Purpose:To develop implementable confocal multispectral imaging (MSI) by adapting data capture and developing novel analysis on a commercially available device. Using an aging mouse model as proof-of-principle confocal MSI was compared against current reference-standard flood-illuminated hyperspectral imaging (HSI). Methods:Mice aged three, five, and eight months old (n = 14-19/group) underwent confocal scanning laser ophthalmoscopy retinal imaging (MultiColor module on Spectralis OCT) at three laser wavelengths returning: blue (BR, 486 nm), green (GR, 518 nm), and infrared reflectance (IR, 815 nm). Images were focused in 2D steps over a range of 20D. Average retinal reflectance was calculated for wavelengths across the dioptric range and corrected for chromatic aberration. Flood-illuminated HSI from 320 to 680 nm was also conducted. Results:MSI post-chromatic aberration correction found a significant interaction effect with age and retinal depth in IR, GR and BR (P < 0.05). The reflectance ratio of short/long wavelength shows a significant interaction effect (BR/IR, P < 0.01; average (BR,GR)/IR, P < 0.05) or trend GR/IR, P = 0.13 with advancing age and retinal depth. HSI also found a decrease in reflectance ratio with age (P < 0.0001), consistent with MSI. The effect size (Cohen's d) between HSI and MSI in the middle retina were comparable (P < 0.05). Conclusions:Confocal MSI can be achieved on a common commercial optical coherence tomography (OCT) device with off the shelf add-on features, and after chromatic aberration correction, enables depth-resolved spectral analysis. This offers advances over flood-illuminated HSI, which does not return depth information and requires custom made or dedicated equipment. Both MSI and HSI detect similar age-related retinal reflectivity changes. This development conceptually parallels the transition from two-dimensional fundus photography to three-dimensional OCT, offering potential enhancements for selected wavelengths.

Cells and other microscopic phase objects can be visualized in the living retina, non-invasively, using non-confocal light detection schemes in adaptive optics scanning light ophthalmoscopes (AOSLOs). There is not yet widespread agreement regarding the origin of image contrast, nor the best way to render multichannel images. Here, we present data to support the interpretation that variations in the intensity of non-confocal images approximate a direct linear mapping of the prismatic deflection of the scanned beam. We advance a simple geometric framework in which local 2D image gradients are used to estimate the spherocylindrical refractive power for each element of the tissue. This framework combines all available information from the non-confocal image channels simultaneously, reducing noise and directional bias. We show that image derivatives can be computed with a scalable, separable gradient operator that minimizes directional errors; this further mitigates noise and directional bias as compared with previous filtering approaches. Strategies to render the output of split-detector gradient operations have been recently described for the visualization of immune cells, blood flow, and photoreceptors; our framework encompasses these methods as rendering astigmatic refractive power. In addition to astigmatic power, we advocate the use of the mean spherical equivalent power, which appears to minimize artifacts even for highly directional micro-structures such as immune cell processes. We highlight examples of positive, negative, and astigmatic power that match expectations according to the known refractive indices and geometries of the relevant structures (for example, a blood vessel filled with plasma acts as a negatively powered cylindrical lens). The examples highlight the benefits of the proposed scheme for the visualization of diverse phase objects including rod and cone inner segments, immune cells near the inner limiting membrane, flowing blood cells, the intravascular cell-free layer, and anatomical details of the vessel wall.

Purpose:To characterize red blood cell (RBC) distribution and associated changes in cell size at capillary junctions in the human neural retina. Methods:Cell-resolved blood flow across 60 capillary junctions in the retina of 6 healthy human subjects was measured, using flood-illumination adaptive optics at 200 to 400 fps. Several empirical RBC partitioning models, including a widely used model developed by Pries et al., were compared for predictive accuracy. Results:We provide updated Pries model coefficients to suit human retinal capillaries. We also propose a simpler linear model that predicts RBC flow distribution based solely on the share of blood flow received by a branch, without reference to the main vessel. Cell size analysis revealed that the average RBC volume in branch vessels was 5.5% lower than in the main vessel (P < 0.05). The reduction in cell volume accompanied a 2.3 µm increase in inter-cell spacing (P < 10-4) and a 3.0% decrease in hematocrit (P < 10-4). These findings support the hypothesis of a net transfer of water from RBCs to plasma as they pass through narrower capillary branches, with potential reabsorption by the time cells enter wider collecting capillaries. Conclusions:This study provides the first noninvasive in vivo characterization of RBC partitioning behavior at capillary junctions in the living human retina. The subtle fluid exchange between cells and plasma may play a role in optimizing oxygen delivery to support immediate metabolic needs.

