Optometry and Vision Sciences - Research Publications
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ItemNo Preview AvailableChanges to the shape, orientation and packing of red cells as a function of retinal capillary size.(Optica Publishing Group, 2024-02-01)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.
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ItemNo Preview AvailableValidation of an automated method for studying retinal capillary blood flow.(Optica Publishing Group, 2024-02-01)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.
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ItemNo Preview AvailableMapping the human parafoveal vascular network to understand flow variability in capillaries.(Public Library of Science (PLoS), 2023)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.
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ItemNo Preview AvailableAdaptive optics imaging in inherited retinal diseases: A scoping review of the clinical literature(ELSEVIER SCIENCE INC, 2024)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.
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ItemNo Preview AvailableAssessment of photoreceptor function with ultrafast retinal densitometry(Optica Publishing Group, 2022-10-01)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.
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ItemScaling the size of perimetric stimuli reduces variability and returns constant thresholds across the visual field(ASSOC RESEARCH VISION OPHTHALMOLOGY INC, 2020-10)The conventional stimulus for standard automated perimetry is fixed in size, giving elevated contrast thresholds and reduced test reliability in the periphery. Here, we test the hypothesis that appropriate scaling of the size of perimetric stimuli will return fixed thresholds and reduced variability across the visual field. We derived frequency-of-seeing (FOS) curves in five healthy subjects at central (3 degrees) and peripheral (27 degrees) locations with a method of constant stimuli (MOCS) using a desktop LCD display. FOS curves for a Goldmann III (GIII) stimulus were compared with those for size scaled spots. To consider clinical translation, we tested a further five healthy subjects (22-24 years) with the Melbourne Rapid Fields (MRF) tablet perimeter at several locations spanning 1 degree to 25 degrees from fixation, deriving FOS curves (MOCS) and also conducting repeated adaptive clinical thresholding to assess intra- and interobserver variability. We found that GIII contrast thresholds were significantly elevated in the periphery compared with the parafovea, with concomitant reduction of FOS slope. Using appropriately size scaled spots, threshold and slope differences between these locations were significantly reduced. FOS data collected with the tablet perimeter confirmed that size scaling confers broad equivalence of the shape of the FOS curve across the visual field. Repeated adaptive thresholding with size scaled stimuli gave relatively constant intra-observer variability across the visual field, which compares favorably with published normative data obtained with the GIII stimulus. The reduced variability will improve signal-to-noise ratio for correct classification of normal visual field test results, whereas the lower contrast thresholds yield greater dynamic range, which should improve the ability to reliably monitor moderate defects.
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ItemNo Preview AvailableImaging relative stasis of the blood column in human retinal capillaries(OPTICAL SOC AMER, 2019-11-01)Capillary flow largely consists of alternating red cells and plasma whose speed oscillates predictably with the cardiac cycle. Superimposed on this regular background are sporadic events potentially disruptive to capillary exchange: the passage of white cells, aggregates of red cells, epochs of sparse haematocrit, or unusually slow flow. Such events are not readily differentiated with velocimetry or perfusion mapping. Here we propose a method to identify these phenomena in retinal capillaries imaged with high frame-rate adaptive optics, by calculating and representing pictorially the autocorrelation of intensity through time at each pixel during short epochs. The phenomena described above manifest as bright regions which transiently appear and propagate across an otherwise dark image. Drawing data from normal subjects and those with Type I diabetes, we demonstrate proof of concept and high sensitivity and specificity of this metric to variations in capillary contents and rate of flow in health and disease. The proposed metric offers a useful adjunct to velocimetry and perfusion mapping in the study of normal and abnormal capillary blood flow.
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ItemOptic nerve tissue displacement during mild intraocular pressure elevation: its relationship to central corneal thickness and corneal hysteresis(WILEY, 2018-07)PURPOSE: To determine the extent to which (1) optic nerve tissue is displaced following mild acute elevation of intraocular pressure, and (2) clinically accessible measures at the anterior eye can be used as a surrogate for such displacements. METHODS: We imaged the optic disc of 21 healthy subjects before and after intraocular pressure (IOP) elevation of ~10 mmHg delivered by ophthalmodynamometry. Steady-state tissue displacement during IOP elevation was assessed axially from OCT data, and laterally from SLO data. Recovery from IOP elevation was assessed by tracking a single vertical B-scan through the cup centre. Anatomical structures were demarcated by three masked clinicians to determine lateral shifts for temporal cup edge and central disc vessels, and axial shifts of disc surface and anterior lamina cribrosa. Spatial maps of deformation were constructed within the demarcated cup and disc to assess within-tissue displacement. Measured displacements were correlated with corneal hysteresis, corneal thickness, and IOP. RESULTS: The temporal cup edge moved more temporally with higher baseline IOP (R2 = 0.33, p = 0.006) and with lesser elevation of IOP (R2 = 0.43, p = 0.001); it moved more superiorly for thinner corneas (R2 = 0.35, p = 0.007). Thinner corneas also produced less within-cup deformation, relative to that of the disc (R2 = 0.39, p = 0.004). Axial displacement of the lamina and lateral displacement of vessels were often substantial (lamina 20 ± 15 μm, range 1-60 μm; vessels 37 ± 25 μm, range 2-102 μm) but did not correlate with measured parameters. Recovery from IOP elevation did not take more than 300-400 ms in any subject. CONCLUSIONS: Mild acute elevation of IOP produces large and rapidly reversible shifts in optic nerve tissue in young, healthy eyes. The resulting degree, direction and spatial distribution of cup movement are associated with IOP status and corneal thickness, but not corneal hysteresis.
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ItemTowards distortion-free imaging of the eye(PUBLIC LIBRARY SCIENCE, 2021-06-10)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.
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ItemRetinal hyperspectral imaging in the 5xFAD mouse model of Alzheimer's disease(NATURE PORTFOLIO, 2021-03-18)Hyperspectral imaging of the retina has recently been posited as a potentially useful form of spectroscopy of amyloid-beta (Aβ) 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 Aβ 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 Aβ in isolated preparations. Hyperspectral imaging was conducted across two preparations of Aβ using a custom built bench ophthalmoscope. In the in vitro condition, 1 mg of purified human Aβ42 was solubilised and left to aggregate for 72 h. This soluble/insoluble Aβ 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 Aβ (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.