Label-Free Imaging of Inflammation at the Level of Single Cells in the Living Human Eye.

Purpose: Putative microglia were recently detected using adaptive optics ophthalmoscopy in healthy eyes. Here we evaluate the use of nonconfocal adaptive optics scanning light ophthalmoscopy (AOSLO) for quantifying the morphology and motility of presumed microglia and other immune cells in eyes with retinal inflammation from uveitis and healthy eyes. Design: Observational exploratory study. Participants: Twelve participants were imaged, including 8 healthy participants and 4 posterior uveitis patients recruited from the clinic of 1 of the authors (M.H.E.). Methods: The Pittsburgh AOSLO imaging system was used with a custom-designed 7-fiber optical fiber bundle for simultaneous confocal and nonconfocal multioffset detection. The inner retina was imaged at several locations at multiple timepoints in healthy participants and uveitis patients to generate time-lapse images. Main Outcome Measures: Microglia and macrophages were manually segmented from nonconfocal AOSLO images, and their morphological characteristics quantified (including soma size, diameter, and circularity). Cell soma motion was quantified across time for periods of up to 30 minutes and their speeds were calculated by measuring their displacement over time. Results: A spectrum of cell morphologies was detected in healthy eyes from circular amoeboid cells to elongated cells with visible processes, resembling activated and ramified microglia, respectively. Average soma diameter was 16.1 ± 0.9 µm. Cell movement was slow in healthy eyes (0.02 µm/sec on average), but macrophage-like cells moved rapidly in some uveitis patients (up to 3 µm/sec). In an eye with infectious uveitis, many macrophage-like cells were detected; during treatment their quantity and motility decreased as vision improved. Conclusions: In vivo adaptive optics ophthalmoscopy offers promise as a potentially powerful tool for detecting and monitoring inflammation and response to treatment at a cellular level in the living eye. Financial Disclosures: Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article.

Comparison between Two Adaptive Optics Methods for Imaging of Individual Retinal Pigmented Epithelial Cells.

The Retinal Pigment Epithelium (RPE) plays a prominent role in diseases such as age-related macular degeneration, but imaging individual RPE cells is challenging due to their high absorption and low autofluorescence emission. The RPE lies beneath the highly reflective photoreceptor layer (PR) and contains absorptive pigments, preventing direct backscattered light detection when the PR layer is intact. Here, we used near-infrared autofluorescence adaptive optics scanning laser ophthalmoscopy (NIRAF AOSLO) and transscleral flood imaging (TFI) in the same healthy eyes to cross-validate these approaches. Both methods revealed a consistent RPE mosaic pattern and appeared to reflect a distribution of fluorophores consistent with findings from histological studies. Interestingly, even in apparently healthy RPE, we observed dynamic changes over months, suggesting ongoing cellular activity or alterations in fluorophore distribution. These findings emphasize the value of NIRAF AOSLO and TFI in understanding RPE morphology and dynamics.

Head stabilization apparatus for high-resolution ophthalmic imaging.

Head movement must be stabilized to enable high-quality data collection from optical instrumentation such as eye trackers and ophthalmic imaging devices. Though critically important for imaging, head stabilization is often an afterthought in the design of advanced ophthalmic imaging systems, and experimental devices often adapt used and/or discarded equipment from clinical devices for this purpose. Alternatively, those seeking the most stable solution possible, including many users of adaptive optics ophthalmoscopy systems, utilize bite bars. Bite bars can provide excellent stability but are time consuming to fabricate, decreasing imaging efficiency, and uncomfortable for many patients, especially the elderly and/or those with prosthodontics such as dentures who may refuse participation in a study that requires one. No commercial vendors specifically offer head mount solutions for experimental ophthalmic imaging devices, resulting in nearly every custom device having a different solution for this commonly encountered problem. Parallelizing the head stabilization apparatus across different custom devices may improve standardization of experimental imaging systems for clinical trials and other multicenter investigations. Here we introduce a head mount design for ophthalmic imaging that is modular, adjustable, and customizable to the constraints of different experimental imaging configurations. The three points of head contact in our solution provide excellent stabilization across a range of head sizes and shapes from small children to adults, and the ease of adjustment afforded by our design minimizes the time to get participants stabilized and comfortable.

