Adaptive Optics Scanning Laser Ophthalmoscope Research Articles

Application of Adaptive Optics in Ophthalmology

The eye, the photoreceptive organ used to perceive the external environment, is of great importance to humans. It has been proven that some diseases in humans are accompanied by fundus changes; therefore, the health status of people may be interpreted from retinal images. However, the human eye is not a perfect refractive system for the existence of ocular aberrations. These aberrations not only affect the ability of human visual discrimination and recognition, but restrict the observation of the fine structures of human eye and reduce the possibility of exploring the mechanisms of eye disease. Adaptive optics (AO) is a technique that corrects optical wavefront aberrations. Once integrated into ophthalmoscopes, AO enables retinal imaging at the cellular level. This paper illustrates the principle of AO in correcting wavefront aberrations in human eyes, and then reviews the applications and advances of AO in ophthalmology, including the adaptive optics fundus camera (AO-FC), the adaptive optics scanning laser ophthalmoscope (AO-SLO), the adaptive optics optical coherence tomography (AO-OCT), and their combined multimodal imaging technologies. The future development trend of AO in ophthalmology is also prospected.

Investigating Biomarkers for USH2A Retinopathy Using Multimodal Retinal Imaging.

Pathogenic mutations in USH2A are a leading cause of visual loss secondary to non-syndromic or Usher syndrome-associated retinitis pigmentosa (RP). With an increasing number of RP-targeted clinical trials in progress, we sought to evaluate the photoreceptor topography underlying patterns of loss observed on clinical retinal imaging to guide surrogate endpoint selection in USH2A retinopathy. In this prospective cross-sectional study, twenty-five patients with molecularly confirmed USH2A-RP underwent fundus autofluorescence (FAF), spectral-domain optical coherence tomography (SD-OCT) and adaptive optics scanning laser ophthalmoscopy (AOSLO) retinal imaging. Analysis comprised measurement of FAF horizontal inner (IR) and outer (OR) hyperautofluorescent ring diameter; SD-OCT ellipsoid zone (EZ) and external limiting membrane (ELM) width, normalised EZ reflectance; AOSLO foveal cone density and intact macular photoreceptor mosaic (IMPM) diameter. Thirty-two eyes from 16 patients (mean age ± SD, 36.0 ± 14.2 years) with USH2A-associated Usher syndrome type 2 (n = 14) or non-syndromic RP (n = 2) met the inclusion criteria. Spatial alignment was observed between IR-EZ and OR-ELM diameters/widths (p < 0.001). The IMPM border occurred just lateral to EZ loss (p < 0.001), although sparser intact photoreceptor inner segments were detected until ELM disruption. EZ width and IR diameter displayed a biphasic relationship with cone density whereby slow cone loss occurred until retinal degeneration reached ~1350 μm from the fovea, beyond which greater reduction in cone density followed. Normalised EZ reflectance and cone density were significantly associated (p < 0.001). As the strongest correlate of cone density (p < 0.001) and best-corrected visual acuity (p < 0.001), EZ width is the most sensitive biomarker of structural and functional decline in USH2A retinopathy, rendering it a promising trial endpoint.

Cone Photoreceptors in Diabetic Patients.

PurposeCones in diabetic patients are at risk due to metabolic and vascular changes. By imaging retinal vessel modeling at high magnification, we reduced its impact on cone distribution measurements. The retinal vessel images and retinal thickness measurements provided information about cone microenvironment.MethodsWe compared cone data in 10 diabetic subjects (28–78 yr) to our published norms from 36 younger and 10 older controls. All subjects were consented and tested in a manner approved by the Indiana University Institutional Review Board, which adhered to the Declaration of Helsinki. Custom adaptive optics scanning laser ophthalmoscopy (AOSLO) was used to image cones and retinal microcirculation. We counted cones in a montage of foveal and temporal retina, using four non-contiguous samples within 0.9–7 deg that were selected for best visibility of cones and least pathology. The data were fit with a two parameter exponential model: ln(cone density) = a * microns eccentricity + b. These results were compared to retinal thickness measurements from SDOCT.ResultsDiabetic cone maps were more variable than in controls and included patches, or unusually bright and dark cones, centrally and more peripherally. Model parameters and total cones within the central 14 deg of the macula differed across diabetic patients. Total cones fell into two groups: similar to normal for 5 vs. less than normal for 2 of 2 younger diabetic subjects and 3 older subjects, low but not outside the confidence limits. Diabetic subjects had all retinal vascular remodeling to varying degrees: microaneurysms; capillary thickening, thinning, or bends; and vessel elongation including capillary loops, tangles, and collaterals. Yet SD-OCT showed that no diabetic subject had a Total Retinal Thickness in any quadrant that fell outside the confidence limits for controls.ConclusionsAOSLO images pinpointed widespread retinal vascular remodeling in all diabetic eyes, but the SDOCT showed no increased retinal thickness. Cone reflectivity changes were found in all diabetic patients, but significantly low cone density in only some. These results are consistent with early changes to neural, glial, or vascular components of the retinal without significant retinal thickening due to exudation.

