Light-evoked deformations in rod photoreceptors, pigment epithelium and subretinal space revealed by prolonged and multilayered optoretinography

Abstract

Phototransduction involves changes in concentration of ions and other solutes within photoreceptors and in subretinal space, which affect osmotic pressure and the associated water flow. Corresponding expansion and contraction of cellular layers can be imaged using optoretinography (ORG), based on phase-resolved optical coherence tomography (OCT). Until now, ORG could reliably detect only photoisomerization and phototransduction in photoreceptors, primarily in cones under bright stimuli. Here, by employing a phase-restoring subpixel motion correction algorithm, which enables imaging of the nanometer-scale tissue dynamics during minute-long recordings, and unsupervised learning of spatiotemporal patterns, we discover optical signatures of the other retinal structures' response to visual stimuli. These include inner and outer segments of rod photoreceptors, retinal pigment epithelium, and subretinal space in general. The high sensitivity of our technique enables detection of the retinal responses to dim stimuli: down to 0.01% bleach level, corresponding to natural levels of scotopic illumination. We also demonstrate that with a single flash, the optoretinogram can map retinal responses across a 12° field of view, potentially replacing multifocal electroretinography. This technique expands the diagnostic capabilities and practical applicability of optoretinography, providing an alternative to electroretinography, while combining structural and functional retinal imaging in the same OCT machine.

Fig. 1. Retinal layers and their dynamics in response to visual stimuli.

a Averaged retinal B-scan (n = 125) from the same location. b Despeckled retinal B-scan, generated by averaging individual B-scans (n = 125) from a neighboring region, allows better resolving photoreceptors’ OS, RPE, and BrM layers. An enlarged view of the magenta dashed box is shown in Fig. 3b. Both scans were acquired after 12 h of dark adaptation. Scale bar: 50 µm. c ORG signals obtained from various hyperreflective bands in the outer retina by taking the BrM as the reference. d ORG signals obtained at various depths in the mixed layer relative to IS/OS, with colors matching the corresponding bars at the bottom right of Fig. 1a. RNFL: retinal nerve fiber layer; GCL: ganglion cell layer; IPL: inner plexiform layer; INL: inner nuclear layer; OPL: outer plexiform layer; ONL: outer nuclear layer; ELM: external limiting membrane; IS/OS: inner segment/outer segment junction; OS: outer segment; RPE: retinal pigment epithelium; BrM: Bruch’s membrane. Source data are provided as a Source Data file.

Fig. 2. Unsupervised clustering in the spatiotemporal feature space.

a Distribution of the signals in the 3D spatiotemporal feature space. Gray dots represent the outliers identified using a distance-based detection method, while remaining data points were labeled in pink. The heatmap shows the distribution density of the remaining data points when projected onto the temporal feature plane. b Dendrogram showing the cluster structure of the remaining phase traces (pink dots in Fig. 2a) in the spatiotemporal feature space, with only the top 100 subclusters displayed. c Clustering the remaining phase traces in the spatiotemporal feature space into three clusters by thresholding the dendrogram along the solid black line. Colored dots denote different groups, and the gray dots denote outliers. d Corresponding representative Type-I and Type-II signals obtained by averaging the individual phase traces within each cluster. The solid lines and color bands denote the mean values and the range of standard deviations. Source data of Fig. 2d are provided as a Source Data file.

Fig. 3. Classification of new phase traces and validation of their origins.

a The trained SVM decision boundaries for the Type-I signal (red) and the Type-II signal (blue). The dots represent phase traces extracted from a new dataset, preprocessed, and projected onto the same 3D feature space. They were classified into Type-I signals (red dots), Type-II signals (blue dots), intermediate phase traces (brown dots), and outliers (gray dots). b An enlarged view of the dashed box in Fig. 1b, with contrast adjustment to enhance the RPE visibility. The histograms of Type-I (red bins) and Type-II (blue bins) signals, fitted by Gaussian functions (solid lines), display the depth distribution of the signals overlaid on top of the despeckled structural image, with a yellow line representing the averaged intensity profile. The colored bars on the left correspond to the depth range from which the signals in Fig. 1d were extracted. c Type-I, Type-II, and SRS signals extracted from a prolonged recording. Source data of Fig. 3c are provided as a Source Data file.

