Abstract
Purpose :
Fluorescence lifetime ophthalmoscopy (FLIO) can distinguish lifetime variations across the normal and diseased fundus, yet the role of individual fluorophores is unclear. Adaptive optics (AO) FLIO enables cellular-scale resolution of unique cell types in distinct retinal layers. Here, we apply in vivo AOFLIO to compare the lifetimes of cellular-scale structures throughout the healthy macaque retina to those of specific retinal fluorophores.
Methods :
In 2 male macaques, AOFLIO was performed using two-photon excited fluorescence (730 nm, ~55 fs, 80 MHz, 7 mW) in a scanning light ophthalmoscope (19 1.1x1.3° regions, 5-18° ecc.). The nerve fiber (NF), ganglion cell (GC), and photoreceptor (PR) layers were imaged. At each pixel, phasor analysis, where the cos and sin Fourier transform of the decay trace is computed at the laser pulse rate, yielded a Cartesian coordinate (g,s). Clusters from in vivo data were compared to a phasor fingerprint of common retinal fluorophores NADH, FAD, elastin, all-trans-retinol (ROL), and all-trans-retinal (RAL) in solution measured in the same system.
Results :
In the phasor fingerprint, each fluorophore had a unique position. Phasor plots of in vivo data suggested contributions from several fluorophores. The artery wall phasor showed a dominance of elastin not seen in veins (Fig 1). Phasors of NF and GC, dominated by metabolites, oriented along the free/bound NADH axis. Consistent with previous work, the PR phasors separated into rods, M/L cones, and S cones. Rods and M/L cones laid on a ROL/RAL axis (Fig 2); M/L cones had a 48±12% mean relative contribution from ROL, significantly higher than in rods (30±16%) possibly due to increased photopigment bleach (paired t-test, p=.02). After a 2 min 730 nm exposure, the S cone lifetime became longer, which may be indicative of oxidative stress. The optic disc edge clustered separately from PR closer to bound NADH, FAD, and elastin (Fig 2).
Conclusions :
The axial and lateral resolution of AOFLIO allows identification of multiple origins of fluorescence in distinct retinal cells and layers. We demonstrated the first in vivo AOFLIO assessment in the inner retina and further characterized the outer retina. Comparing phasor signatures throughout the retina to those of known fluorophores establishes a basis for assessing subcellular molecular pathways that are expected to shift in disease.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.