Longitudinal Adaptive Optics Scanning Laser Ophthalmoscopy Reveals Regional Variation in Cone and Rod Photoreceptor Loss in Stargardt Disease.

PURPOSE: To investigate the temporal sequence of changes in the photoreceptor cell mosaic in patients with Stargardt disease type 1 (STGD1), using adaptive optics scanning laser ophthalmoscopy (AOSLO). METHODS: Two brothers with genetically confirmed STGD1 underwent comprehensive eye exams, spectral-domain optical coherence tomography (SD-OCT), fundus auto fluorescence (FAF) and AOSLO imaging 3 times over the course of 28 months. Confocal images of the cones and rods were obtained from the central fovea to 10 degrees inferiorly. Photoreceptors were counted in sampling windows at 100 µm intervals of 200 µm × 200 µm for cones and 50 µm × 50 µm for rods, using custom cell marking software with manual correction. Photoreceptor density and spacing were measured and compared across imaging sessions using one-way ANOVA. RESULTS: AOSLO revealed the younger brother had a 30% decline in foveal cone density after 8 months, followed by complete loss of foveal cones at 28 months; the older brother had no detectable foveal cones at baseline. In the peripheral macula, cone and rod spacings were greater than normal in both patients. The ratio of the cone spacing to rod spacing was greater than normal across all eccentricities, with a greater divergence closer to the foveal center. CONCLUSIONS: Cone cell loss may be an early pathogenetic step in Stargardt disease. AOSLO provides the capability to track individual photoreceptor changes longitudinally in Stargardt disease. SUMMARY STATEMENT: The pathogenetic mechanism of Stargardt disease remains poorly understood. We used high resolution AOSLO to track the progression of the disease and found cone cell loss may be an early pathogenetic step in Stargardt disease.

Temporal Changes in Fixational Eye Movements After Concussion in Adolescents and Adults: Preliminary Findings.

Concussions often involve ocular impairment and symptoms such as convergence insufficiency, accommodative insufficiency, blurred vision, diplopia, eye strain, and pain. Current clinical assessments of ocular function and symptoms rely on subjective symptom reporting and/or involve lengthy administration time. More objective, brief assessments of ocular function following concussion are warranted. The purpose of this study was to evaluate changes in fixational eye movements (FEMs) and their association with clinical outcomes including recovery time, symptoms, cognitive and vestibular/ocular motor impairment. Thirty-three athletes (13-27 years of age; 54.5% female) within 21 days of a diagnosed concussion participated in the study. A tracking scanning laser ophthalmoscope (TSLO) evaluated FEMs metrics during fixation on a center and corner target. Participants completed symptom (Post-Concussion Symptom Scale [PCSS]), cognitive (Immediate Post-concussion Assessment and Cognitive Testing [ImPACT], and Vestibular/Ocular Motor Screening (VOMS) evaluations. All measures were administered at the initial visit and following medical clearance, which was defined as clinical recovery. Changes in FEMs were calculated using paired-samples t tests. Linear regression (LR) models were used to evaluate the association of FEMs with clinical recovery. Pearson product-moment correlations were used to evaluate the associations among FEMs and clinical outcomes. On the center task, changes across time were supported for average microsaccade amplitude (p = 0.005; Cohen's d = 0.53), peak velocity of microsaccades (p = 0.01; d = 0.48), peak acceleration of microsaccades (p = 0.02; d = 0.48), duration of microsaccade (p < 0.001; d = 0.72), and drift vertical (p = 0.017; d = -0.154). The LR model for clinical recovery was significant (R2 = 0.37; p = 0.023) and retained average instantaneous drift amplitude (ß = 0.547) and peak acceleration of microsaccade (ß = 0.414). On the corner task, changes across time were supported for drift proportion (p = 0.03; d = 0.43). The LR model to predict clinical recovery was significant (R2 = 0.85; p = 0.004) and retained average amplitude of microsaccades (ß = 2.66), peak velocity of microsaccades (ß = -15.11), peak acceleration of microsaccades (ß = 12.56), drift horizontal (ß = 7.95), drift vertical (ß = 1.29), drift amplitude (ß = -8.34), drift proportion (ß = 0.584), instantaneous drift direction (ß = -0.26), and instantaneous drift amplitude (ß = 0.819). FEMs metrics were also associated with reports of nausea and performance within the domain of visual memory. The FEMs metric were also associated with PCSS, ImPACT, and VOMS clinical concussion outcomes, with the highest magnitude correlations between average saccade amplitude and VOMS symptoms of nausea and average instantaneous drift speed and ImPACT visual memory, respectively. FEMs metrics changed across time following concussion, were useful in predicting clinical recovery, and were correlated with clinical outcomes. FEMs measurements may provide objective data to augment clinical assessments and inform prognosis following this injury.

