Longitudinal in vivo Ca2+ imaging reveals dynamic activity changes of diseased retinal ganglion cells at the single-cell level.

Retinal ganglion cells (RGCs) are heterogeneous projection neurons that convey distinct visual features from the retina to brain. Here, we present a high-throughput in vivo RGC activity assay in response to light stimulation using noninvasive Ca2+ imaging of thousands of RGCs simultaneously in living mice. Population and single-cell analyses of longitudinal RGC Ca2+ imaging reveal distinct functional responses of RGCs and unprecedented individual RGC activity conversions during traumatic and glaucomatous degeneration. This study establishes a foundation for future in vivo RGC function classifications and longitudinal activity evaluations using more advanced imaging techniques and visual stimuli under normal, disease, and neural repair conditions. These analyses can be performed at both the population and single-cell levels using temporal and spatial information, which will be invaluable for understanding RGC pathophysiology and identifying functional biomarkers for diverse optic neuropathies.

Fundus topographical distribution patterns of ocular toxoplasmosis.

BACKGROUND: To establish topographic maps and determine fundus distribution patterns of ocular toxoplasmosis (OT) lesions. METHODS: In this retrospective study, patients who presented with OT to ophthalmology clinics from four countries (Argentina, Turkey, UK, USA) were included. Size, shape and location of primary (1°)/recurrent (2°) and active/inactive lesions were converted into a two-dimensional retinal chart by a retinal drawing software. A final contour map of the merged image charts was then created using a custom Matlab programme. Descriptive analyses were performed. RESULTS: 984 lesions in 514 eyes of 464 subjects (53% women) were included. Mean area of all 1° and 2° lesions was 5.96±12.26 and 5.21±12.77 mm2, respectively. For the subset group lesions (eyes with both 1° and 2° lesions), 1° lesions were significantly larger than 2° lesions (5.52±6.04 mm2 vs 4.09±8.90 mm2, p=0.038). Mean distances from foveola to 1° and 2° lesion centres were 6336±4267 and 5763±3491 µm, respectively. The majority of lesions were found in temporal quadrant (p<0.001). Maximum overlap of all lesions was at 278 µm inferotemporal to foveola. CONCLUSION: The 1° lesions were larger than 2° lesions. The 2° lesions were not significantly closer to fovea than 1° lesions. Temporal quadrant and macular region were found to be densely affected underlining the vision threatening nature of the disease.

Structural and Functional Changes in Non-Paraneoplastic Autoimmune Retinopathy.

BACKGROUND: To describe longitudinal changes in patients with non-paraneoplastic autoimmune retinopathy (npAIR) by utilizing different diagnostic modalities/tests. METHODS: The index study is a retrospective longitudinal review of sixteen eyes of eight patients from a tertiary care eye hospital diagnosed with npAIR. Multiple diagnostic modalities such as wide-angle fundus photography (WAFP), WA fundus autofluorescence (WAFAF), spectral-domain optical coherence tomography (SD-OCT), Goldmann visual field (GVF) perimetry, microperimetry (MP), electrophysiologic testing, and adaptive optics scanning laser ophthalmoscopy (AOSLO) were reviewed and analyzed. RESULTS: At the baseline visits, anomalies were detected by multimodal diagnostic tests on all patients. Subjects were followed up for a median duration of 11.5 [3.0-18.7] months. Structural changes at the baseline were detected in 14 of 16 (87.5%) eyes on WAFP and WAFAF and 13 of 16 (81.2%) eyes on SD-OCT. Eight of the ten (80%) eyes that underwent AOSLO imaging depicted structural changes. Functional changes were detected in 14 of 16 (87.5%) eyes on GVF, 15 of 16 (93.7%) eyes on MP, and 11 of 16 (68.7%) eyes on full-field electroretinogram (ff-ERG). Multifocal electroretinogram (mf-ERG) and visual evoked potential (VEP) tests were performed in 14 eyes, of which 12 (85.7%) and 14 (100%) of the eyes demonstrated functional abnormalities, respectively, at baseline. Compared to all the other structural diagnostic tools, AOSLO had a better ability to demonstrate deterioration in retinal microstructures occurring at follow-ups. Functional deterioration at follow-up was detected on GVF in 8 of 10 (80%) eyes, mf-ERG in 4 of 8 (50%) eyes, and MP in 7 of 16 (43.7%) eyes. The ff-ERG and VEP were stable in the majority of cases at follow-up. CONCLUSIONS: The utilization of multimodal imaging/tests in the diagnosing and monitoring of npAIR patients can aid in identifying anomalous changes over time. Analysis of both the anatomical and functional aspects by these devices can be supportive of detecting the changes early in such patients. AOSLO shows promise as it enables the capture of high-resolution images demonstrating quantifiable changes to retinal microstructure.

