Adaptive Optics Scanning Ophthalmoscopy with Annular Pupils

Annular apodization of the illumination and/or imaging pupils of an adaptive optics scanning light ophthalmoscope (AOSLO) for improving transverse resolution was evaluated using three different normalized inner radii (0.26, 0.39 and 0.52). In vivo imaging of the human photoreceptor mosaic at 0.5 and 10° from fixation indicates that the use of an annular illumination pupil and a circular imaging pupil provides the most benefit of all configurations when using a one Airy disk diameter pinhole, in agreement with the paraxial confocal microscopy theory. Annular illumination pupils with 0.26 and 0.39 normalized inner radii performed best in terms of the narrowing of the autocorrelation central lobe (between 7 and 12%), and the increase in manual and automated photoreceptor counts (8 to 20% more cones and 11 to 29% more rods). It was observed that the use of annular pupils with large inner radii can result in multi-modal cone photoreceptor intensity profiles. The effect of the annular masks on the average photoreceptor intensity is consistent with the Stiles-Crawford effect (SCE). This indicates that combinations of images of the same photoreceptors with different apodization configurations and/or annular masks can be used to distinguish cones from rods, even when the former have complex multi-modal intensity profiles. In addition to narrowing the point spread function transversally, the use of annular apodizing masks also elongates it axially, a fact that can be used for extending the depth of focus of techniques such as adaptive optics optical coherence tomography (AOOCT). Finally, the positive results from this work suggest that annular pupil apodization could be used in refractive or catadioptric adaptive optics ophthalmoscopes to mitigate undesired back-reflections

Non-common Path Aberration Correction in an Adaptive Optics Scanning Ophthalmoscope

The correction of non-common path aberrations (NCPAs) between the imaging and wavefront sensing channel in a confocal scanning adaptive optics ophthalmoscope is demonstrated. NCPA correction is achieved by maximizing an image sharpness metric while the confocal detection aperture is temporarily removed, effectively minimizing the monochromatic aberrations in the illumination path of the imaging channel. Comparison of NCPA estimated using zonal and modal orthogonal wavefront corrector bases provided wavefronts that differ by ~λ/20 in root-mean-squared (~λ/30 standard deviation). Sequential insertion of a cylindrical lens in the illumination and light collection paths of the imaging channel was used to compare image resolution after changing the wavefront correction to maximize image sharpness and intensity metrics. Finally, the NCPA correction was incorporated into the closed-loop adaptive optics control by biasing the wavefront sensor signals without reducing its bandwidth

First-order Design of a Reflective Viewfinder for Adaptive Optics Ophthalmoscopy

Adaptive optics (AO) ophthalmoscopes with small fields of view have limited clinical utility. We propose to address this problem in reflective instruments by incorporating a viewfinder pupil relay designed by considering pupil and image centering and conjugation. Diverting light from an existing pupil optical relay to the viewfinder relay allows switching field of view size. Design methods that meet all four centering and conjugation conditions using either a single concave mirror or with two concave mirrors forming an off-axis afocal telescope are presented. Two different methods for calculating the focal length and orientation of the concave mirrors in the afocal viewfinder relay are introduced. Finally, a 2.2 × viewfinder mode is demonstrated in an AO scanning light ophthalmoscope

\u3cem\u3eIn vivo\u3c/em\u3e Dark-field Imaging of the Retinal Pigment Epithelium Cell Mosaic

Non-invasive reflectance imaging of the human RPE cell mosaic is demonstrated using a modified confocal adaptive optics scanning light ophthalmoscope (AOSLO). The confocal circular aperture in front of the imaging detector was replaced with a combination of a circular aperture 4 to 16 Airy disks in diameter and an opaque filament, 1 or 3 Airy disks thick. This arrangement reveals the RPE cell mosaic by dramatically attenuating the light backscattered by the photoreceptors. The RPE cell mosaic was visualized in all 7 recruited subjects at multiple retinal locations with varying degrees of contrast and cross-talk from the photoreceptors. Various experimental settings were explored for improving the visualization of the RPE cell boundaries including: pinhole diameter, filament thickness, illumination and imaging pupil apodization, unmatched imaging and illumination focus, wavelength and polarization. None of these offered an obvious path for enhancing image contrast. The demonstrated implementation of dark-field AOSLO imaging using 790 nm light requires low light exposures relative to light safety standards and it is more comfortable for the subject than the traditional autofluorescence RPE imaging with visible light. Both these factors make RPE dark-field imaging appealing for studying mechanisms of eye disease, as well as a clinical tool for screening and monitoring disease progression

