Measuring red blood cell shape in the human retina.

The free diameter of a red blood cell generally exceeds the lumen diameter of capillaries in the central nervous system, requiring significant cellular deformation. However, the deformations undertaken are not well established under natural conditions due to the difficulty in observing corpuscular flow in vivo. Here we describe a novel, to the best of our knowledge, method to noninvasively study the shape of red blood cells as they traverse the narrow capillary networks of the living human retina, using high-speed adaptive optics. One hundred and twenty-three capillary vessels were analyzed in three healthy subjects. For each capillary, image data were motion-compensated and then averaged over time to reveal the appearance of the blood column. Data from hundreds of red blood cells were used to profile the average cell in each vessel. Diverse cellular geometries were observed across lumens ranging from 3.2 to 8.4 µm in diameter. As capillaries narrowed, cells transitioned from rounder to more elongated shapes and from being counter-aligned to aligned with the axis of flow. Remarkably, in many vessels the red blood cells maintained an oblique orientation relative to the axis of flow.

Does vision therapy for visual information processing improve academic performance? A randomised clinical trial.

PURPOSE: 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. METHODS: 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. RESULTS: 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). CONCLUSIONS: 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.

Direct measurement of pulse wave propagation in capillaries of the human retina.

With each contraction of the heart's left ventricle, a pulse pressure wave surges into the aorta and propagates throughout the vascular tree. The pulse wave drives blood flow forward. Its passage is complex, but it passes more quickly through non-compliant, or stiff, vessels, providing an important signpost of cardiovascular disease. The transparent media of the eye allow direct and non-invasive measurement of this phenomenon within the microvasculature of neural tissue. However, previous estimates differ over three orders of magnitude. Here, we used high spatiotemporal resolution adaptive optics imaging to directly track the pulse wave within individual retinal capillaries in three human subjects. Across 74 unique capillary segments, pulse wave velocity averaged 6.4±0.5mm/sec (mean±SEM). There was large variation between vessels; the slowest pulse wave was at most 0.8 mm/sec and the fastest at least 17.6 mm/sec. In 44% of vessels, the pulse wave traveled upstream, in the opposite direction to flow, suggesting wave reflection from downstream collecting junctions.

Recovering the appearance of the capillary blood column from under-sampled flow data.

The regular spacing of cells in capillary flow results in spurious cell trajectories if the sampling rate is too low. This makes it difficult to identify cells, even if the velocity is known. Here, we demonstrate a software method to overcome this problem and validate it using high frame rate data with known velocity, which is downsampled to produce aliasing. The method assumes high spatial sampling, constant velocity over short epochs, and an incompressible blood column. Data in successive frames are shifted along the capillary tube axis according to the flow velocity, faithfully rendering cells and plasma. The velocity estimate, required as input to this procedure, can be obtained from either a) the blind optimization of a simple heuristic, or b) a recently proposed velocimetry algorithm, which appears to extend the aliasing limit.

Are you sure? The relationship between response certainty and performance in visual detection using a perimetry-style task.

Conventional psychophysical methods ignore the degree of confidence associated with each response. We compared the psychometric function for detection with that for "absolute certainty" in a perimetry-style task, to explore how knowledge of response certainty might aid the estimation of detection thresholds. Five healthy subjects performed a temporal 2-AFC detection task, indicating on each trial whether they were "absolutely certain." The method of constant stimuli was used to characterize the shape of the two psychometric functions. Four eccentricities spanning central and peripheral vision were tested. Where possible, conditions approximated those of the Humphrey Field Analyzer (spot size, duration, background luminance, test locations). Based on the empirical data, adaptive runs (ZEST) were simulated to predict the likely improvement in efficiency obtained by collecting certainty information. Compared to detection, threshold for certainty was 0.5 to 1.0 dB worse, and slope was indistinguishable across all eccentricities tested. A simple two-stage model explained the threshold difference; under this model, psychometric functions for detection and for certainty-given-detection are the same. Exploiting this equivalence is predicted to reduce the number of trials required to achieve a given level of accuracy by approximately 30% to 40%. The chances of detecting a spot and the chances of certainty-given-detection were approximately the same in young, healthy subjects. This means, for example, that a spot detected at threshold was labeled as "certainly" detected approximately half the time. The collection of certainty information could be used to improve the efficiency of estimation of detection thresholds.