Autofluorescent hyperreflective foci on infrared autofluorescence adaptive optics ophthalmoscopy in central serous chorioretinopathy.

Purpose: To test the hypothesis that hyperreflective foci in central serous chorioretinopathy (CSCR) are autofluorescent and may represent macrophages that have engulfed outer retinal fluorophores from the retinal pigment epithelium (RPE) and photoreceptors. Methods: Enrolled subjects underwent spectral domain and swept-source optical coherence tomography, adaptive optics flood-illumination, and adaptive optics scanning laser ophthalmoscopy (AOSLO), including near-infrared autofluorescence (AO-IRAF). For the AO-IRAF imaging, retinal fluorophores were excited using 795 nm light and collected in an emission band from 814 to 850 nm. Results: In 2 of 3 eyes, a hyperautofluorescent signal was detected with an elliptical shape and punctate, granular aspects surrounded by a hypoautofluorescent halo. The size of these structures in the active case was measured to be 17 ± 4 µm in diameter, with at least 45 individual hyperautofluorescent foci identified from the AO-IRAF montage in the active stage of patient 2. In the asymptomatic case there were fewer structures visible (∼10) and their size was smaller (11 ± 4 µm). These hyper-AF foci were colocalized with hyperreflective foci on OCT and visible in simultaneously acquired confocal AOSLO images in active stage. The hyperautofluorescent foci in the patient with active CSCR disappeared coincident with clinical resolution. Conclusion and importance: We show here the first AO-IRAF images from patients with CSCR, demonstrating hyper-autofluorescent punctate foci, colocalized with hyper-reflective foci on confocal AOSLO images and in OCT. The autofluorescence of these foci may be driven by the accumulation of photoreceptor and RPE fluorophores within macrophages during the active stage of the disease.

MULTIMODAL IMAGING OF MULTIFOCAL CHOROIDITIS WITH ADAPTIVE OPTICS OPHTHALMOSCOPY.