Distinct Patterns of Choroidal Lesions in Punctate Inner Choroidopathy and Multifocal Choroiditis Determined by Heatmap Analysis.

PURPOSE: A heatmap analysis of choroidal lesions in patients with punctate inner choroidopathy (PIC) or multifocal choroiditis (MFC) with or without uveitis was performed to determine if there were any distinguishing features among these uveitic entities. METHODS: Retrospective review of medical records was conducted at the Byers Eye Institute, Stanford. Fundus photographs were masked and placed on a standardized template. Lesions were identified and heatmaps were generated in a standardized fashion. RESULTS: 30 eyes were identified with PIC or MFC. Heatmap analysis revealed three distinct patterns of fundus lesions: posterior, peripheral, and combined. All patients with PIC had the posterior pattern. Patients with MFC had the peripheral or combined pattern, and all patients with MFC with uveitis had the combined pattern. CONCLUSION: Three patterns of fundus lesions were identified in patients with PIC and MFC. PIC and MFC may represent two separate disease entities with distinct phenotypes of choroidal lesions.

Reflectance adaptive optics findings in a patient with Vogt-Koyanagi-Harada disease.

Purpose: To describe the reflectance adaptive optics scanning laser ophthalmoscopy (AOSLO) findings in different stages of Vogt-Koyanagi-Harada (VKH) disease and correlate them to visual gain post treatment. Confocal (cAOSLO) and non-confocal split-detector AOSLO (sdAOSLO) were used to assess longitudinally the status of the photoreceptors in a patient with VKH managed on corticosteroid and immunomodulatory therapy. Observation: A 32-year-old Japanese American female presented with a 2-week history of blurred vision in both eyes (OU) and worsening headache previously diagnosed as a case of VKH and treated with high dose oral prednisone. At the time of presentation, though vision was improving, and frank serous retinal detachments were absent, spectral domain optical coherence tomography (SD-OCT) showed presence of residual subretinal fluid with disruption of the photoreceptor inner segments and outer segments (IS/OS) involving OU. The photoreceptor mosaic at the foveal center appeared very sparse with large areas devoid of visible photoreceptors on cAOSLO, in agreement with the SD-OCT data. sdAOSLO imaging over the same location shows a higher number of contiguous photoreceptors. After imaging, the patient was started on mycophenolate mofetil as steroid-sparing long-term therapy. Three months later, visual acuity improved to 20/20 OU, and SD-OCT showed almost complete resolution of subretinal fluid with significant improvement of the IS/OS SD-OCT signal, OU. cAOSLO imaging revealed a contiguous photoreceptor mosaic without gaps and of normal appearance. Conclusions and Importance: VKH patients may demonstrate transient photoreceptor abnormalities on SD-OCT and cAOSLO imaging. sdAOSLO imaging revealed intact photoreceptor segments in areas that appeared as voids on cAOSLO, which later showed structural recovery on SD-OCT and cAOSLO. Therefore, sdAOSLO may predict potential for improvement in patients wherein there appears to be photoreceptor loss in cAOSLO and/or SD-OCT.

Imaging of vitreous cortex hyalocyte dynamics using non-confocal quadrant-detection adaptive optics scanning light ophthalmoscopy in human subjects.

Vitreous cortex hyalocytes are resident macrophage cells that help maintain the transparency of the media, provide immunosurveillance, and respond to tissue injury and inflammation. In this study, we demonstrate the use of non-confocal quadrant-detection adaptive optics scanning light ophthalmoscopy (AOSLO) to non-invasively visualize the movement and morphological changes of the hyalocyte cell bodies and processes over 1-2 hour periods in the living human eye. The average velocity of the cells 0.52 ± 0.76 µm/min when sampled every 5 minutes and 0.23 ± 0.29 µm/min when sampled every 30 minutes, suggesting that the hyalocytes move in quick bursts. Understanding the behavior of these cells under normal physiological conditions may lead to their use as biomarkers or suitable targets for therapy in eye diseases such as diabetic retinopathy, preretinal fibrosis and glaucoma.

Insights into Sickle Cell Disease through the Retinal Microvasculature: Adaptive Optics Scanning Light Ophthalmoscopy Correlates of Clinical OCT Angiography.

