Quantification of Human Photoreceptor-Retinal Pigment Epithelium Macular Topography with Adaptive Optics-Optical Coherence Tomography

Abstract

Photoreceptors (PRs) and retinal pigment epithelial (RPE) cells form a functional unit called the PR-RPE complex. The PR-RPE complex plays a critical role in maintaining retinal homeostasis and function, and the quantification of its structure and topographical arrangement across the macula are important for understanding the etiology, mechanisms, and progression of many retinal diseases. However, the three-dimensional cellular morphology of the PR-RPE complex in living human eyes has not been completely described due to limitations in imaging techniques. We used the cellular resolution and depth-sectioning capabilities of a custom, high-speed Fourier domain mode-locked laser-based adaptive optics-optical coherence tomography (FDML-AO-OCT) platform to characterize human PR-RPE complex topography across the temporal macula from eleven healthy volunteers. With the aid of a deep learning algorithm, key metrics were extracted from the PR-RPE complex of averaged AO-OCT volumes including PR and RPE cell density, PR outer segment length (OSL), and PR/RPE ratio. We found a tight grouping among our cohort for PR density, with a mean (±SD) value of 53,329 (±8106) cells/mm² at 1° decreasing to 8669 (±737) cells/mm² at 12°. We observed a power function relationship between eccentricity and both PR density and PR/RPE ratio. We found similar variability in our RPE density measures, with a mean value of 7335 (±681) cells/mm² at 1° decreasing to 5547 (±356) cells/mm² at 12°, exhibiting a linear relationship with a negative slope of -123 cells/mm² per degree. OSL monotonically decreased from 33.3 (±2.4) µm at 1° to 18.0 (±1.8) µm at 12°, following a second-order polynomial relationship. PR/RPE ratio decreased from 7.3 (±0.9) µm at 1° to 1.5 (±0.1) µm at 12°. The normative data from this investigation will help lay a foundation for future studies of retinal pathology.

Keywords: adaptive optics; optical coherence tomography; photoreceptor; photoreceptor outer segment; retinal pigment epithelium.

Figure 1

AO-OCT image acquisition example for a 33-year-old volunteer (8195). (A) Spectralis SLO image with nine AO-OCT imaging regions overlaid (black border). (B) Cross-sectional AO-OCT B-scan view of the PR-RPE complex along the temporal raphe at the region denoted by a white dashed line in (A). (C) Montage of RPE mosaics with AO-OCT. (D) Magnified 250 × 250 µm subregions of PR (top) and RPE (bottom) mosaic from three selected locations at fovea, 6T, and 12T. PR en face images were generated by average intensity projection across the inner segment/outer segment junction (IS/OS) and cone outer segment tip (COST) layers. The whole montage of PR and RPE of all study volunteers are provided in the Supplementary Materials. Scale bars = 200 µm.

Figure 2

AO-OCT image processing and analysis approach for extracting outer retinal cell morphology. (A) Spectralis SLO image of a 32-year-old volunteer (1060) with AO-OCT imaging region overlay (black border). (B) AO-OCT montage of RPE mosaics showing 13 selected ROIs where PR-RPE complex quantification was performed. Magenta- and cyan-colored regions delineate areas used for cone photoreceptor and RPE quantification, respectively. For each ROI, the PR and RPE en face images are segmented and extracted from the average AO-OCT volume. Representative PR and RPE quantification results at (C) 3T, (D) 7T, and (E) 11T. For (C–E), images in rows from the left to right show RPE mosaic, Voronoi map with each RPE cell marked in cyan color, PR mosaic with cell centers marked in magenta color, superimposed RPE Voronoi map and PR locations, and segmented PR outer segments at the B-scans marked with the dashed white lines.

Figure 3

AO-OCT measurement of PR-RPE complex morphology with retinal eccentricity. (A) Cone PR density, (B) RPE density, (C) cone PR OSL, and (D) PR/RPE ratio. The black symbols are the mean values for our study cohort. The solid black lines represent the power/linear/polynomial fits to the mean data using Pearson’s correlation coefficient.

Figure 4

Reproducibility of AO-OCT PR-RPE complex measurements. (A–C) PR density, OSL, and RPE density measurements for three AO-OCT imaging sessions (light blue/orange symbols) and mean ± SD (dark-blue/brown symbol) for seven volunteers with overall cohort mean (solid line) and SD (dashed line). (D–F) Plot of normalized value ($x/\overline{x}$) vs. x, where $\overline{x}$ denotes mean value, illustrating the spread of repeat measurements for PR density (d), OSL, and RPE density (d) measurements. Dashed lines indicate overall cohort ±SD.

Figure 5

AO-OCT measurement of RPE density as compared to (A) in vivo studies [17,18,20,21,22,23,26,27,30] and (B) ex vivo studies [39,40,41,42,43,44,45,46]. The dashed lines denote ± SD from the mean.

Figure 6

Comparison of AO-OCT measurements with published literature for (A) cone PR density [29], (B) PR/RPE ratio [21,22,27,30], and (C) PR OSL [27,47,48]. The solid line denotes mean values and the dashed lines represent ±SD for the entire study cohort.