PURPOSE: To describe longitudinal, anatomical, and functional alterations caused by inflammatory and neovascular lesions of idiopathic multifocal choroiditis/punctate inner choroidopathy using adaptive optics imaging and microperimetry. METHODS: Longitudinal case study using multiple imaging modalities, including spectral-domain optical coherence tomography, fluorescein angiography, indocyanine green angiography, optical coherence tomography angiography, flood illumination adaptive optics, and microperimetry. RESULTS: A 21-year-old myopic Asian man presented with blurred vision in the right eye. Clinical examination was notable for an isolated hypopigmented, perifoveal lesion in each eye. Multimodal imaging showed inflammatory lesions in the outer retina, retina pigment epithelium, and inner choroid lesions of both eyes. The right eye additionally exhibited active Type-2 macular neovascularization with loss of cone mosaic regularity that was associated with reduced sensitivity on microperimetry. The clinical picture was consistent with multifocal choroiditis/punctate inner choroidopathy. The patient was treated with oral steroids and three injections of intravitreal bevacizumab in the right eye. After therapy, imaging showed reestablishment of the cone mosaic on flood illumination adaptive optics and improvement in sensitivity on microperimetry. CONCLUSION: Adaptive optics imaging and microperimetry may detect biomarkers that help to characterize the nature and activity of multifocal choroiditis lesions and to help monitor response to therapy. With timely intervention, structural abnormalities in the outer retina and choroid can be treated, and anatomical improvements precede improvements in visual function.

Design of a radial multi-offset detection pattern for in vivo phase contrast imaging of the inner retina in humans.

Previous work has shown that multi-offset detection in adaptive optics scanning laser ophthalmoscopy (AOSLO) can be used to image transparent cells such as retinal ganglion cells (RGCs) in monkeys and humans. Though imaging in anesthetized monkeys with high light levels produced high contrast images of RGCs, images from humans failed to reach the same contrast due to several drawbacks in the previous dual-wavelength multi-offset approach. Our aim here was to design and build a multi-offset detection pattern for humans at safe light levels that could reveal transparent cells in the retinal ganglion cell layer with a contrast and acquisition time approaching results only previously obtained in monkeys. Here, we present a new single-wavelength solution that allows for increased light power and eliminates problematic chromatic aberrations. Then, we demonstrate that a radial multi-offset detection pattern with an offset distance of 8-10 Airy Disk Diameter (ADD) is optimal to detect photons multiply scattered in all directions from weakly reflective retinal cells thereby enhancing their contrast. This new setup and image processing pipeline led to improved imaging of inner retinal cells, including the first images of microglia with multi-offset imaging in AOSLO.

Near infrared autofluorescence imaging of retinal pigmented epithelial cells using 663 nm excitation.

PURPOSE: Fundus autofluorescence (AF) using adaptive optics scanning laser ophthalmoscopy (AOSLO) enables morphometric analysis of individual retinal pigmented epithelial (RPE) cells. However, only a few excitation wavelengths in the visible and near-infrared have been evaluated. Visible light excitation (<600 nm) presents additional safety hazards and is uncomfortable for patients. Near-infrared excitation (>700 nm) overcomes those problems but introduces others, including decreased AF signal and cone signatures that obscure RPE structure. Here we investigated the use of an intermediate wavelength, 663 nm, for excitation and compared it to 795 nm. METHODS: Subjects were imaged using AOSLO equipped with a detection channel to collect AF emission between 814 and 850 nm. Two light sources (663 and 795 nm) were used to excite the retinal fluorophores. We recorded 90 s videos and registered them with custom software to integrate AF images for analysis. RESULTS: We imaged healthy eyes and an eye with pattern dystrophy. Similar AF microstructures were detected with each excitation source, despite ~4 times lower excitation power with 663 nm. The signal-to-noise values showed no meaningful difference between 663 nm and 795 nm excitation and a similar trend was observed for image contrast between the two excitation wavelengths. CONCLUSIONS: Lower light levels can be used with shorter wavelength excitation to achieve comparable images of the microstructure of the RPE as have been obtained using higher light levels at longer wavelengths. Further experiments are needed to fully characterize AF across spectrum and determine the optimal excitation and emission bandwidths that balance efficiency, patient comfort, and efficacy.

Choriocapillaris: Fundamentals and advancements.