Recent attention has been given to immune cells near the inner limiting membrane (ILM) of the retina due to their potential use as a biomarker or therapeutic target. Non-invasive phase contrast imaging via optical coherence tomography or non-confocal adaptive optics scanning light ophthalmoscopy has allowed investigations into cellular dynamics and morphology. The cells have been variously referred to as microglia, hyalocytes, or ILM macrophages, with no consensus yet reached as to their identity. Here, we employed AOSLO with an improved non-confocal detection scheme to investigate motile immune cells and their relation, if any, to nearby retinal vasculature. Image series were systematically acquired to observe cells over time (30-60 minutes) at different depth planes (9.2 µm separation), in 3 healthy human subjects, within 2°x2° fields between 8° and 14° temporally and 0° and 8° superior from fixation. A total of 77 cell somas and their process tips were labelled, with a further 20 cells tracked with higher frequency to explore moment-to-moment variations. Cell somas were separated from the nearest blood vessel by 39.1 ± 17.3 µm (mean ± standard deviation). The cells occupied an effective monolayer, averaging 25.0 ± 15.7 µm anterior to the nearest vessel and with no cell observed posterior to a vessel. Compared to cell distributions known from histology, our observations suggest that only a subset of resident immune cells is amenable to phase contrast imaging. Monte Carlo simulations were carried out to determine whether immune cells are located differently from random within their monolayer; we found that cells did not co-localize with the nearby vasculature (p > 0.05), but did have a strong tendency to avoid each other (p ∼ 10-12). We note, however, that cell processes (often rapidly extending and contracting) are often long enough to reach the closest vessels, averaging 34.8 ± 11.5 µm in length with a maximum observed extension of 101.9 µm. Finally, soma speeds averaged 0.52 ± 0.49 µm/min when followed every 5 minutes, consistent with previous reports. A sub-analysis with data collected every 12-15 seconds demonstrated an almost eight-fold increase in measured speed (averaging 4.04 ± 1.47 µm/min), reflecting the stop-start nature of cellular motility.

Purpose:To characterize corneal immune cell morphodynamics and nerve features, and define the in vivo immune landscape in older adults with human immunodeficiency virus (HIV) receiving antiretroviral therapy (ART), relative to healthy age-matched adults. Methods:In this cross-sectional study, 16 HIV-positive individuals receiving ART and 15 age-matched controls underwent ocular surface examinations and functional in vivo confocal microscopy (Fun-IVCM). Time-lapsed videos were created to analyze corneal immune cells (T cells, dendritic cells [DCs], macrophages). Subclinical indicators of corneal health (sensory nerve and endothelial cell features), clinical ocular surface findings, and tear cytokines (analyzed using multiplex bead-based immunoassay) were compared between groups. Results:Participants comprised mostly males (HIV 71 ± 5 years; male:female 15:1; controls 67 ± 6 years; 14:1). The HIV-positive group showed less T-cell motility at the corneal whorl relative to the control group (P = 0.01), and region-dependent differences in T-cell speed (P = 0.001) and DC area (P < 0.001). The HIV-positive group showed greater central corneal nerve fiber width (P = 0.004) and larger endothelial cells (P = 0.02). Clinical findings, corneal immune cell densities, and tear cytokine profiles were similar between groups. Conclusions:Among older individuals with well-controlled HIV infection and clinically-normal ocular surface health, this study identifies subclinical group differences in corneal immune cells (potentially indicative of a heightened, pro-inflammatory activation state in the peripheral cornea) and corneal endothelial cell morphology that parallel those in chronic inflammatory disease. This study demonstrates the utility of Fun-IVCM to evaluate subclinical immune cell features in systemic disease, which could inform the future identification of biomarkers in immune-related conditions.

