and inferior), and in a 64 YO with drusen. Using commercial SPCImage (Becker & Hickl GmbH) and custom software, images of IRAF emission timing data were registered and fit with a two-component exponential decay (a₁e^-t/T1 + a₂e^-t/T₂) from which T_m = a₁T₁ + a₂T₂ was calculated. The mean FL parameters were determined for each image. ANOVAs were used to compare FL components across eccentricity and age. Repeatability between 2 visits in 4 subjects was assessed by intraclass correlation (ICC).

Results : RPE cells were resolved at all locations in 5 subjects. Across all young healthy eyes T_m increased from fovea to perifovea (p = 0.02; 64 ± 18 ps fovea, 70 ± 7 ps 2°, 103 ± 7 ps 8°, 110 ± 5 ps 12°; Fig 1). Similar trends were observed for T₁, T₂, and a₂. All parameters were repeatable between 2 visits (ICC: 0.88 T_m, 0.75 T₁, 0.65 T₂, 0.98 a₁). This sample size showed no effect of age. In a subject with drusen, at the hyper-AF region T_m was longer (111 ± 14 ps) than in older healthy eyes (67 ± 38 ps) at similar eccentricity.

Conclusions : IR-AOFLIO of in vivo human RPE cellular mosaic is a new, repeatable measure for detection of alterations in RPE composition. The lower lifetime near the fovea compared to perifovea in healthy subjects may be attributed to higher melanin composition (a₁) at the fovea or variations in oxidation state of melanin. Variations observed in the presence of drusen suggest that IR-AOFLIO is a promising new in vivo tool with high potential to measure biomarkers of RPE cellular health and disease.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.