Purpose We employed in vivo, 1.0-µm axial resolution visible-light optical coherence tomography (OCT) and ex vivo electron microscopy (EM) to investigate three subcellular features in the mouse outer retina: reflectivity oscillations inner to band 1 (study 1); hyperreflective band 2, attributed to the ellipsoid zone or inner segment/outer segment (IS/OS) junction (study 2); and the hyperreflective retinal pigment epithelium (RPE) within band 4 (study 3). Methods Pigmented (C57BL/6J, n = 10) and albino (BALB/cJ, n = 3) mice were imaged in vivo. Enucleated eyes were processed for light and electron microscopy. Using well-accepted reference surfaces, we compared micrometer-scale axial reflectivity of visible-light OCT with subcellular organization, as revealed by 9449 annotated EM organelles and features across four pigmented eyes. Results In study 1, outer nuclear layer reflectivity peaks coincided with valleys in heterochromatin clump density (−0.34 ± 2.27 µm limits of agreement [LoA]). In study 2, band 2 depth on OCT and IS/OS junction depth on EM agreed (−0.57 ± 0.76 µm LoA), with both having similar distributions. In study 3, RPE electron dense organelle distribution did not agree with reflectivity in C57BL/6J mice, with OCT measures of RPE thickness exceeding those of EM (2.09 ± 0.89 µm LoA). Finally, RPE thickness increased with age in pigmented mice (slope = 0.056 µm/mo; P = 6.8 × 10 −7 ). Conclusions Visible-light OCT bands arise from subcellular organization, enabling new measurements in mice. Quantitative OCT–EM comparisons may be confounded by hydration level, particularly in the OS and RPE. Caution is warranted in generalizing results to other species.

Purpose We employ visible light optical coherence tomography (OCT) to investigate the relationship between the myoid, ellipsoid, and band 2 in the living human retina. Rather than refute existing theories, we aim to reveal new bands and better delineate the structures at hand. Methods An upgraded spectral/Fourier domain visible light OCT prototype, with 1.0-µm axial resolution, imaged 13 eyes of 13 young adult human subjects (23–40 years old) without a history of ocular pathology. The external limiting membrane (band 1) and band 2 edges were segmented. Reflectivity was examined along the inner segment (IS), defined as extending from band 1 to the band 2 center, and within band 2 itself. Results Images highlight a nearly continuously resolved extrafoveal internal limiting membrane, the peripheral single-cell thick ganglion cell layer, and the peripheral photoreceptor axonal fiber layer, a peripheral division of band 2 into bands 2a and 2b, and a reflectivity-based division of the IS into “m” and “e” zones. Discussion Topography and transverse intensity variations of the outermost band 2b suggest an association with rods. The “m” and “e” zone border is consistent with the myoid–ellipsoid boundary, even recapitulating the well-documented distribution of mitochondria throughout the IS at the foveal center. Theories of outer retinal reflectivity in OCT must adequately explain these observations. Translational Relevance Findings support that band 2 does partially overlap with the ellipsoid in transversally averaged OCT images due to photoreceptor IS length dispersion but argue that the inner ellipsoid must be inner to band 2, as suggested by prior quantitative measurements.

Near-infrared Optical Coherence Tomography (OCT) can image bands of alternating reflectivity that delineate the major layers of the retina in living subjects. With the latest technical developments, visible light OCT has taken this capability even further, revealing fine sub-bands and strata within conventional NIR OCT bands. Here we performed visible light OCT study of the retina in a cross-section of mice with varying ages. C57BL/6J, BALB/cJ, and a selection of mutant mice and controls (aged 1.15-20 months) were imaged with 1.0 micrometer axial resolution visible light OCT while under isoflurane anesthesia. Retinal layers and sub-layers were analyzed through a combination of manual and automated segmentation procedures. In agreement with previous studies, we found a decrease in outer nuclear layer (ONL) thickness with age. In addition, we found age-related changes in two closely-associated visible light OCT features. First, a hyporeflective band at the outer edge of the ONL was found to thin with age. Second, a moderately reflective band inner to the ONL was found to thin with age. Histological correlations suggest that outer ONL reflectivity oscillations, or striations, arise from the nuclei being arranged into rows, and that the moderately reflective band inner to the ONL arises from rod spherules. The concomitant age-related thinning of these visible light OCT features, along with the ONL, strengthens these hypothesized associations. These observations also suggest that changes in the organization of the ONL accompany age-related thinning, and that photoreceptor loss can be studied at the level of the soma and at the synapse by visible light OCT in vivo.

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