Purpose: Clinical OCT angiography (OCTA) of the retinal microvasculature offers a quantitative correlate to systemic disease burden and treatment efficacy in sickle cell disease (SCD). The purpose of this study was to use the higher resolution of adaptive optics scanning light ophthalmoscopy (AOSLO) to elucidate OCTA features of parafoveal microvascular compromise identified in SCD patients. Design: Case series of 11 SCD patients and 1 unaffected control. Participants: A total of 11 eyes of 11 SCD patients (mean age, 33 years; range, 23-44; 8 female, 3 male) and 1 eye of a 34-year-old unaffected control. Methods: Ten sequential 3 × 3 mm parafoveal OCTA full vascular slab scans were obtained per eye using a commercial spectral domain OCT system (Avanti RTVue-XR; Optovue). These were used to identify areas of compromised perfusion near the foveal avascular zone (FAZ), designated as regions of interest (ROIs). Immediately thereafter, AOSLO imaging was performed on these ROIs to examine the cellular details of abnormal perfusion. Each participant was imaged at a single cross-sectional time point. Additionally, 2 of the SCD patients were imaged prospectively 2 months after initial imaging to study compromised capillary segments across time and with treatment. Main Outcome Measures: Detection and characterization of parafoveal perfusion abnormalities identified using OCTA and resolved using AOSLO imaging. Results: We found evidence of abnormal blood flow on OCTA and AOSLO imaging among all 11 SCD patients with diverse systemic and ocular histories. Adaptive optics scanning light ophthalmoscopy imaging revealed a spectrum of phenomena, including capillaries with intermittent blood flow, blood cell stasis, and sites of thrombus formation. Adaptive optics scanning light ophthalmoscopy imaging was able to resolve single sickled red blood cells, rouleaux formations, and blood cell-vessel wall interactions. OCT angiography and AOSLO imaging were sensitive enough to document improved retinal perfusion in an SCD patient 2 months after initiation of oral hydroxyurea therapy. Conclusions: Adaptive optics scanning light ophthalmoscopy imaging was able to reveal the cellular details of perfusion abnormalities detected using clinical OCTA. The synergy between these clinical and laboratory imaging modalities presents a promising avenue in the management of SCD through the development of noninvasive ocular biomarkers to prognosticate progression and measure the response to systemic treatment.

Wavefront sensorless adaptive optics ophthalmoscopy in the human eye.

Wavefront sensor noise and fidelity place a fundamental limit on achievable image quality in current adaptive optics ophthalmoscopes. Additionally, the wavefront sensor 'beacon' can interfere with visual experiments. We demonstrate real-time (25 Hz), wavefront sensorless adaptive optics imaging in the living human eye with image quality rivaling that of wavefront sensor based control in the same system. A stochastic parallel gradient descent algorithm directly optimized the mean intensity in retinal image frames acquired with a confocal adaptive optics scanning laser ophthalmoscope (AOSLO). When imaging through natural, undilated pupils, both control methods resulted in comparable mean image intensities. However, when imaging through dilated pupils, image intensity was generally higher following wavefront sensor-based control. Despite the typically reduced intensity, image contrast was higher, on average, with sensorless control. Wavefront sensorless control is a viable option for imaging the living human eye and future refinements of this technique may result in even greater optical gains.

Comparison of confocal and non-confocal split-detection cone photoreceptor imaging.

Quadrant reflectance confocal and non-confocal scanning light ophthalmoscope images of the photoreceptor mosaic were recorded in a subject with congenital achromatopsia (ACHM) and a normal control. These images, captured with various circular and annular apertures, were used to calculate split-detection images, revealing two cone photoreceptor contrast mechanisms. The first contrast mechanism, maximal in the non-confocal 5.5-10 Airy disk diameter annular region, is unrelated to the cone reflectivity in confocal or flood illumination imaging. The second mechanism, maximal for confocal split-detection, is related to the cone reflectivity in confocal or flood illumination imaging that originates from the ellipsoid zone and/or inner-outer segment junction. Seeking to maximize image contrast, split-detection images were generated using various quadrant detector combinations, with opposite (diagonal) quadrant detectors producing the highest contrast. Split-detection generated with the addition of adjacent quadrant detector pairs, shows lower contrast, while azimuthal split-detection images, calculated from adjacent quadrant detectors, showed the lowest contrast. Finally, the integration of image pairs with orthogonal split directions was used to produce images in which the photoreceptor contrast does not change with direction.

Novel Foveal Features Associated With Vision Impairment in Multiple Sclerosis.