The choriocapillaris is the innermost structure of the choroid that directly nourishes the retinal pigment epithelium and photoreceptors. This article provides an overview of its hemovasculogenesis development to achieve its final architecture as a lobular vasculature, and also summarizes the current histological and molecular knowledge about choriocapillaris and its dysfunction. After describing the existing state-of-the-art tools to image the choriocapillaris, we report the findings in the choriocapillaris encountered in the most frequent retinochoroidal diseases including vascular diseases, inflammatory diseases, myopia, pachychoroid disease spectrum disorders, and glaucoma. The final section focuses on the development of imaging technology to optimize visualization of the choriocapillaris as well as current treatments of retinochoroidal disorders that specifically target the choriocapillaris. We conclude the article with pertinent unanswered questions and future directions in research for the choriocapillaris.

A New Method for Visualizing Drusen and Their Progression in Flood-Illumination Adaptive Optics Ophthalmoscopy.

Purpose: Drusen are dynamic sub-RPE deposits that are risk factors for late-stage age-related macular degeneration (AMD). Here we show a new imaging method using flood-illumination adaptive optics (FIAO) that reveal drusen with high contrast and resolution. Methods: A fovea-centered 4° × 4° FIAO image and eight surrounding images with gaze displaced by ±2° vertically and horizontally were acquired. Clinical color fundus and spectral-domain optical coherence tomography were acquired for clinical grading and comparison. Custom software registered overlapping FIAO images and fused the data statistically to generate a fovea-centered 4° × 4° gaze-dependent image. Our dataset included 15 controls (aged 31-72) and 182 eyes from 104 AMD patients (aged 56-92), graded as either normal aging (n = 7), and early (n = 12), intermediate (n = 108) and late AMD (n = 42); 27 had subretinal drusenoid deposits (SDDs), and 83 were imaged longitudinally. Results: No gaze varying structures were detected in young eyes. In aging eyes with no evidence of age-related changes, putative drusen <20 µm in diameter were visible. Gaze-dependent images revealed more drusen and many smaller drusen than visible in color fundus images. Longitudinal images showed expansion and fusion of drusen. SDDs were lower contrast, and RPE atrophy did not yield a consistent signal. Conclusions: Gaze-dependent imaging in a commercially available FIAO fundus camera combined with image registration and postprocessing permits visualization of drusen and their progression with high contrast and resolution. Translational Relevance: This new technique offers promise as a robust and sensitive method to detect, map, quantify, and monitor the dynamics of drusen in aging and AMD.

Fixational eye movements following concussion.

The purpose of this study was to evaluate fixational eye movements (FEMs) with high spatial and temporal resolution following concussion, where oculomotor symptoms and impairments are common. Concussion diagnosis was determined using current consensus guidelines. A retinal eye-tracking device, the tracking scanning laser ophthalmoscope (TSLO), was used to measure FEMs in adolescents and young adults following a concussion and in an unaffected control population. FEMs were quantified in two fixational paradigms: (1) when fixating on the center, or (2) when fixating on the corner of the TSLO imaging raster. Fixational saccade amplitude in recent concussion patients (≤ 21 days) was significantly greater, on average, in the concussion group (mean = 1.03°; SD = 0.36°) compared with the controls (mean = 0.82°; SD = 0.31°), when fixating on the center of the imaging raster (t = 2.87, df = 82, p = 0.005). These fixational saccades followed the main sequence and therefore also had greater peak velocity (t = 2.86, df = 82, p = 0.006) and peak acceleration (t = 2.80, df = 82, p = 0.006). These metrics significantly differentiated concussed from controls (AUC = 0.67-0.68, minimum p = 0.005). No group differences were seen for the drift metrics in either task or for any of the FEMs metrics in the corner-of-raster fixation task. Fixational saccade amplitudes were significantly different in the concussion group, but only when fixating on the center of the raster. This task specificity suggests that task optimization may improve differentiation and warrants further study. FEMs measured in the acute-to-subacute period of concussion recovery may provide a quick (<3 minutes), objective, sensitive, and accurate ocular dysfunction assessment. Future work should assess the impact of age, mechanism of injury, and post-concussion recovery on FEM alterations following concussion.