\u3cem\u3eIn vivo\u3c/em\u3e Imaging of Human Cone Photoreceptor Inner Segments

Purpose. An often overlooked prerequisite to cone photoreceptor gene therapy development is residual photoreceptor structure that can be rescued. While advances in adaptive optics (AO) retinal imaging have recently enabled direct visualization of individual cone and rod photoreceptors in the living human retina, these techniques largely detect strongly directionally-backscattered (waveguided) light from normal intact photoreceptors. This represents a major limitation in using existing AO imaging to quantify structure of remnant cones in degenerating retina. Methods. Photoreceptor inner segment structure was assessed with a novel AO scanning light ophthalmoscopy (AOSLO) differential phase technique, that we termed nonconfocal split-detector, in two healthy subjects and four subjects with achromatopsia. Ex vivo preparations of five healthy donor eyes were analyzed for comparison of inner segment diameter to that measured in vivo with split-detector AOSLO. Results. Nonconfocal split-detector AOSLO reveals the photoreceptor inner segment with or without the presence of a waveguiding outer segment. The diameter of inner segments measured in vivo is in good agreement with histology. A substantial number of foveal and parafoveal cone photoreceptors with apparently intact inner segments were identified in patients with the inherited disease achromatopsia. Conclusions. The application of nonconfocal split-detector to emerging human gene therapy trials will improve the potential of therapeutic success, by identifying patients with sufficient retained photoreceptor structure to benefit the most from intervention. Additionally, split-detector imaging may be useful for studies of other retinal degenerations such as AMD, retinitis pigmentosa, and choroideremia where the outer segment is lost before the remainder of the photoreceptor cell

Noninvasive imaging of the thirteen-lined ground squirrel photoreceptor mosaic.

Ground squirrels are an increasingly important model for studying visual processing, retinal circuitry, and cone photoreceptor function. Here, we demonstrate that the photoreceptor mosaic can be longitudinally imaged noninvasively in the 13-lined ground squirrel (Ictidomys tridecemlineatus) using confocal and nonconfocal split-detection adaptive optics scanning ophthalmoscopy using 790 nm light. Photoreceptor density, spacing, and Voronoi analysis are consistent with that of the human cone mosaic. The high imaging success rate and consistent image quality in this study reinforce the ground squirrel as a practical model to aid drug discovery and testing through longitudinal imaging on the cellular scale

Comparison of Adaptive Optics Scanning Light Ophthalmoscopic Fluorescein Angiography and Offset Pinhole Imaging

Recent advances to the adaptive optics scanning light ophthalmoscope (AOSLO) have enabled finer in vivo assessment of the human retinal microvasculature. AOSLO confocal reflectance imaging has been coupled with oral fluorescein angiography (FA), enabling simultaneous acquisition of structural and perfusion images. AOSLO offset pinhole (OP) imaging combined with motion contrast post-processing techniques, are able to create a similar set of structural and perfusion images without the use of exogenous contrast agent. In this study, we evaluate the similarities and differences of the structural and perfusion images obtained by either method, in healthy control subjects and in patients with retinal vasculopathy including hypertensive retinopathy, diabetic retinopathy, and retinal vein occlusion. Our results show that AOSLO OP motion contrast provides perfusion maps comparable to those obtained with AOSLO FA, while AOSLO OP reflectance images provide additional information such as vessel wall fine structure not as readily visible in AOSLO confocal reflectance images. AOSLO OP offers a non-invasive alternative to AOSLO FA without the need for any exogenous contrast agent

Effects of Intraframe Distortion on Measures of Cone Mosaic Geometry from Adaptive Optics Scanning Light Ophthalmoscopy