Contributed Session III: The benefits of naturally moving over stabilized stimuli for acuity increase with longer presentation times

Longer stimulus durations aid in resolving fine spatial detail (Graham & Cook, 1937) - an effect partially attributed to fixational eye movements (FEM; Rucci et al., 2007, Anderson et al., 2020). While FEM do not appear to impact acuity at short presentations (6-300 ms; Tulunay-Keesey & Jones, 1976), recent work has shown that stabilizing stimuli on the retina, eliminating the effects of FEM, impedes acuity at longer durations (750 ms; Ratnam et al., 2017). At intermediate durations, the interplay between acuity and stimulus motion on the retina is not well understood. We used an adaptive optics scanning laser ophthalmoscope to stabilize stimuli on the retina at 1 degree from fixation. A 4 AFC tumbling E staircase was used to find the minimum angle of resolution (MAR) for 100, 375 and 750 ms presentations of unstabilized stimuli. The MAR-duration relationship followed an asymptotic shape, improving from 100 ms (mean MAR ± SEM: 1.84 ± 0.17 arcmin) to 375 ms (1.45 ± 0.09) but not to 750 ms (1.48 ± 0.08). Next, performance (% correct) on stabilized and unstabilized stimuli was measured at each duration's MAR. For stabilized stimuli, we measured performance as a ratio relative to unstabilized performance and found that it degraded with increasing stimulus duration (1.1 ± 0.01, 0.91 ± 0.07, 0.65 ± 0.11 for 100, 375, and 750 ms durations, respectively). These results suggest that retinal stabilization may disrupt the mechanisms by which integration over time enhances visual acuity.

Patches of Dysflective Cones in Eyes With No Known Disease.

PurposeTo characterize the structure and function of patches of dysflective cones in the foveal region of subjects with normal vision and no known pathology. Dysflective cones are cones that have little or no reflective properties in optical coherence tomography (OCT) or adaptive optics scanning laser ophthalmoscope (AOSLO) images yet exhibit measurable function.MethodsAOSLO images were surveyed for the presence of hyporeflective cone patches, and subjects were brought back for imaging to determine the changes in the hyporeflective region. Adaptive optics microperimetry (AOMP) was used to assess the function of hyporeflective patches in four subjects to determine that they did, in fact, contain dysflective cones. AOMP utilized a stimulus size of less than 1 arcmin to measure thresholds inside and outside the hyporeflective region.ResultsNineteen out of 47 individuals retrospectively reviewed had one or more regions with hyporeflective cone patches in one or both eyes. Ten subjects with hyporeflective cone patches were brought back for imaging. Seven of the 10 had resolved at follow up, and in three subjects new hyporeflective patches appeared in a different location. All AOMP-measured subjects had measurable function in the dysflective cone region. Three out of four subjects showed no difference in light sensitivity in the dysflective region compared to adjacent areas, and one subject showed a 3× reduction in sensitivity in the area.ConclusionsPatches of dysflective cone have been identified in subjects with normal vision and no known pathology, and we have observed instances where dysflective cones in these subjects regain normal reflective properties.

Longitudinal In Vivo Changes in Radial Peripapillary Capillaries and Optic Nerve Head Structure in Non-Human Primates With Early Experimental Glaucoma.