Multimodal Imaging of Torpedo Maculopathy With Fluorescence Adaptive Optics Imaging of Individual Retinal Pigmented Epithelial Cells.

Torpedo maculopathy (TM) is a rare congenital defect of the retinal pigment epithelium (RPE). The RPE is often evaluated clinically using fundus autofluorescence (AF), a technique that visualizes RPE structure at the tissue level from the intrinsic AF of RPE fluorophores. TM lesions typically emit little or no AF, but this macroscopic assessment is unable to resolve the RPE cells, leaving the organization of the RPE cell mosaic in TM unknown. We used fluorescence adaptive optics scanning laser ophthalmoscopy (AOSLO) to show here for the first time the microscopic cellular-level structural alterations to the RPE cell mosaic in TM that underlie the tissue-level changes seen in conventional clinical imaging. We evaluated two patients with TM using conventional clinical imaging techniques and adaptive optics (AO) infrared autofluorescence (IRAF) in AOSLO. Confocal AOSLO revealed relatively normal cones outside the TM lesion but altered cone appearance within it and along its margins in both patients. We quantified cone topography and RPE cell morphometry from the fovea to the margin of the lesion in case 1 and found cone density to be within the normal range across the locations imaged. However, RPE morphometric analysis revealed disrupted RPE cells outside the margin of the lesion; the mean RPE cell area was greater than two standard deviations above the normative range up to approximately 1.5 mm from the lesion margin. Similar morphometric changes were seen to individual RPE cells in case 2. Multi-modal imaging with AOSLO reveals that RPE cells are abnormal in TM well beyond the margins of the characteristic TM lesion boundary defined with conventional clinical imaging. Since the TM fovea appears to be fully formed, with normal cone packing, it is possible that the congenital RPE defect in TM occurs relatively late in retinal development. This work demonstrates how cellular level imaging of the RPE can provide new insight into RPE pathologies, particularly for rare conditions such as TM.

Strip-based digital image registration for distortion minimization and robust eye motion measurement from scanned ophthalmic imaging systems.

Retinal image-based eye motion measurement from scanned ophthalmic imaging systems, such as scanning laser ophthalmoscopy, has allowed for precise real-time eye tracking at sub-micron resolution. However, the constraints of real-time tracking result in a high error tolerance that is detrimental for some eye motion measurement and imaging applications. We show here that eye motion can be extracted from image sequences when these constraints are lifted, and all data is available at the time of registration. Our approach identifies and discards distorted frames, detects coarse motion to generate a synthetic reference frame and then uses it for fine scale motion tracking with improved sensitivity over a larger area. We demonstrate its application here to tracking scanning laser ophthalmoscopy (TSLO) and adaptive optics scanning light ophthalmoscopy (AOSLO), and show that it can successfully capture most of the eye motion across each image sequence, leaving only between 0.1-3.4% of non-blink frames untracked, while simultaneously minimizing image distortions induced from eye motion. These improvements will facilitate precise measurement of fixational eye movements (FEMs) in TSLO and longitudinal tracking of individual cells in AOSLO.

Spatial-frequency-based image reconstruction to improve image contrast in multi-offset adaptive optics ophthalmoscopy.

Off-axis detection methods in adaptive optics (AO) ophthalmoscopy can enhance image contrast of translucent retinal structures such as cone inner segments and retinal ganglion cells. Here, we propose a 2D optical model showing that the phase contrast produced by these methods depends on the offset orientation. While one axis provides an asymmetric light distribution, hence high phase contrast, the perpendicular axis provides a symmetric one, thus substantially lower contrast. We support this model with in vivo human data acquired with a multi-offset AO scanning light ophthalmoscope. Then, using this finding, we provide a post-processing method, named spatial-frequency-based image reconstruction, to optimally combine images from different off-axis detector orientations, significantly increasing the structural cellular contrast of in vivo human retinal neurons such as cone inner segment, putative rods, and retinal ganglion cells.