Purpose: To characterize the effects of intraframe distortion due to involuntary eye motion on measures of cone mosaic geometry derived from adaptive optics scanning light ophthalmoscope (AOSLO) images. Methods: We acquired AOSLO image sequences from 20 subjects at 1.0, 2.0, and 5.08 temporal from fixation. An expert grader manually selected 10 minimally distorted reference frames from each 150-frame sequence for subsequent registration. Cone mosaic geometry was measured in all registered images (n ¼ 600) using multiple metrics, and the repeatability of these metrics was used to assess the impact of the distortions from each reference frame. In nine additional subjects, we compared AOSLO-derived measurements to those from adaptive optics (AO)-fundus images, which do not contain system-imposed intraframe distortions. Results: We observed substantial variation across subjects in the repeatability of density (1.2%–8.7%), inter-cell distance (0.8%–4.6%), percentage of six-sided Voronoi cells (0.8%–10.6%), and Voronoi cell area regularity (VCAR) (1.2%–13.2%). The average of all metrics extracted from AOSLO images (with the exception of VCAR) was not significantly different than those derived from AO-fundus images, though there was variability between individual images. Conclusions: Our data demonstrate that the intraframe distortion found in AOSLO images can affect the accuracy and repeatability of cone mosaic metrics. It may be possible to use multiple images from the same retinal area to approximate a ‘‘distortionless’’ image, though more work is needed to evaluate the feasibility of this approach. Translational Relevance: Even in subjects with good fixation, images from AOSLOs contain intraframe distortions due to eye motion during scanning. The existence of these artifacts emphasizes the need for caution when interpreting results derived from scanning instruments

Photoreceptor Inner Segment Morphology in Best Vitelliform Macular Dystrophy

PURPOSE To characterize outer retina structure in best vitelliform macular dystrophy (BVMD) and to determine the effect of macular lesions on overlying and adjacent photoreceptors. METHODS Five individuals with BVMD were followed prospectively with spectral domain optical coherence tomography and confocal and nonconfocal split-detector adaptive optics scanning light ophthalmoscopy (AOSLO). The AOSLO cone photoreceptor mosaic images were obtained within and around retinal lesions. Cone density was measured inside and outside lesions. In 2 subjects, densities were compared with published measurements acquired ∼2.5 years before. One subject was imaged 3 times over a 5-month period. RESULTS The AOSLO imaging demonstrated that photoreceptor morphology within BVMD retinal lesions was highly variable depending on the disease stage, with photoreceptor structure present even in advanced disease. The AOSLO imaging was repeatable even in severe disease over short-time and long-time intervals. Photoreceptor density was normal in retinal areas immediately adjacent to lesions and stable over ∼2.5 years. Mobile disk-like structures possibly representing subretinal macrophages were also observed. CONCLUSION Combined confocal and nonconfocal split-detector AOSLO imaging reveals substantial variability within clinical lesions in all stages of BVMD. Longitudinal cellular photoreceptor imaging could prove a powerful tool for understanding disease progression and monitoring emerging therapeutic treatment response in inherited degenerations such as BVMD

\u3cem\u3eIn vivo\u3c/em\u3e Imaging of Human Retinal Microvasculature Using Adaptive Optics Scanning Light Ophthalmoscope Fluorescein Angiography

The adaptive optics scanning light ophthalmoscope (AOSLO) allows visualization of microscopic structures of the human retina in vivo. In this work, we demonstrate its application in combination with oral and intravenous (IV) fluorescein angiography (FA) to the in vivo visualization of the human retinal microvasculature. Ten healthy subjects ages 20 to 38 years were imaged using oral (7 and/or 20 mg/kg) and/or IV (500 mg) fluorescein. In agreement with current literature, there were no adverse effects among the patients receiving oral fluorescein while one patient receiving IV fluorescein experienced some nausea and heaving. We determined that all retinal capillary beds can be imaged using clinically accepted fluorescein dosages and safe light levels according to the ANSI Z136.1-2000 maximum permissible exposure. As expected, the 20 mg/kg oral dose showed higher image intensity for a longer period of time than did the 7 mg/kg oral and the 500 mg IV doses. The increased resolution of AOSLO FA, compared to conventional FA, offers great opportunity for studying physiological and pathological vascular processes