Purpose:To present a first principle-based, high-fidelity computational model for predicting full three-dimensional (3D) and time-resolved retinal microvascular hemodynamics taking into consideration the flow and deformation of individual blood cells. Methods:The computational model is a 3D fluid-structure interaction model based on combined finite volume/finite element/immersed-boundary methods. Three in silico microvascular networks are built from high-resolution in vivo motion contrast images of the superficial capillary plexus in the parafoveal region of the human retina. The maximum tissue area represented in the model is approximately 500 × 500 µm2, and vessel lumen diameters ranged from 5.5 to 25 µm covering capillaries, arterioles, and venules. Blood is modeled as a suspension of individual blood cells, namely, erythrocytes (RBC), leukocytes (WBC), and platelets in plasma. An accurate and detailed biophysical modeling of each blood cell and their flow-induced deformation is considered. A physiological, pulsatile boundary condition corresponding to an average cardiac cycle of 0.9 second is used. Results:Detailed quantitative data and analysis of 3D retinal microvascular hemodynamics are presented, and their relationship to RBC flow dynamics is illustrated. Blood velocity is shown to have temporal oscillations superimposed on the background pulsatile variation, which arise because of the way RBCs partition at vascular junctions, causing repeated clogging and unclogging of vessels. Temporal variations in RBC velocity and hematocrit are anti-correlated in a given vessel, but their time-averaged distributions are positively correlated across the network. Whole blood velocity is 65% to 85% of RBC velocity, with the discrepancy related to the formation of an RBC-free region, adjacent to the vascular endothelium and typically 0.8 to 1.8 µm thick. The 3D velocity and RBC concentration profiles are shown to be oppositely skewed with respect to each other, because of the way that RBCs "hug" the apex of each bifurcation. RBC deformation is predicted to have biphasic behavior with respect to vessel diameter, with minimal cell length for vessels approximately 7 µm in diameter. The wall shear stress (WSS) exhibits a strongly 3D distribution with local regions of high value and gradient spanning a range of 10 to 80 dyn/cm2. WSS is highest where there is faster flow, greater curvature of the vessel wall, capillary bifurcations, and at locations of RBC crowding and associated thinning of the cell-free layer. Conclusions:This study highlights the usefulness of high-fidelity cell-resolved modeling to obtain accurate and detailed 3D, time-resolved retinal hemodynamic parameters that are not readily available through noninvasive imaging approaches. The results presented are expected to complement and enhance the interpretation of in vivo data, as well as open new avenues to study retinal hemodynamics in health and disease.

Retinal hyperspectral imaging (HSI) is a non-invasive in vivo approach that has shown promise in Alzheimer’s disease. Parkinson’s disease is another neurodegenerative disease where brain pathobiology such as alpha-synuclein and iron overaccumulation have been implicated in the retina. However, it remains unknown whether HSI is altered in in vivo models of Parkinson’s disease, whether it differs from healthy aging, and the mechanisms which drive these changes. To address this, we conducted HSI in two mouse models of Parkinson’s disease across different ages; an alpha-synuclein overaccumulation model (hA53T transgenic line M83, A53T) and an iron deposition model (Tau knock out, TauKO). In comparison to wild-type littermates the A53T and TauKO mice both demonstrated increased reflectivity at short wavelengths ~ 450 to 600 nm. In contrast, healthy aging in three background strains exhibited the opposite effect, a decreased reflectance in the short wavelength spectrum. We also demonstrate that the Parkinson’s hyperspectral signature is similar to that from an Alzheimer’s disease model, 5xFAD mice. Multivariate analyses of HSI were significant when plotted against age. Moreover, when alpha-synuclein, iron or retinal nerve fibre layer thickness were added as a cofactor this improved the R2 values of the correlations in certain groups. This study demonstrates an in vivo hyperspectral signature in Parkinson’s disease that is consistent in two mouse models and is distinct from healthy aging. There is also a suggestion that factors including retinal deposition of alpha-synuclein and iron may play a role in driving the Parkinson’s disease hyperspectral profile and retinal nerve fibre layer thickness in advanced aging. These findings suggest that HSI may be a promising translation tool in Parkinson’s disease.