PurposeThere is conflicting evidence regarding whether a loss of radial peripapillary capillaries (RPCs) precedes neuronal loss in glaucoma. We examined the time course of in vivo changes in RPCs, optic nerve head (ONH) structure, and retinal nerve fiber layer thickness (RNFLT) in experimental glaucoma (EG).MethodsSpectral domain optical coherence tomography images were acquired before and approximately every two weeks after inducing unilateral EG in nine rhesus monkeys to quantify mean anterior lamina cribrosa surface depth (ALCSD), minimum rim width (MRW), and RNFLT. Perfused RPC density was measured from adaptive optics scanning laser ophthalmoscope images acquired on the temporal half of the ONH. The time of first significant change was quantified as when values fell and remained outside of the 95% confidence interval established from control eyes.ResultsMean ALCSD and/or MRW were the first parameters to change in eight EG eyes. RPC density changed first in the ninth. At their first points of change, mean ALCSD posteriorly deformed by 100.2 ± 101.2 µm, MRW thinned by 82.3 ± 65.9 µm, RNFLT decreased by 25 ± 14 µm, and RPC density decreased by 4.5 ± 2.1%. RPC density decreased before RNFL thinning in 5 EG eyes. RNFLT decreased before RPC density decreased in two EG eyes, whereas two EG eyes had simultaneous changes.ConclusionsIn most EG eyes, RPC density decreased before (or simultaneous with) a change in RNFLT, suggesting that vascular factors may play a role in axonal loss in some eyes in early glaucoma.

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

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

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

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

Fixational eye movements in passive versus active sustained fixation tasks.

Human fixational eye movements are so small and precise that high-speed, accurate tools are needed to fully reveal their properties and functional roles. Where the fixated image lands on the retina and how it moves for different levels of visually demanding tasks is the subject of the current study. An Adaptive Optics Scanning Laser Ophthalmoscope (AOSLO) was used to image, track and present a variety of fixation targets (Maltese cross, disk, concentric circles, Vernier and tumbling-E letter) to healthy subjects. During these different passive (static) or active (discriminating) tasks under natural eye motion, the landing position of the target on the retina was tracked in space and time over the retinal image directly with high spatial (<1 arcmin) and temporal (960 Hz) resolution. We computed both the eye motion and the exact trajectory of the fixated target's motion over the retina. We confirmed that compared to passive tasks, active tasks elicited a partial inhibition of microsaccades, leading to longer drift periods compensated by larger corrective saccades. Consequently, the overall fixation stability during active tasks was on average 57% larger than during passive tasks. The preferred retinal locus of fixation was the same for each task and did not coincide with the location of the peak cone density.

The retinal and perceived locus of fixation in the human visual system.

Due to the dramatic difference in spatial resolution between the central fovea and the surrounding retinal regions, accurate fixation on important objects is critical for humans. It is known that the preferred retinal location (PRL) for fixation of healthy human observers rarely coincides with the retinal location with the highest cone density. It is not currently known, however, whether the PRL is consistent within an observer or is subject to fluctuations and, moreover, whether observers’ subjective fixation location coincides with the PRL. We studied whether the PRL changes between days. We used an adaptive optics scanning laser ophthalmoscope to project a Maltese cross fixation target on an observer's retina and continuously imaged the exact retinal location of the target. We found that observers consistently use the same PRL across days, regardless of how much the PRL is displaced from the cone density peak location. We then showed observers small stimuli near the visual field location on which they fixated, and the observers judged whether or not the stimuli appeared in fixation. Observers’ precision in this task approached that of fixation itself. Observers based their judgment on both the visual scene coordinates and the retinal location of the stimuli. We conclude that the PRL in a normally functioning visual system is fixed, and observers use it as a reference point in judging stimulus locations.

Novel Foveal Features Associated With Vision Impairment in Multiple Sclerosis.

PurposeTo characterize scattering and hyperreflective features in the foveal avascular zone of people with multiple sclerosis (MS) using adaptive optics scanning laser ophthalmoscopy (AOSLO) and to evaluate their relationship with visual function and MS disease characteristics.MethodsTwenty subjects with MS underwent confocal reflectance and non-confocal split-detection AOSLO foveal imaging. Peripapillary retinal nerve fiber layer thickness was measured using optic nerve optical coherence tomography. Blood pressure, intraocular pressure (IOP), and best-corrected high-contrast visual acuity (HCVA) and low-contrast visual acuity (LCVA) were measured. AOSLO images were graded to determine the presence and characteristics of distinct structures.ResultsTwo distinct structures were seen in the avascular zone of the foveal pit. Hyperreflective puncta, present in 74% of eyes, were associated with IOP and blood pressure. Scattering features, observed in 58% of eyes, were associated with decreased HCVA and LCVA, as well as increased MS duration and disability, but were not associated with retinal nerve fiber layer thickness. Hyperreflective puncta and scattering features were simultaneously present in 53% of eyes.ConclusionsHyperreflective puncta were associated with parameters affecting ophthalmic perfusion, but they were not associated with MS disease parameters. Scattering features were associated with parameters corresponding to advanced MS, suggesting that they may be related to disease progression. Scattering features were also correlated with reduced visual function independent from ganglion cell injury, suggesting the possibility of a novel ganglion cell–independent mechanism of impaired vision in people with MS.

