Characterizing cone photoreceptors with outer segments that generate multiple reflections

Abstract

Purpose : The cone photoreceptor outer segment (OS) has garnered much interest in high-resolution retinal imaging studies as it is the site of phototransduction and is sensitive to numerous retinal diseases afflicting photoreceptors. Bright reflections from the OS end tips—inner-segment/outer-segment junction (IS/OS) and cone outer segment tip (COST)—facilitate OS length and diameter measurements as well as functional measurements (optoretinogram, ORG), both routinely measured with adaptive optics (AO) OCT. However, many cone photoreceptors have additional reflections posterior to IS/OS that complicate identifying the true COST. This causes errors in OS length and ORG measurements and requires cells to be discarded from analyses. Here, we develop a method based on the cone ORG to identify the true COST reflection of these cones.

Methods : Six normal subjects were imaged with AO-OCT at 3.7° temporal retina with 1°×0.8° field of view and 5-s duration. At 2.5 s, a flash stimulus was delivered to the retina from one of three LEDs at 637 nm, 528 nm, and 450 nm. For each cone, we recorded the depths of all reflections between IS/OS and retinal pigment epithelium (RPE) and measured wavelength-dependent changes in optical path length difference (ΔOPL). We then selected cones with multiple reflections to determine the relationship between ΔOPL response and reflection depth.

Results : In six subjects, 2010 out of 6644 (30.2%) cones had multiple reflections between IS/OS and RPE. S cones exhibited the largest prevalence of multiple OS reflections (42.9%) compared with M (20.8%) and L (17.5%) cones. Examining the cones with multiple OS reflections, we found a positive correlation between ΔOPL response and reflection depth (p < 0.001) followed by an abrupt decrease in response at deeper reflections. The positive correlation is consistent with OS lengthening and implies that its deepest reflection corresponds to the true COST. Deeper reflections of reduced response are indicative of RPE. The correlation was stronger in S cones (R² = 0.75) than in M (R² = 0.34) and L (R² = 0.15) cones. For cones whose true COST was different from the brightest reflection between IS/OS and RPE, our method’s estimate of OS length and ΔOPL response differ by 39 ± 21% and 30 ± 24% respectively from those measured using the conventional method.

Conclusions : We can identify COST in cone photoreceptors that generate multiple outer segment reflections.

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