Purpose: To investigate neurovascular function in eyes with age-related macular degeneration (AMD). Methods: Subjects with bilateral large drusen (intermediate AMD) and healthy controls >= 50 years old were recruited. The vasculature within the central 6 x 6-mm retinal area was captured using optical coherence tomography angiography (OCTA) and segmented to return superficial plexus, deep plexus, choriocapillaris, and choroid. OCTA scans were acquired without flicker light stimulation (conventional OCTA) and during flicker light stimulation to increase retinal activity and metabolic demand (functional OCTA). Vascular area density (VAD) and the vascular reactivity index (VRI; change in VAD induced by flicker stimulation) were determined and compared between control and AMD eyes. Results: Thirty-five subjects (19 AMD cases and 16 healthy controls) participated in the study. In healthy eyes, flicker stimulation induced an increase in VAD (positive VRI, vasodilation) in the superficial plexus (P < 0.001) and deep plexus (P < 0.001). There was a trend for increased VAD in the choriocapillaris (P = 0.077), but there was no change in the choroid (P = 0.654). In AMD eyes, there was no change in VAD in response to flicker stimulation in any of the vascular layers examined (P >= 0.294). Linear mixed models confirmed that AMD was associated with a reduced VRI in the superficial plexus (P < 0.001) and deep plexus (P < 0.001). Conclusions: Eyes with large drusen show a reduction in retinal vascular reactivity compared to healthy eyes, which suggests that there is impairment of retinal neurovascular function in intermediate AMD. Translational Relevance: Functional OCTA could be used to study neurovascular function in retinal diseases.

Two major approaches for tracking cellular motion across a range of biological tissues are the manual labelling of cells, and automated analysis of spatiotemporal information represented in a kymograph. Here we compare these two approaches for the measurement of retinal capillary flow, a particularly noisy application due to the low intrinsic contrast of single red blood cells (erythrocytes). Image data were obtained using a flood-illuminated adaptive optics ophthalmoscope at 750 nm, allowing the acquisition of flow information over several cardiac cycles which provided key information in evaluating tracking accuracy. Our results show that in addition to being much faster, the automated method is more accurate in the face of rapid flow and reduced image contrast. This study represents the first validation of commonly used kymograph approaches to capillary flow analysis.

In vivo confocal microscopy (IVCM) is a widely used technique for imaging the cornea of the eye with a confocal scanning light ophthalmoscope. Cellular resolution and high contrast are achieved without invasive procedures, suiting the study of living humans. However, acquiring useful image data can be challenging due to the incessant motion of the eye, such that images are typically limited by noise and a restricted field of view. These factors affect the degree to which the same cells can be identified and tracked over time. To redress these shortcomings, here we present a data acquisition protocol together with the details of a free, open-source software package written in Matlab. The software package automatically registers and processes IVCM videos to significantly improve contrast, resolution, and field of view. The software also registers scans acquired at progressive time intervals from the same tissue region, producing a time-lapsed video to facilitate visualization and quantification of individual cell dynamics (e.g., motility and dendrite probing). With minimal user intervention, to date, this protocol has been employed to both cross-sectionally and longitudinally assess the dynamics of immune cells in the human corneal epithelium and stroma, using a technique termed functional in vivo confocal microscopy (Fun-IVCM) in 68 eyes from 68 participants. Using the custom software, registration of 'sequence scan' data was successful in 97% of videos acquired from the corneal epithelium and 93% for the corneal stroma. Creation of time-lapsed videos, in which the averages from single videos were registered across time points, was successful in 93% of image series for the epithelium and 75% of image series for the stroma. The reduced success rate for the stroma occurred due to practical difficulties in finding the same tissue between time points, rather than due to errors in image registration. We also present preliminary results showing that the protocol is well suited to in vivo cellular imaging in the retina with adaptive optics scanning laser ophthalmoscopy (AOSLO). Overall, the approach described here substantially improves the efficiency and consistency of time-lapsed video creation to enable non-invasive study of cell dynamics across diverse tissues in the living eye.