Automated cone photoreceptor cell identification in confocal adaptive optics scanning laser ophthalmoscope images based on object detection

Cone photoreceptor cell identification is important for the early diagnosis of retinopathy. In this study, an object detection algorithm is used for cone cell identification in confocal adaptive optics scanning laser ophthalmoscope (AOSLO) images. An effectiveness evaluation of identification using the proposed method reveals precision, recall, and [Formula: see text]-score of 95.8%, 96.5%, and 96.1%, respectively, considering manual identification as the ground truth. Various object detection and identification results from images with different cone photoreceptor cell distributions further demonstrate the performance of the proposed method. Overall, the proposed method can accurately identify cone photoreceptor cells on confocal adaptive optics scanning laser ophthalmoscope images, being comparable to manual identification.

Reliable Single-image Denoising for Adaptive Optics Scanning Laser Ophthalmoscopy

A reliable single-image denoising method is presented for adaptive optics scanning laser ophthalmoscopy. This method firstly averaged multiple images and then used the averaged image as the reference to adjust the parameters of the filtering process that was subsequently applied to other individual images. Six filtering methods, including the mean, median, Gaussian, fast adaptive nonlocal synthetic aperture radar despeckling, K-single value decomposition, and block matching and three-dimensional filtering, were utilized. The effectiveness of our method was verified based on the comparison of sets of images without and with parameter adjustments. Furthermore, we applied the same parameter settings as those obtained from the filter adjustments of another adaptive optics scanning laser ophthalmoscope image acquired by the same instrument. The filtered images showed that the parameter-adjusted filters work well on other images, which is helpful for improving the image quality of adaptive optics scanning laser ophthalmoscope images.

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

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

Automated Cone Cell Identification on Adaptive Optics Scanning Laser Ophthalmoscope Images Based on TV-L1 Optical Flow Registration and K-Means Clustering

Cone cell identification is essential for diagnosing and studying eye diseases. In this paper, we propose an automated cone cell identification method that involves TV-L1 optical flow estimation and K-means clustering. The proposed algorithm consists of the following steps: image denoising based on TV-L1 optical flow registration, bias field correction, cone cell identification based on K-means clustering, duplicate identification removal, identification based on threshold segmentation, and merging of closed identified cone cells. Compared with manually labelled ground-truth images, the proposed method shows high effectiveness with precision, recall, and F1 scores of 93.10%, 94.97%, and 94.03%, respectively. The method performance is further evaluated on adaptive optics scanning laser ophthalmoscope images obtained from a healthy subject with low cone cell density and subjects with either diabetic retinopathy or acute zonal occult outer retinopathy. The evaluation results demonstrate that the proposed method can accurately identify cone cells in subjects with healthy retinas and retinal diseases.

Template free eye motion correction for scanning systems.

Scanning imaging systems are susceptible to image warping in the presence of target motion occurring within the time required to acquire an individual image frame. In this Letter, we introduce the use of a dual raster scanning approach to correct for motion distortion without the need for prior knowledge of the undistorted image. In the dual scanning approach, the target is imaged simultaneously with two imaging beams from the same imaging system. The two imaging beams share a common pupil but have a spatial shift between the beams on the imaging plane. The spatial shift can be used to measure high speed events, because it measures an identical region at two different times within the time required for acquisition of a single frame. In addition, it provides accurate spatial information, since two different regions on the target are imaged simultaneously, providing an undistorted estimate of the spatial relation between regions. These spatial and temporal relations accurately measure target motion. Data from adaptive optics scanning laser ophthalmoscope (AOSLO) imaging of the human retina are used to demonstrate this technique. We apply the technique to correct the shearing of retinal images produced by eye motion. Three control subjects were measured while imaging different retinal layers and retinal locations to qualify the effectiveness of the algorithm. Since the time shift between channels is readily adjustable, this method can be tuned to match different imaging situations. The major requirement is the need to separate the two images; in our case, we used different near infrared spectral regions and dichroic filters.

