From Soma to Synapse: Imaging Age-Related Rod Photoreceptor Changes in the Mouse with Visible Light OCT.

Purpose: Although the outer nuclear layer (ONL) and outer plexiform layer (OPL) each exhibit a complex internal organization, near-infrared OCT depicts both as monolithic bands. Here, using visible light OCT in the C57BL/6J mouse retina, sublaminar age-related changes in photoreceptor features were imaged and interpreted. These features were (1) oscillations in reflectivity, or striations, in the ONL and (2) a moderately reflective subband in the OPL. Design: Cross-sectional study. Participants: Pigmented mice (C57BL/6J, n = 14). Methods: A 1.0-µm axial resolution visible light spectral/Fourier domain OCT system was used for in vivo retinal imaging. Light and electron microscopy were performed ex vivo. Linear mixed effects models or regression were employed for statistical analysis. Main Outcome Measures: Comparison of OCT subbands with corresponding histological features, as well as quantification of subband thickness and reflectivity. Results: Corresponding histological comparisons confirm that striations in the ONL arise from the rowlike arrangement of photoreceptor nuclei and reveal that the moderately reflective OPL subband arises from rod spherules. Compression of outer ONL striations with age suggests changes in soma organization. Thinning of the moderately reflective OPL subband with age supports a reduction of synapses in the OPL. Critically, the ONL somas are tightly correlated with the purported spherule layer but not with the rest of the OPL. Conclusions: Visible light OCT imaging of the mouse OPL resolves postsynaptic and synaptic differences. Visible light OCT can study rod photoreceptor changes from the soma to the synapse in the living mouse retina. Financial Disclosures: Proprietary or commercial disclosure may be found after the references.

Visible Light Optical Coherence Tomography Reveals the Relationship of the Myoid and Ellipsoid to Band 2 in Humans.

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.

Subcellular Comparison of Visible-Light Optical Coherence Tomography and Electron Microscopy in the Mouse Outer Retina.

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.

In vivo Morphometry of Inner Plexiform Layer (IPL) Stratification in the Human Retina With Visible Light Optical Coherence Tomography.

From the bipolar cells to higher brain visual centers, signals in the vertebrate visual system are transmitted along parallel on and off pathways. These two pathways are spatially segregated along the depth axis of the retina. Yet, to our knowledge, there is no way to directly assess this anatomical stratification in vivo. Here, employing ultrahigh resolution visible light Optical Coherence Tomography (OCT) imaging in humans, we report a stereotyped reflectivity pattern of the inner plexiform layer (IPL) that parallels IPL stratification. We characterize the topography of this reflectivity pattern non-invasively in a cohort of normal, young adult human subjects. This proposed correlate of IPL stratification is accessible through non-invasive ocular imaging in living humans. Topographic variations should be carefully considered when designing studies in development or diseases of the visual system.

Visible Light Optical Coherence Tomography (OCT) Quantifies Subcellular Contributions to Outer Retinal Band 4.

Purpose: To use visible light optical coherence tomography (OCT) to investigate subcellular reflectivity contributions to the outermost (4th) of the retinal hyperreflective bands visualized by current clinical near-infrared (NIR) OCT. Methods: Visible light OCT, with 1.0 µm axial resolution, was performed in 28 eyes of 19 human subjects (21-57 years old) without history of ocular pathology. Two foveal and three extrafoveal hyperreflective zones were consistently depicted within band 4 in all eyes. The two outermost hyperreflective bands, occasionally visualized by NIR OCT, were presumed to be the retinal pigment epithelium (RPE) and Bruch's membrane (BM). RPE thickness, BM thickness, and RPE interior reflectivity were quantified topographically across the macula. Results: A method for correcting RPE multiple scattering tails was found to both improve the Gaussian goodness-of-fit for the BM intensity profile and reduce the coefficient of variation of BM thickness in vivo. No major topographical differences in macular BM thickness were noted. RPE thickness decreased with increasing eccentricity. Visible light OCT signal intensity in the RPE was weighted to the apical side and attenuated more across the RPE in the fovea than peripherally. Conclusions: Morphometry of the presumed RPE and BM bands is consistent with known anatomy. Weighting of RPE reflectivity toward the apical side suggests that melanosomes are the predominant contributors to RPE backscattering and signal attenuation in young eyes. Translational Relevance: By enabling morphometric analysis of the RPE and BM, visible light OCT deciphers the main reflectivity contributions to outer retinal band 4, commonly visualized by commercial OCT systems.

