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.

Efficacy of CRISPR-Based Gene Editing in a Sickle Cell Disease Patient as Measured through the Eye.

Sickle cell disease (SCD) exists on a phenotypic spectrum with variable genetic expressivity, making it difficult to assess an individual patient's risk of complications at any particular point in time. Current and emerging SCD treatments, including CRISPR-based gene editing, result in a variable proportion of affected red blood cells (RBCs) still vulnerable to sickling. Clinical serological indicators of disease such as hemoglobin, indirect bilirubin, and reticulocyte count and clinical metrics including number of emergency department visits and hospitalizations over time often fall short in their ability to objectively quantify ischemic disease activity and efficacy of treatments. Clearly, better clinical biomarkers are needed. The rapidly developing field of oculomics leverages the transparent nature of the ocular tissue to directly study the retinal microvasculature in order to characterize the status of systemic diseases. In this case report, we demonstrate the ability of optical coherence tomography angiography (OCT-A) to detect and measure micro-occlusive events within the retinal capillary bed before and after RBC exchange transfusion and following CRISPR-based gene editing, as an indicator of systemic ischemic disease activity and measure of treatment efficacy. The implications of these findings are discussed.

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.

Quantification of intermittent retinal capillary perfusion in sickle cell disease.

Pathophysiology of sickle cell disease (SCD) features intermittent vaso-occlusion of microcirculatory networks that facilitate ischemic damage. Past research has, however, relied on static images to characterize this active disease state. This study develops imaging metrics to more fully capture dynamic vascular changes, quantifying intermittent retinal capillary perfusion in unaffected controls and SCD patients using sequential optical coherence tomography angiography (OCT-A) scans. The results reveal significant dynamic variation of capillary perfusion in SCD patients compared to controls. This measurement of vaso-occlusive burden in patients would provide utility in monitoring of the disease state and in evaluating treatment efficacy.

Fundus albipunctatus photoreceptor microstructure revealed using adaptive optics scanning light ophthalmoscopy.

PURPOSE: Fundus albipunctatus is an inherited cause of congenital stationary night blindness. The objective of this report is to describe structural changes occurring in a macular phenotype of a novel RDH5 mutation producing fundus albipunctatus using high-resolution in vivo imaging. A 62-year-old male with longstanding night blindness underwent imaging and genetic evaluation. High-resolution images of the photoreceptor mosaic were compared to those of a healthy subject. Results of a comprehensive ophthalmic evaluation and genetic testing with imaging including fundus photography, spectral-domain optical coherence tomography (OCT), fluorescein angiography (FA), OCT angiography (OCT-A), and adaptive optics scanning light ophthalmoscopy (AOSLO) are described. OBSERVATIONS: The patient presented with visual acuity of 20/25 in both eyes and longstanding poor dark adaptation. Anterior segment examination was unremarkable. Fundoscopy revealed well circumscribed bilateral perifoveal mottling and atrophy in both eyes. Discrete white-yellow flecks were present beyond the vascular arcades extending to the far periphery. Genetic testing revealed a novel compound heterozygous RDH5 mutation (c.388C > T, p.Gln130*; c.665T > C, p.Leu222Pro). OCT demonstrated perifoveal photoreceptor and outer retinal irregularities, which corresponded to a window defect with late staining on FA. OCT-A demonstrated normal retinal vasculature with patchy areas of non-perfusion in the choriocapillaris. Macular abnormalities in both eyes were imaged using AOSLO to assess cone and rod photoreceptor architecture. While clinical features are consistent with a primary rod disorder, confocal AOSLO showed a paucity of normal cones with a small spared central island in both eyes. Rods appeared larger and more irregular throughout the macula. Non-confocal split detection AOSLO imaging revealed the presence of cone inner segments in dark regions of confocal imaging, indicating some degree of photoreceptor preservation. CONCLUSIONS AND IMPORTANCE: The AOSLO imaging of this particular macular phenotype of fundus albipunctatus demonstrates some of the structural photoreceptor abnormalities that occur in this condition, adding insight to the variable presentation of RDH5 retinopathy. The presence of preserved inner segment architecture suggests the possibility that gene therapy could play a future role in treating this condition.

Megahertz-rate optical coherence tomography angiography improves the contrast of the choriocapillaris and choroid in human retinal imaging.

Angiographic imaging of the human eye with optical coherence tomography (OCT) is becoming an increasingly important tool in the scientific investigation and clinical management of several blinding diseases, including age-related macular degeneration and diabetic retinopathy. We have observed that OCT angiography (OCTA) of the human choriocapillaris and choroid with a 1.64 MHz A-scan rate swept-source laser yields higher contrast images as compared to a slower rate system operating at 100 kHz. This result is unexpected because signal sensitivity is reduced when acquisition rates are increased, and the incident illumination power is kept constant. The contrast of angiography images generated by acquiring multiple sequential frames and calculating the variation caused by blood flow, however, appears to be improved significantly when lower-contrast images are taken more rapidly. To demonstrate that the acquisition rate plays a role in the quality improvement, we have imaged five healthy subjects with a narrow field of view (1.2 mm) OCTA imaging system using two separate swept-source lasers of different A-line rates and compared the results quantitatively using the radially-averaged power spectrum. The average improvement in the contrast is 23.0% (+/-7.6%). Although the underlying cause of this enhancement is not explicitly determined here, we speculate that the higher-speed system suppresses the noise contribution from eye motion in subjects and operates with an inter-scan time that better discriminates the flow velocities present in the choroid and choriocapillaris. Our result informs OCT system developers on the merits of ultrahigh-speed acquisition in functional imaging applications.