Purpose: To characterize scattering and hyperreflective features in the foveal avascular zone of people with multiple sclerosis (MS) using adaptive optics scanning laser ophthalmoscopy (AOSLO) and to evaluate their relationship with visual function and MS disease characteristics. Methods: Twenty subjects with MS underwent confocal reflectance and non-confocal split-detection AOSLO foveal imaging. Peripapillary retinal nerve fiber layer thickness was measured using optic nerve optical coherence tomography. Blood pressure, intraocular pressure (IOP), and best-corrected high-contrast visual acuity (HCVA) and low-contrast visual acuity (LCVA) were measured. AOSLO images were graded to determine the presence and characteristics of distinct structures. Results: Two distinct structures were seen in the avascular zone of the foveal pit. Hyperreflective puncta, present in 74% of eyes, were associated with IOP and blood pressure. Scattering features, observed in 58% of eyes, were associated with decreased HCVA and LCVA, as well as increased MS duration and disability, but were not associated with retinal nerve fiber layer thickness. Hyperreflective puncta and scattering features were simultaneously present in 53% of eyes. Conclusions: Hyperreflective puncta were associated with parameters affecting ophthalmic perfusion, but they were not associated with MS disease parameters. Scattering features were associated with parameters corresponding to advanced MS, suggesting that they may be related to disease progression. Scattering features were also correlated with reduced visual function independent from ganglion cell injury, suggesting the possibility of a novel ganglion cell-independent mechanism of impaired vision in people with MS.

A correction algorithm to simultaneously control dual deformable mirrors in a woofer-tweeter adaptive optics system.

We present a direct slope-based correction algorithm to simultaneously control two deformable mirrors (DMs) in a woofer-tweeter adaptive optics system. A global response matrix was derived from the response matrices of each deformable mirror and the voltages for both deformable mirrors were calculated simultaneously. This control algorithm was tested and compared with a 2-step sequential control method in five normal human eyes using an adaptive optics scanning laser ophthalmoscope. The mean residual total root-mean-square (RMS) wavefront errors across subjects after adaptive optics (AO) correction were 0.128 +/- 0.025 microm and 0.107 +/- 0.033 microm for simultaneous and 2-step control, respectively (7.75-mm pupil). The mean intensity of reflectance images acquired after AO convergence was slightly higher for 2-step control. Radially-averaged power spectra calculated from registered reflectance images were nearly identical for all subjects using simultaneous or 2-step control. The correction performance of our new simultaneous dual DM control algorithm is comparable to 2-step control, but is more efficient. This method can be applied to any woofer-tweeter AO system.

Sub-Airy Confocal Adaptive Optics Scanning Ophthalmoscopy.

PURPOSE: To demonstrate the viability of improving transverse image resolution in reflectance scanning adaptive optics ophthalmoscopy using sub-Airy disk confocal detection. METHODS: The foveal cone mosaic was imaged in five human subjects free of known eye disease using two custom adaptive optics scanning light ophthalmoscopes (AOSLOs) in reflectance with 7.75 and 4.30 mm pupil diameters. Confocal pinholes of 0.5, 0.6, 0.8, and 1.0 Airy disk diameters (ADDs) were used in a retinal conjugate plane before the light detector. Average cone photoreceptor intensity profile width and power spectrum were calculated for the resulting images. Detected energy using a model eye was recorded for each pinhole size. RESULTS: The cone photoreceptor mosaic is better resolved with decreasing confocal pinhole size, with the high spatial frequency content of the images enhanced in both the large- and small-pupil AOSLOs. The average cone intensity profile width was reduced by ∼15% with the use of a 0.5 ADD pinhole when compared to a 1.0 ADD, with an accompanying reduction in signal greater than a factor of four. CONCLUSIONS: The use of sub-Airy disk confocal pinhole detection without increasing retinal light exposure results in a substantial improvement in image resolution at the cost of larger than predicted signal reduction. TRANSLATIONAL RELEVANCE: Improvement in transverse resolution using sub-Airy disk confocal detection is a practical and low-cost approach that is applicable to all point- and line-scanning ophthalmoscopes, including optical coherence tomographers.

In Vivo Changes in Lamina Cribrosa Microarchitecture and Optic Nerve Head Structure in Early Experimental Glaucoma.