DeepLab and Bias Field Correction Based Automatic Cone Photoreceptor Cell Identification with Adaptive Optics Scanning Laser Ophthalmoscope Images

The identification of cone photoreceptor cells is important for early diagnosing of eye diseases. We proposed automatic deep‐learning cone photoreceptor cell identification on adaptive optics scanning laser ophthalmoscope images. The proposed algorithm is based on DeepLab and bias field correction. Considering manual identification as reference, our algorithm is highly effective, achieving precision, recall, and F1 score of 96.7%, 94.6%, and 95.7%, respectively. To illustrate the performance of our algorithm, we present identification results for images with different cone photoreceptor cell distributions. The experimental results show that our algorithm can achieve accurate photoreceptor cell identification on images of human retinas, which is comparable to manual identification.

The Anatomical, Functional and Perceived Location of the Fovea in the Human Visual System

Due to the dramatic difference in spatial resolution between the central fovea and the surrounding retinal regions, accurate fixation on important objects is critical for human visual behavior. It is known that the preferred retinal location for fixation (PRL) of healthy human observers does not exactly coincide with the retinal location with the highest cone density. It is not currently known, however, whether the PRL is consistent within an observer or subject to fluctuations and, moreover, whether observers’ subjective fixation location coincides with the PRL. We studied whether the PRL changes between days. We used an adaptive optics scanning laser ophthalmoscope to project a Maltese cross fixation target on an observer’s retina, and continuously imaged the exact retinal location of the target, while the observers fixated on it. We found that observers consistently use the same PRL across days, regardless of how much the PRL is displaced from the cone density peak location. We then showed observers small stimuli near the visual field location they were asked to fixate on, and the observers judged whether the stimuli appeared along their line of sight (i.e., in fixation) or not. Observers’ precision in this task approached that of fixation itself. Observers based their judgement on both the external world coordinates and the retinal location of the stimuli. We conclude that, for a monocular fixation task, the PRL in a normally functioning visual system is fixed, and observers are relatively well aware of its location.

Topographic Relationship between Telangiectasia and Cone Mosaic Disruption in Macular Telangiectasia Type 2.

In this cross-sectional observational study, we investigated the relationship between photoreceptor layer disruption and telangiectasia in patients diagnosed with early stage macular telangiectasia type 2 (MacTel). A total of 31 eyes (17 patients) with MacTel were imaged with adaptive optics scanning laser ophthalmoscopy (AOSLO) and optical coherence tomography angiography (OCTA). Confocal AOSLO was used to visualize dark regions of nonwaveguiding outer segments, which we refer to as “photoreceptor lesions”. En-face OCTA images of the deep capillary plexus (DCP) were used in conjunction with confocal AOSLO to evaluate the topographic relationship between areas of capillary telangiectasias and photoreceptor lesions. Among seven eyes with early stage MacTel (stage 0–2 based on OCT), we identified ten photoreceptor lesions, all of which were located within parafoveal quadrants containing DCP telangiectasia on OCTA. Seven of the lesions corresponded to the intact ellipsoid zone on spectral-domain OCT (SD-OCT), and three of these also corresponded to the intact interdigitation zone. This work demonstrates a topographic relationship between AOSLO photoreceptor lesions and DCP telangiectasias, and it also suggests that these lesions with normal SD-OCT appearance may represent areas of photoreceptors at risk for dysfunction. Thus, confocal AOSLO may have a meaningful role in detecting early photoreceptor abnormalities in eyes with MacTel.

Full-field flicker evoked changes in parafoveal retinal blood flow

When retinal activity is increased by exposure to dynamic visual stimuli, blood vessels dilate and the flow of blood within vessels increases to meet the oxygen and glucose demands of the neurons. This relationship is termed ‘neurovascular coupling’ and it is critical for regulating control of the human retinal vasculature. In this study, we used a recently developed technique based on a dual-beam adaptive optics scanning laser ophthalmoscope to measure changes in red blood cell velocities, vessel diameter, and flow in interconnected small parafoveal retinal vessels (< 50 µm) of nine healthy participants. A full-field flicker stimulus was presented onto the retina to induce a vascular response to neural activity. Flicker stimulation increased blood velocity, vessel diameter, and therefore flow in arterioles, capillaries, and venules in all nine subjects. ANOVA and post hoc t-test showed significant increases in velocity and flow in arterioles and venules. These measurements indicate that the mechanism of neurovascular coupling systematically affects the vascular response in small retinal vessels in order to maintain hemodynamic regulation in the retina when exposed to visual stimulation, in our case flicker. Our findings may provide insight into future investigations on the impairments of neurovascular coupling from vascular diseases such as diabetic mellitus.