The influence of retinal image motion on the perceptual grouping of temporally asynchronous stimuli.

Briefly presented stimuli can reveal the lower limit of retinal-based perceptual stabilization mechanisms. This is demonstrated in perceptual grouping of temporally asynchronous stimuli, in which alternate row or column elements of a regular grid are presented over two successive display frames with an imperceptible temporal offset. The grouping phenomenon results from a subtle shift between alternate grid elements due to incomplete compensation of small, fixational eye movements occurring between the two presentation frames. This suggests that larger retinal shifts should amplify the introduced shifts between alternate grid elements and improve grouping performance. However, large shifts are necessarily absent in small eye movements. Furthermore, shifts follow a random walk, making the relationship between shift magnitude and performance difficult to explore systematically. Here, we established a systematic relationship between retinal image motion and perceptual grouping by presenting alternate grid elements (untracked) during smooth pursuit of known velocities. Our results show grouping performance to improve in direct proportion to pursuit velocity. Any potential compensation by extraretinal signals (e.g., efference copy) does not seem to occur.

Impact of fetal growth and preterm birth on the retinal microvasculature in mid-adulthood.

OBJECTIVE: We hypothesized that preterm birth and being born SGA would be associated with changes in retinal microvascular architecture and that these changes would be more marked among those born preterm. We further hypothesized that these microvascular changes would correlate with early markers of CVD in mid-adulthood. METHODS: The Cardiovascular Risk in Young Finns Study included randomly selected children from 5 Finnish University cities. Retinal microvascular architecture of participants born preterm, born at term and SGA and a control group born at term and AGA were compared (aged 34-49 years). RESULTS: In participants born preterm, arteriolar tortuosity (×10(2)) was higher-means (standard error), 0.06 (0.01) versus 0.04 (0.01), p = 0.001, arteriolar length (pixels) were greater-644.9 (35.9) versus 591.7 (33.5), p = 0.007 and arteriolar diameters (pixels) were narrower-19.9 (0.4) versus 20.3 (0.3), p = 0.034 compared to participants born AGA, after adjustment. In participants born SGA, only arteriolar tortuosity was higher-0.05 (0.01) versus 0.04 (0.01), p = 0.074 compared to participants born AGA. CONCLUSION: This study demonstrated that being born SGA and in particular preterm birth are associated with changes in retinal microvascular architecture. The prenatal and immediate postnatal environment may contribute to the mechanisms.

Impact of blood pressure on retinal microvasculature architecture across the lifespan: the Young Finns Study.

OBJECTIVE: The present study examined the impact of BP from childhood to mid-adulthood on retinal microvascular architecture. METHODS: The Cardiovascular Risk in Young Finns Study included children aged 3-18 years, from five Finnish University cities, with participants chosen randomly from the national population registrar from those areas. The age of participants included in the current analyses in childhood (1980) ranged from three to nine years and in mid-adulthood (2011) ranged from 34 to 40 years (complete data n = 657). Measures of retinal microvasculature architecture measured in adulthood included diameters, tortuosity, lengths, and LDR. RESULTS: Regression analysis showed a strong negative association between childhood systolic BP and adult arteriolar diameter (standardized regression coefficient [ß] -0.300; p < 0.001) and with change in systolic BP from childhood to adulthood (ß = -0.249; p < 0.001). For arteriolar tortuosity, there was a strong positive association between childhood systolic BP and adult arteriolar tortuosity (ß = 0.154; p < 0.001) and no association with change in systolic BP from childhood to adulthood (ß = 0.072; p = 0.110). CONCLUSIONS: High BP in childhood and increased BP from childhood to adulthood impacts on retinal microvascular architecture in mid-adulthood.

Analysis of contrast and motion signals generated by human blood constituents in capillary flow.