The lamina cribrosa likely plays an important role in retinal ganglion cell axon injury in glaucoma. We sought to (1) better understand optic nerve head (ONH) structure and anterior lamina cribrosa surface (ALCS) microarchitecture between fellow eyes of living, normal non-human primates and (2) characterize the time-course of in vivo structural changes in the ONH, ALCS microarchitecture, and retinal nerve fiber layer thickness (RNFLT) in non-human primate eyes with early experimental glaucoma (EG). Spectral domain optical coherence tomography (SDOCT) images of the ONH were acquired cross-sectionally in six bilaterally normal rhesus monkeys, and before and approximately every two weeks after inducing unilateral EG in seven rhesus monkeys. ONH parameters and RNFLT were quantified from segmented SDOCT images. Mean ALCS pore area, elongation and nearest neighbor distance (NND) were quantified globally, in sectors and regionally from adaptive optics scanning laser ophthalmoscope images. In bilaterally normal monkeys, ONH parameters were similar between fellow eyes with few inter-eye differences in ALCS pore parameters. In EG monkeys, an increase in mean ALCS Depth (ALCSD) was the first structural change measured in 6 of 7 EG eyes. A decrease in mean minimum rim width (MRW) simultaneously accompanied this early change in 4 of 6 EG eyes and was the first structural change in the 7th EG eye. Mean ALCS pore parameters were among the first or second changes measured in 4 EG eyes. Mean ALCS pore area and NND increased in superotemporal and temporal sectors and in central and peripheral regions at the first time-point of change in ALCS pore geometry. RNFLT and/or mean ALCS radius of curvature were typically the last parameters to initially change. Survival analyses found mean ALCSD was the only parameter to significantly show an initial change prior to the first measured loss in RNFLT across EG eyes.

3D modeling to characterize lamina cribrosa surface and pore geometries using in vivo images from normal and glaucomatous eyes.

En face adaptive optics scanning laser ophthalmoscope (AOSLO) images of the anterior lamina cribrosa surface (ALCS) represent a 2D projected view of a 3D laminar surface. Using spectral domain optical coherence tomography images acquired in living monkey eyes, a thin plate spline was used to model the ALCS in 3D. The 2D AOSLO images were registered and projected onto the 3D surface that was then tessellated into a triangular mesh to characterize differences in pore geometry between 2D and 3D images. Following 3D transformation of the anterior laminar surface in 11 normal eyes, mean pore area increased by 5.1 ± 2.0% with a minimal change in pore elongation (mean change = 0.0 ± 0.2%). These small changes were due to the relatively flat laminar surfaces inherent in normal eyes (mean radius of curvature = 3.0 ± 0.5 mm). The mean increase in pore area was larger following 3D transformation in 4 glaucomatous eyes (16.2 ± 6.0%) due to their more steeply curved laminar surfaces (mean radius of curvature = 1.3 ± 0.1 mm), while the change in pore elongation was comparable to that in normal eyes (-0.2 ± 2.0%). This 3D transformation and tessellation method can be used to better characterize and track 3D changes in laminar pore and surface geometries in glaucoma.

Reproducibility of measuring lamina cribrosa pore geometry in human and nonhuman primates with in vivo adaptive optics imaging.

PURPOSE: The ability to consistently resolve lamina cribrosa pores in vivo has applications in the study of optic nerve head and retinal disease mechanisms. Repeatability was assessed in imaging laminar pores in normal living eyes with a confocal adaptive optics scanning laser ophthalmoscope (AOSLO). METHODS: Reflectance images (840 nm) of the anterior lamina cribrosa were acquired using the AOSLO in four or more different sessions in two normal rhesus monkey eyes and three normal human eyes. Laminar pore areas, elongations (ratio of major to minor axes of the best-fit ellipse) and nearest neighbor distances were calculated for each session. Measurement repeatability was assessed across sessions. RESULTS: Pore areas ranged from 90 to 4365 µm(2) in monkeys and 154 to 6637 µm(2) in humans. Mean variabilities in measuring pore area and elongation (i.e., mean of the standard deviation of measurements made across sessions for the same pores) were 50 µm(2) (6.1%) and 0.13 (6.7%), respectively, in monkeys and 113 µm(2) (8.3%) and 0.17 (7.7%), respectively, in humans. Mean variabilities in measuring nearest neighbor distances were 1.93 µm (5.2%) in monkeys and 2.79 µm (4.1%) in humans. There were no statistically significant differences in any pore parameters across sessions (ANOVA, P > 0.05). CONCLUSIONS: The anterior lamina cribrosa was consistently imaged in vivo in normal monkey and human eyes. The small intersession variability in normal pore geometry suggests that AOSLO imaging could be used to measure and track changes in laminar pores in vivo during glaucomatous progression.