Visible light OCT improves imaging through a highly scattering retinal pigment epithelial wall.

Here we provide a counter-example to the conventional wisdom in biomedical optics that longer wavelengths aid deeper imaging in tissue. Specifically, we investigate visible light optical coherence tomography of Bruch's membrane (BM) in the non-pathologic eyes of humans and two mouse strains. Surprisingly, we find that shorter visible wavelengths improve the visualization of BM in pigmented eyes, where it is located behind a highly scattering layer of melanosomes in the retinal pigment epithelium (RPE). Monte Carlo simulations of radiative transport suggest that, while absorption and scattering are higher at shorter wavelengths, detected multiply scattered light from the RPE is preferentially attenuated relative to detected backscattered light from the BM.

Incoherent excess noise spectrally encodes broadband light sources.

Across optics and photonics, excess intensity noise is often considered a liability. Here, we show that excess noise in broadband supercontinuum and superluminescent diode light sources encodes each spectral channel with unique intensity fluctuations, which actually serve a useful purpose. Specifically, we report that excess noise correlations can both characterize the spectral resolution of spectrometers and enable cross-calibration of their wavelengths across a broad bandwidth. Relative to previous methods that use broadband interferometry and narrow linewidth lasers to characterize and calibrate spectrometers, our approach is simple, comprehensive, and rapid enough to be deployed during spectrometer alignment. First, we employ this approach to aid alignment and reduce the depth-dependent degradation of the sensitivity and axial resolution in a spectrometer-based optical coherence tomography (OCT) system, revealing a new outer retinal band. Second, we achieve a pixel-to-pixel correspondence between two otherwise disparate spectrometers, enabling a robust comparison of their respective measurements. Thus, excess intensity noise has useful applications in optics and photonics.

Water wavenumber calibration for visible light optical coherence tomography.

SIGNIFICANCE: Visible light optical coherence tomography (OCT) is emerging for spectroscopic and ultrahigh resolution imaging, but challenges remain. Depth-dependent dispersion limits retinal image quality and current correction approaches are cumbersome. Inconsistent group refractive indices during image reconstruction also limit reproducibility. AIM: To introduce and evaluate water wavenumber calibration (WWC), which corrects depth-dependent dispersion and provides an accurate depth axis in water. APPROACH: Enabled by a visible light OCT spectrometer configuration with a 3- to 4-dB sensitivity roll-off over 1 mm in air across a 90-nm bandwidth, we determine the spectral phase of a 1-mm water cell, an affine function of water wavenumber. Via WWC, we reconstruct visible light OCT human retinal images with 1.3-µm depth resolution in water. RESULTS: Images clearly reveal Bruch's membrane, inner plexiform layer lamination, and a thin nerve fiber layer in the temporal parafovea. WWC halves the processing time, while achieving the same image definition as an assumption-free gold standard approach, suggesting that water wavenumber is a suitable proxy for tissue wavenumber. WWC also provides a depth axis in water without explicitly assuming a group refractive index. CONCLUSIONS: WWC is a simple method that helps to realize the full potential of visible light OCT.

Improving visible light OCT of the human retina with rapid spectral shaping and axial tracking.

Visible light optical coherence tomography (OCT) theoretically provides finer axial resolution than near-infrared OCT for a given wavelength bandwidth. To realize this potential in the human retina in vivo, the unique technical challenges of visible light OCT must be addressed. We introduce three advances to further the performance of visible light OCT in the human retina. First, we incorporate a grating light valve spatial light modulator (GLV-SLM) spectral shaping stage to modify the source spectrum. This enables comfortable subject alignment with a red light spectrum, and image acquisition with a broad "white light" spectrum, shaped to minimize sidelobes. Second, we develop a novel, Fourier transform-free, software axial motion tracking algorithm with fast, magnetically actuated stage to maintain near-optimal axial resolution and sensitivity in the presence of eye motion. Third, we implement spatially dependent numerical dispersion compensation for the first time in the human eye in vivo. In vivo human retinal OCT images clearly show that the inner plexiform layer consists of 3 hyper-reflective bands and 2 hypo-reflective bands, corresponding with the standard anatomical division of the IPL. Wavelength-dependent images of the outer retina suggest that, beyond merely improving the axial resolution, shorter wavelength visible light may also provide unique advantages for visualizing Bruch's membrane.