The flow of individual corpuscles through retinal capillaries may now be observed noninvasively by using adaptive optics (AO). To explore their imaging properties, we imaged retinal capillary flow in two healthy subjects at 593 nm with a flood-based AO ophthalmoscope, at a variety of retinal locations and levels of defocus. The image intensity of red cells and plasma depends upon capillary depth relative to focus: red cells appear brighter than background, and plasma darker, for capillaries posterior to focus. The reverse is true for capillaries anterior to focus. Contrast reversals were obtained over 0.05 D (∼14 µm), which are well within the typical undulations in depth of retinal capillaries. We relate these observations to phase-contrast defocusing microscopy. This defocusing effect confounds flow measurements, which rely on correlation of image intensity between successive locations along the same capillary, a requirement made further difficult by high physiological variability in flow. Peak correlation was maintained >0.25 over a distance of 22±15 µm (roughly the spacing between red cells) and over a duration of 154±49 ms (roughly eight times the temporal period between red cells). We provide a 2D correlogram approach that significantly improves robustness in the face of optical and physiological variability, compared to the traditional spatiotemporal plot, without requiring additional data.

Changes to the shape, orientation and packing of red cells as a function of retinal capillary size.

The free diameter of a red blood cell exceeds the lumen diameter of capillaries in the central nervous system, requiring significant deformation of cells. However the deformations undertaken in vivo are not well established due to the difficulty in observing cellular capillary flow in living human tissue. Here, we used high resolution adaptive optics imaging to non-invasively track 17,842 red blood cells in transit through 121 unique capillary segments of diameter 8 µm or less in the retina of 3 healthy human subjects. Within each vessel, a 2D en face profile was generated for the "average cell", whose shape was then inferred in 3D based on the key assumption of a circular capillary cross-section. From this we estimated the average volume, surface area, orientation, and separation between red cells within each capillary tube. Our results showed a network filtration effect, whereby narrower vessels were more likely to contain smaller cells (defined by surface area, which is thought not to vary during a cell's passage through the vascular system). A bivariate linear model showed that for larger cells in narrower vessels: cells re-orient themselves to align with the flow axis, their shape becomes more elongated, there are longer gaps between successive cells, and remarkably, that cell volume is less which implies the ejection of water from cells to facilitate capillary transit. Taken together, these findings suggest that red cells pass through retinal capillaries with some reluctance. A biphasic distribution for cell orientation and separation was evident, indicating a "tipping point" for vessels narrower than approx. 5 µm. This corresponds closely to the typical capillary lumen diameter, and may maximize sensitivity of cellular flow to small changes in diameter. We suggest that the minimization of unnecessary oxygen exchange, and hence of damage via reactive oxygen pathways, may have provided evolutionary pressure to ensure that capillary lumens are generally narrower than red blood cells.

Validation of an automated method for studying retinal capillary blood flow.

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.

Adaptive optics imaging in inherited retinal diseases: A scoping review of the clinical literature.

Adaptive optics (AO) imaging enables direct, objective assessments of retinal cells. Applications of AO show great promise in advancing our understanding of the etiology of inherited retinal disease (IRDs) and discovering new imaging biomarkers. This scoping review systematically identifies and summarizes clinical studies evaluating AO imaging in IRDs. Ovid MEDLINE and EMBASE were searched on February 6, 2023. Studies describing AO imaging in monogenic IRDs were included. Study screening and data extraction were performed by 2 reviewers independently. This review presents (1) a broad overview of the dominant areas of research; (2) a summary of IRD characteristics revealed by AO imaging; and (3) a discussion of methodological considerations relating to AO imaging in IRDs. From 140 studies with AO outcomes, including 2 following subretinal gene therapy treatments, 75% included fewer than 10 participants with AO imaging data. Of 100 studies that included participants' genetic diagnoses, the most common IRD genes with AO outcomes are CNGA3, CNGB3, CHM, USH2A, and ABCA4. Confocal reflectance AO scanning laser ophthalmoscopy was the most reported imaging modality, followed by flood-illuminated AO and split-detector AO. The most common outcome was cone density, reported quantitatively in 56% of studies. Future research areas include guidelines to reduce variability in the reporting of AO methodology and a focus on functional AO techniques to guide the development of therapeutic interventions.

Retinal hyperspectral imaging in mouse models of Parkinson's disease and healthy aging.

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.

Mapping the human parafoveal vascular network to understand flow variability in capillaries.