PURPOSE. Capillary flow plays an important role in the nourishment and maintenance of healthy neural tissue and can be observed directly and non-invasively in the living human retina. Despite their importance, patterns of normal capillary flow are not well understood due to limitations in spatial and temporal resolution of imaging data. METHODS. Capillary flow characteristics were studied in the retina of three healthy young individuals using a high-resolution adaptive optics ophthalmoscope. Imaging with frame rates of 200 to 300 frames per second was sufficient to capture details of the single-file flow of red blood cells in capillaries over the course of about 3 seconds. RESULTS. Erythrocyte velocities were measured from 72 neighboring vessels of the parafoveal capillary network for each subject. We observed strong variability among vessels within a given subject, and even within a given imaged field, across a range of capillary flow parameters including maximum and minimum velocities, pulsatility, abruptness of the systolic peak, and phase of the cardiac cycle. The observed vari-ability was not well explained by "local" factors such as the vessel diameter, tortuosity, length, linear cell density, or hematocrit of the vessel. Within a vessel, a moderate relation between the velocities and hematocrit was noted, suggesting a redistribution of plasma between cells with changes in flow. CONCLUSIONS. These observations advance our fundamental understanding of normal capillary physiology and raise questions regarding the potential role of network-level effects in explaining the observed flow heterogeneity.

To determine whether a typical vision therapy (VT) programme designed to improve visual information processing (VIP) skills is effective in improving these skills and/or academic performance. We used a double-blind, randomised clinical trial to compare VIP VT to placebo training. Participating schools referred a sample of 579 early primary school children identified as being within the lower third of their class for literacy. From the referred sample, we identified 247 children eligible to participate (passed visions and auditory processing screening, and VIP performance <34th percentile), 94 of whom participated. Matching IQ, school grade and sex was achieved by sorting hierarchically on these values and then alternately allocating to VT or placebo groups. Both programmes ran for 10 weeks and consisted of 33 h working at home and 4 h working in office. The VT programme was indicative of that employed in Australian paediatric optometry practices, with the placebo programme containing similar activities, except targeting skills within a child's competencies and with specific VIP development activities removed. The main outcome measures were score change on three standardised educational tests (reading comprehension, spelling and mathematics) and six VIP tests, both immediately post-intervention (PI) and 6 months later. Sixty-nine children completed the programmes. The VT programme produced no significant improvement in the three educational tests or in five of the six VIP tests compared to the control. The VT programme improved visual sequential memory (VSM) by a moderate amount compared to the control (Cohen's d = 0.57 and 0.52, immediately PI and at 6 months, respectively: p < 0.03 and p < 0.02). The VIP and academic performance benefits from a VT programme were largely identical to those from a control programme, both immediately and 6-month PI. Placebo effects and general effects such as improvements in executive function and/or regression-to-the-mean could be mistaken for specific programme effectiveness.

ABSTRACT Clinical relevance The use of chloroquine or hydroxychloroquine can lead to both acute and chronic changes to both retinal structure and function. Background Chloroquine (CQ) and hydroxychloroquine (HCQ) have the potential for retina toxicity. The acute impact of short-term drug exposure (2-4 weeks) on in vivo retinal structure and function and assess whether short wavelength light exposure further exacerbates any structural and functional changes was assessed in a murine model. Methods Adult C57BL/6 J mice received intraperitoneal injection of vehicle or hydroxychloroquine (10 mg/kg) 3 times per week for 2 or 4 weeks, or chloroquine for 4 weeks (10 mg/kg). Over this period, animals were exposed to room light (8 hours) or short-wavelength light 4 hours per day (4 hours of normal room light) for 5 days each week. Retinal changes were assessed using electroretinography (ERG), in vivo optical coherence tomography (OCT) imaging. Results Short-term low-dose HCQ and CQ treatment led to RPE thickening and elongation of photoreceptors. These structural changes were associated with a no dysfunction in the case of HCQ treatments and widespread functional changes (photoreceptor sensitivity, bipolar cell amplitude and oscillatory potential amplitude) in the case of CQ treatment. Exposure to low intensity short-wavelength light does not appear to alter the effect of HCQ or CQ. Conclusions HCQ and CQ treatment has acute effects on both retinal structure and function, effects that were not exacerbated by short wavelength light exposure. Whether chronic short wavelength light exposure exacerbates these changes require further study.