Reduced foveal cone density in early idiopathic macular telangiectasia.

OBJECTIVE: Idiopathic macular telangiectasia (MacTel) is considered primarily a vascular disease affecting juxtafoveal retinal capillaries. However, recent evidence suggests that neuronal changes may occur early in disease development. We used high-resolution adaptive optics retinal imaging to elucidate the foveal cone photoreceptor changes at a cellular level in patients with MacTel. METHODS AND ANALYSIS: We used adaptive optics scanning light ophthalmoscopy (AOSLO) to evaluate the foveal cone photoreceptors in the less-affected eye of patients with asymmetric MacTel. AOSLO images of cone photoreceptors were obtained in a 4°×4° area centred on the foveola. Individual cone positions were identified within a 2°×2° area centred on the fovea, using semiautomatic cone marking software with manual correction, permitting calculation of a map of cone density. RESULTS: In all participants, one eye was affected with MacTel, the fellow eye was clinically normal or near normal, with visual acuity of 20/25 or better and subtle angiographic leakage. The foveal cone mosaics were continuous with tight packing and cones exhibited normal reflectivity. However, cone density was significantly lower for all participants (mean=80 733 cones/mm2) within 0.5° than the cone density previously reported for normal eyes. CONCLUSIONS: Foveal cone density is lower than normal in the clinically less-affected eyes of patients with asymmetric MacTel. This suggests that cone photoreceptor loss may precede classic obvious vascular changes in idiopathic MacTel.

Laplacian feature detection and feature alignment for multimodal ophthalmic image registration using phase correlation and Hessian affine feature space.

Advances in multimodal imaging have revolutionized diagnostic and treatment monitoring in ophthalmic practice. In multimodal ophthalmic imaging, geometric deformations are inevitable and they contain inherent deformations arising from heterogeneity in the optical characteristics of imaging devices and patient related factors. The registration of ophthalmic images under such conditions is challenging. We propose a novel technique that overcomes these challenges, using Laplacian feature, Hessian affine feature space and phase correlation, to register blue autofluorescence, near-infrared reflectance and color fundus photographs of the ocular posterior pole with high accuracy. Our validation analysis - that used current feature detection and extraction techniques (speed-up robust features (SURF), a concept of wind approach (KAZE), and fast retina keypoint (FREAK)), and quantitative measures (Sørensen-Dice coefficient, Jaccard index, and Kullback-Leibler divergence scores) - showed that our approach has significant merit in registering multimodal images when compared with a mix-and-match SURF-KAZE-FREAK benchmark approach. Similarly, our evaluation analysis that used a state-of-the-art qualitative measure - the mean registration error (MRE) - showed that the proposed approach is significantly better than the mix-and-match SURF-KAZE-FREAK benchmark approach, as well as a cutting edge image registration technique - Linear Stack Alignment with SIFT (scale-invariant feature transform) - in registering multimodal ophthalmic images.

Microstructure of the retinal pigment epithelium near-infrared autofluorescence in healthy young eyes and in patients with AMD.

Retinal pigmented epithelial (RPE) cells are essential for maintaining normal visual function, especially in their role in the visual cycle, and are thought to be one of the first cell classes affected by age-related macular degeneration (AMD). Clinical imaging systems routinely evaluate the structure of the RPE at the tissue level, but cellular level information may provide valuable RPE biomarkers of health, aging and disease. In this exploratory study, participants were imaged with 795 nm excitation in adaptive optics scanning laser ophthalmoscopy (AOSLO) to observe the microstructure of the near-infrared autofluorescence (AO-IRAF) from the RPE layer in healthy retinas and patients with AMD. The expected hexagonal mosaic of RPE cells was only sometimes seen in normal eyes, while AMD patients exhibited highly variable patterns of altered AO-IRAF. In some participants, AO-IRAF structure corresponding to cones was observed, as we have demonstrated previously. In some AMD patients, marked alterations in the pattern of AO-IRAF could be seen even in areas where the RPE appeared relatively normal in clinical imaging modalities, such as spectral domain optical coherence tomography (SD-OCT). AO-IRAF imaging using AOSLO offers promise for better detection and understanding of early RPE changes in the course of AMD, potentially before clinical signs appear.