Functional retinal imaging using adaptive optics swept-source OCT at 1.6 MHz.

Objective optical assessment of photoreceptor function may permit earlier diagnosis of retinal disease than current methods such as perimetry, electrophysiology, and clinical imaging. In this work, we describe an adaptive optics (AO) optical coherence tomography (OCT) system designed to measure functional responses of single cones to visible stimuli. The OCT subsystem consisted of a raster-scanning Fourier-domain mode-locked laser that acquires A scans at 1.64 MHz with a center wavelength of 1063 nm and an AO system operating in closed-loop. Analysis of serial volumetric images revealed phase changes of cone photoreceptors consistent with outer segment elongation and proportional to stimulus intensity, as well as other morphological changes in the outer segment and retinal pigment epithelium.

Challenges and advantages in wide-field optical coherence tomography angiography imaging of the human retinal and choroidal vasculature at 1.7-MHz A-scan rate.

We present noninvasive, three-dimensional, depth-resolved imaging of human retinal and choroidal blood circulation with a swept-source optical coherence tomography (OCT) system at 1065-nm center wavelength. Motion contrast OCT imaging was performed with the phase-variance OCT angiography method. A Fourier-domain mode-locked light source was used to enable an imaging rate of 1.7 MHz. We experimentally demonstrate the challenges and advantages of wide-field OCT angiography (OCTA). In the discussion, we consider acquisition time, scanning area, scanning density, and their influence on visualization of selected features of the retinal and choroidal vascular networks. The OCTA imaging was performed with a field of view of 16 deg (5 mm×5 mm) and 30 deg (9 mm×9 mm). Data were presented in en face projections generated from single volumes and in en face projection mosaics generated from up to 4 datasets. OCTA imaging at 1.7 MHz A-scan rate was compared with results obtained from a commercial OCTA instrument and with conventional ophthalmic diagnostic methods: fundus photography, fluorescein, and indocyanine green angiography. Comparison of images obtained from all methods is demonstrated using the same eye of a healthy volunteer. For example, imaging of retinal pathology is presented in three cases of advanced age-related macular degeneration.

The Properties of Outer Retinal Band Three Investigated With Adaptive-Optics Optical Coherence Tomography.

Purpose: Optical coherence tomography's (OCT) third outer retinal band has been attributed to the zone of interdigitation between RPE cells and cone outer segments. The purpose of this paper is to investigate the structure of this band with adaptive optics (AO)-OCT. Methods: Using AO-OCT, images were obtained from two subjects. Axial structure was characterized by measuring band 3 thickness and separation between bands 2 and 3 in segmented cones. Lateral structure was characterized by correlation of band 3 with band 2 and comparison of their power spectra. Band thickness and separation were also measured in a clinical OCT image of one subject. Results: Band 3 thickness ranged from 4.3 to 6.4 µm. Band 2 correlations ranged between 0.35 and 0.41 and power spectra of both bands confirmed peak frequencies that agree with histologic density measurements. In clinical images, band 3 thickness was between 14 and 19 µm. Measurements of AO-OCT of interband distance were lower than our corresponding clinical OCT measurements. Conclusions: Band 3 originates from a structure with axial extent similar to a single surface. Correlation with band 2 suggests an origin within the cone photoreceptor. These two observations indicate that band 3 corresponds predominantly to cone outer segment tips (COST). Conventional OCT may overestimate both the thickness of band 3 and outer segment length.

Comparison of amplitude-decorrelation, speckle-variance and phase-variance OCT angiography methods for imaging the human retina and choroid.

We compared the performance of three OCT angiography (OCTA) methods: speckle variance, amplitude decorrelation and phase variance for imaging of the human retina and choroid. Two averaging methods, split spectrum and volume averaging, were compared to assess the quality of the OCTA vascular images. All data were acquired using a swept-source OCT system at 1040 nm central wavelength, operating at 100,000 A-scans/s. We performed a quantitative comparison using a contrast-to-noise (CNR) metric to assess the capability of the three methods to visualize the choriocapillaris layer. For evaluation of the static tissue noise suppression in OCTA images we proposed to calculate CNR between the photoreceptor/RPE complex and the choriocapillaris layer. Finally, we demonstrated that implementation of intensity-based OCT imaging and OCT angiography methods allows for visualization of retinal and choroidal vascular layers known from anatomic studies in retinal preparations. OCT projection imaging of data flattened to selected retinal layers was implemented to visualize retinal and choroidal vasculature. User guided vessel tracing was applied to segment the retinal vasculature. The results were visualized in a form of a skeletonized 3D model.

Crosstalk rejection in parallel optical coherence tomography using spatially incoherent illumination with partially coherent sources.

The continuing improvement of high-speed area-scan cameras has made possible the construction of parallel optical coherence tomography (OCT) systems that are competitive with the fastest demonstrated swept-source OCT systems. Unfortunately, when imaging through turbid media using a partially coherent source, parallel OCT suffers resolution loss from coherent multiple scattering, a phenomenon known as crosstalk. We demonstrate the use of a full-field OCT system employing multimode fiber in the illumination arm to reduce the spatial coherence of a partially coherent source. By reducing the spatial coherence area below the system's lateral resolution, we create a spatial coherence gate that rejects these multiply scattered photons. We quantify the image quality and resolution improvement of this method by comparing images of a USAF test chart acquired beneath turbid phantoms using both coherent and incoherent illumination and computing the resulting modulation transfer functions. We demonstrate the feasibility of this method for imaging biological specimens by imaging a Drosophila melanogaster sample.