Fig. 4. Responses of the outer segment (OS) and subretinal space (SRS) in different conditions.

a Representative traces of the light-evoked responses in photoreceptor OS and in SRS. b Amplitude (Amp) and latency (Lat) of the OS response, and slope of the SRS expansion as a function of stimulus intensity on scotopic background (n = 12). c Same as a function of the background illuminance (n = 15). If the regeneration of rhodopsins is neglected, the 5-min background illumination at 6 × 10⁴ photons/(μm² s) would bleach 20.3% rhodopsins. For box plots, horizontal bar: mean value, box edges: 25 and 75 percentiles, whiskers: 1.5 × standard deviations (SDs). Source data are provided as a Source Data file.

Fig. 5. Representative en-face maps of outer segment (OS) and subretinal space (SRS) signals.

a A volumetric scan covered a 12° field of view, with the structural contrast in gray and angiographic contrast highlighted in red. b The OS and SRS signals at selected time points. Locations blocked by large blood vessels are outlined by dashed lines. c Spatiotemporal evolution of the OS and SRS signals over the entire FOV. Each curve presents the average response from a 1.2° × 0.48° (x × y) area. Scale bar: 100 μm. Source data of Fig. 5c are provided as a Source Data file.

Fig. 6. Deformation of cellular layers over time and comparison of ORG with ERG.

a Representative dynamics of the deformations of the rod outer segment (OS), inner segment (IS), retinal pigment epithelium (RPE), and subretinal space (SRS) after a 1 ms green stimulus at 0.26% bleach level. b The expansion rate of the OS (red curve) and RPE (blue curve) averaged across 5 measurements. c, d Zoom-in view of the orange boxed data in (a, b), respectively. e An example electroretinography (ERG) trace in response to a white flash, where the a-wave and c-wave are labeled. Modified from ref. . OPL change was converted into physical deformation with a refractive index of 1.41. Source data are provided as a Source Data file.

Fig. 7. System setup, stimulus scheme, acquisition protocols and image registration.

a A spectral-domain OCT was used to image the posterior segment of rat eyes. A line-scan camera interfaced with the spectrometer was used to acquire the interference fringes. A function generator synchronized the line-scan camera acquisition, galvo scanner rotation, and flash timing. L1-L6: doublet lenses. CL: condenser lens. GS: galvo scanner. Filter spectral window: 500 ± 5 nm. b Three acquisition protocols were used for ORG imaging: repeated B-scans (Protocol 1: 1000 A-scans per B-scan, 200 B-scans per second, Protocol 2: 1000 A-scans per B-scan, 25 B-scans per second) and repeated volumes (Protocol 3: 1000 A-scans per B-scan, 25 B-scans per volume, 8 volumes per second). The details of acquisition and stimulation protocols were listed in Supplementary Table 3. c Time-elapsed intensity (upper) and phase (lower) M-scans without correction, when no light stimulus was delivered to the retina. Scale bar: 500 ms (horizontal), 100 µm (vertical). d The subpixel-level bulk motion estimated by locating the peak of the upsampled cross-correlation map between repeated B-scans. Scale bar: 500 ms (horizontal), 2 µm (vertical). e Corresponding time-elapsed intensity (upper) and phase (lower) M-scans after the phase-restoring subpixel motion correction. Scale bar: 500 ms (horizontal), 100 µm (vertical). Gray and pink arrows in (c) and (e) label loci of the outer retina and the choroid, respectively. Source data of Fig. 7d are provided as a Source Data file.