High-Resolution Adaptive Optics in Vivo Autofluorescence Imaging in Stargardt Disease.

Importance: Targeting the early pathogenic steps in Stargardt disease type 1 (STGD1) is critical to advance our understanding of this condition and to develop potential therapies. Lipofuscin precursors may accumulate within photoreceptors, leading to photoreceptor damage and preceding retinal pigment epithelial (RPE) cell death. Fluorescence adaptive optics scanning light ophthalmoscopy can provide autofluorescence (AF) images in vivo with microscopic resolution to elucidate the cellular origin of AF abnormalities in STGD1. Objective: To study the spatial distribution of photoreceptor, RPE, and AF abnormalities in patients with STGD1 at a cellular level. Design, Setting, and Participants: Cross-sectional study using fluorescence adaptive optics scanning light ophthalmoscopy to compare the cones, rods, and RPE cells between 3 patients with STGD1 and 1 control individual. Imaging sessions were conducted at the University of Rochester. Further image analyses were performed at Beijing Tongren Eye Center and the University of Pittsburgh. Data were collected from August 2015 to February 2016, and analysis began in March 2016. Main Outcomes and Measures: Structural appearance of cones, rods, and AF structures at different retinal locations. Results: Two women and 1 man with macular atrophy phenotype of STGD1 and visual acuity loss ranging from 20/30 to 20/150 and 1 woman without STGD1 with 20/20 visual acuity were analyzed. Cone and rod spacing was increased in all 3 patients at all locations where photoreceptors were detectable; most cones had a dark appearance. Autofluorescence was low contrast but contained structures consistent with RPE cells in the periphery. In the transition zone peripheral to the foveal atrophic lesion, the structural pattern of AF was more consistent with photoreceptors than RPE cells. The microscopic AF was disrupted within areas of clinically detectable atrophy. Conclusions and Relevance: Adaptive optics high-resolution images of cones, rods, and RPE cells at the leading disease front of STGD1 macular atrophy show an AF pattern that appears to colocalize with photoreceptors or may result from a combination of AF signals from both RPE cells and photoreceptors. This in vivo observation is consistent with histologic reports of fluorescence arising from photoreceptors in STGD1. The detection of bisretinoid accumulation in the photoreceptors may represent an early pathologic step in STGD1 and can provide an in vivo imaging tool to act as a biomarker of disease progression.

Cellular-scale evaluation of induced photoreceptor degeneration in the living primate eye.

Progress is needed in developing animal models of photoreceptor degeneration and evaluating such models with longitudinal, noninvasive techniques. We employ confocal scanning laser ophthalmoscopy, optical coherence tomography (OCT) and high-resolution retinal imaging to noninvasively observe the retina of non-human primates with induced photoreceptor degeneration. Photoreceptors were imaged at the single-cell scale in three modalities of adaptive optics scanning light ophthalmoscopy: traditional confocal reflectance, indicative of waveguiding; a non-confocal offset aperture technique visualizing scattered light; and two-photon excited fluorescence, the time-varying signal of which, at 730 nm excitation, is representative of visual cycle function. Assessment of photoreceptor structure and function using these imaging modalities revealed a reduction in retinoid production in cone photoreceptor outer segments while inner segments appeared to remain present. Histology of one retina confirmed loss of outer segments and the presence of intact inner segments. This unique combination of imaging modalities can provide essential, clinically-relevant information on both the structural integrity and function of photoreceptors to not only validate models of photoreceptor degeneration but potentially evaluate the efficacy of future cell and gene-based therapies for vision restoration.