Capillary flow is known to be non-homogenous between vessels and variable over time, for reasons that are poorly understood. The local properties of individual vessels have been shown to have limited explanatory power in this regard. This exploratory study investigates the association of network-level properties such as vessel depth, branch order, and distance from the feeding arteriole with capillary flow. Detailed network connectivity analysis was undertaken in 3 healthy young subjects using flood-illuminated adaptive optics retinal imaging, with axial depth of vessels determined via optical coherence tomography angiography. Forty-one out of 70 vessels studied were of terminal capillary type, i.e. fed from an arterial junction and drained by a venous junction. Approximately half of vessel junctions were amenable to fitting with a model of relative branch diameters, with only a few adhering to Murray's Law. A key parameter of the model (the junction exponent) was found to be inversely related to the average velocity (r = -0.59, p = 0.015) and trough velocity (r = -0.67, p = 0.004) in downstream vessels. Aspects of cellular flow, such as the minimum velocity, were also moderately correlated (r = 0.46, p = 0.009) with distance to the upstream feeding arteriole. Overall, this study shows that capillary network topology contributes significantly to the flow variability in retinal capillaries in human eyes. Understanding the heterogeneity in capillary flow is an important first step before pathological flow states can be properly understood. These results show that flow within capillary vessels is not affected by vessel depths but significantly influenced by the upstream feeder distance as well as the downstream vessel junction exponents, but there remains much to be uncovered regarding healthy capillary flow.

Flow Heterogeneity and Factors Contributing to the Variability in Retinal Capillary Blood Flow.

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 variability 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.

Effect of hydroxychloroquine or chloroquine and short wavelength light on in vivo retinal function and structure in mouse eyes.

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.

Assessment of photoreceptor function with ultrafast retinal densitometry.

The optical density of visual pigment can be measured by imaging the dark-adapted eye while bleaching with visible light. This measurement can be made for individual photoreceptor cells using adaptive optics; however, activation of the phototransduction cascade imparts rapid changes in phase that modulate the signal via optical interference. This limits utility because data must be averaged over many experimental runs. Here we used a "flood" illuminated adaptive optics system at 4000 fps, bright light to achieve rapid bleaching, and broad illumination bandwidth to mitigate interference effects. Data were super-resolved using the natural motion of the eye to overcome the reduced pixel resolution of the ultrafast camera. This approach was applied to classify the trichromatic cone photoreceptor mosaic at a single fixation locus within the foveal region of 3 healthy subjects. Subjects were dark adapted for 6 minutes to replenish cone photopigment. This was followed either directly by imaging at 555 ± 50 nm, or by first pre-adapting the retina to 700 nm light to preferentially deplete "L" cone pigment. A total of 3,252 cones were classified as either "S", "M", or "L" type based on clustering of the intensity data observed under these two conditions. Mean classification probability ranged from 99.3 to 99.8%, with individual cell probabilities exceeding 95% in 97.0 to 99.2% of cones. Accuracy of cone classification peaked when using the first 10-30 ms of data, with significant reductions in accuracy noted with the inclusion of data from later times. Our results show that rapid bleaching and data acquisition significantly improve the robustness of cell-resolved densitometry.

Optimizing Retinal Thermofusion in Retinal Detachment Repair: Achieving Instant Adhesion without Air Tamponade.

Purpose: Rhegmatogenous retinal detachment repair by intraoperative sealing of the tear without a tamponade agent should enable faster restoration of vision and resumption of normal activities. It avoids the need for further surgery in the case of silicone oil endotamponade. This study evaluated the retinal thermofusion (RTF) retinopexy method of subretinal space dehydration before photocoagulation to create an instantaneous intraoperative retina reattachment in a preclinical model. Design: Preclinical study. Participants: Twenty Dutch Belt, pigmented rabbits that underwent RTF repair after experimental retinal detachment. Methods: This ex vivo model quantified adhesion force between the retina and underlying retinal pigment epithelium and choroid after treatment of 1 retinal edge using postmortem porcine or human retina (6 × 12 mm). We compared (1) control, (2) laser photocoagulation alone, (3) dehydration alone, and (4) dehydration followed by photocoagulation (RTF). Optimized parameters for RTF were then applied in the in vivo rabbit model of retinal detachment. Animals were followed up for 14 days. Main Outcome Measures: For this ex vivo model, we measured adhesion force and related this to tissue temperature. For the in vivo study, we assessed retinal attachment using funduscopy and histologic analysis. Results: The ex vivo model showed that RTF repair produced significantly higher adhesion force than photocoagulation alone independent of dehydration method: warm (60° C) high airflow (50-70 ml/minute) or using laser wavelengths targeting water absorption peaks (1470 or 1940 nm) with coaxial low airflow (10-20 ml/minute). The latter approach produced a smaller footprint of dehydration. Application of RTF (1940-nm laser with coaxial airflow) in an in vivo retinal detachment model in rabbit eyes resulted in immediate retinal adhesion, achieving forces similar to those in the ex vivo experiments. Retinal thermofusion repair resulted in stable reattachment of the retina over the 2-week follow-up period. Conclusions: We showed that a short preliminary dehydrating laser treatment of a retinal tear margin before traditional laser photocoagulation creates an immediate intraoperative waterproof retinopexy adhesion independent of tamponade and a wound-healing response. This approach potentially will allow rapid postoperative recovery regardless of the tear location and improved vision.

Towards distortion-free imaging of the eye.

The high power of the eye and optical components used to image it result in "static" distortion, remaining constant across acquired retinal images. In addition, raster-based systems sample points or lines of the image over time, suffering from "dynamic" distortion due to the constant motion of the eye. We recently described an algorithm which corrects for the latter problem but is entirely blind to the former. Here, we describe a new procedure termed "DIOS" (Dewarp Image by Oblique Shift) to remove static distortion of arbitrary type. Much like the dynamic correction method, it relies on locating the same tissue in multiple frames acquired as the eye moves through different gaze positions. Here, the resultant maps of pixel displacement are used to form a sparse system of simultaneous linear equations whose solution gives the common warp seen by all frames. We show that the method successfully handles torsional movement of the eye. We also show that the output of the previously described dynamic correction procedure may be used as input for this new procedure, recovering an image of the tissue that is, in principle, a faithful replica free of any type of distortion. The method could be extended beyond ocular imaging, to any kind of imaging system in which the image can move or be made to move across the detector.

Retinal hyperspectral imaging in the 5xFAD mouse model of Alzheimer's disease.

Hyperspectral imaging of the retina has recently been posited as a potentially useful form of spectroscopy of amyloid-beta (Aß) protein in the eyes of those with Alzheimer's disease (AD). The concept of using the retina as a biomarker for AD is an attractive one, as current screening tools for AD are either expensive or inaccessible. Recent studies have investigated hyperspectral imaging in Aß models however these studies have been in younger mice. Here we characterised hyperspectral reflectance profile in 6 to 17 months old 5xFAD mice and compare this to Aß in isolated preparations. Hyperspectral imaging was conducted across two preparations of Aß using a custom built bench ophthalmoscope. In the in vitro condition, 1 mg of purified human Aß42 was solubilised and left to aggregate for 72 h. This soluble/insoluble Aß mixture was then imaged by suspending the solution at a pipette tip and compared against phosphate buffered saline (PBS) control (n = 10 ROIs / group). In the in vivo condition, a 5xFAD transgenic mouse model was used and retinae were imaged at the age of 6 (n = 9), 12 (n = 9) and 17 months (n = 8) with age matched wildtype littermates as control (n = 12, n = 13, n = 15 respectively). In the vitro condition, hyperspectral imaging of the solution showed greater reflectance compared with vehicle (p < 0.01), with the greatest differences occurring in the short visible spectrum (< 500 nm). In the in vivo preparation, 5xFAD showed greater hyperspectral reflectance at all ages (6, 12, 17 months, p < 0.01). These differences were noted most in the short wavelengths at younger ages, with an additional peak appearing at longer wavelengths (~ 550 nm) with advancing age. This study shows that the presence of Aß (soluble/insoluble mixture) can increase the hyperspectral reflectance profile in vitro as well as in vivo. Differences were evident in the short wavelength spectrum (< 500 nm) in vitro and were preserved when imaged through the ocular media in the in vivo conditions. With advancing age a second hump around ~ 550 nm became more apparent. Hyperspectral imaging of the retina does not require the use of contrast agents and is a potentially useful and non-invasive biomarker for AD.