Ethan A. Rossi

The relationship between visual resolution and cone spacing in the human fovea

Visual resolution decreases rapidly outside of the foveal center. The anatomical and physiological basis for this reduction is unclear. We used simultaneous adaptive optics imaging and psychophysical testing to measure cone spacing and resolution across the fovea, and found that resolution was limited by cone spacing only at the foveal center. Immediately outside of the center, resolution was worse than cone spacing predicted and better matched the sampling limit of midget retinal ganglion cells.

Imaging retinal mosaics in the living eye

Adaptive optics imaging of cone photoreceptors has provided unique insight into the structure and function of the human visual system and has become an important tool for both basic scientists and clinicians. Recent advances in adaptive optics retinal imaging instrumentation and methodology have allowed us to expand beyond cone imaging. Multi-wavelength and fluorescence imaging methods with adaptive optics have allowed multiple retinal cell types to be imaged simultaneously. These new methods have recently revealed rod photoreceptors, retinal pigment epithelium (RPE) cells, and the smallest retinal blood vessels. Fluorescence imaging coupled with adaptive optics has been used to examine ganglion cells in living primates. Two-photon imaging combined with adaptive optics can evaluate photoreceptor function non-invasively in the living primate retina.

In vivo imaging of retinal pigment epithelium cells in age related macular degeneration.

Morgan and colleagues demonstrated that the RPE cell mosaic can be resolved in the living human eye non-invasively by imaging the short-wavelength autofluorescence using an adaptive optics (AO) ophthalmoscope. This method, based on the assumption that all subjects have the same longitudinal chromatic aberration (LCA) correction, has proved difficult to use in diseased eyes, and in particular those affected by age-related macular degeneration (AMD). In this work, we improve Morgans method by accounting for chromatic aberration variations by optimizing the confocal aperture axial and transverse placement through an automated iterative maximization of image intensity. The increase in image intensity after algorithmic aperture placement varied depending upon patient and aperture position prior to optimization but increases as large as a factor of 10 were observed. When using a confocal aperture of 3.4 Airy disks in diameter, images were obtained using retinal radiant exposures of less than 2.44 J/cm(2), which is ~22 times below the current ANSI maximum permissible exposure. RPE cell morphologies that were strikingly similar to those seen in postmortem histological studies were observed in AMD eyes, even in areas where the pattern of fluorescence appeared normal in commercial fundus autofluorescence (FAF) images. This new method can be used to study RPE morphology in AMD and other diseases, providing a powerful tool for understanding disease pathogenesis and progression, and offering a new means to assess the efficacy of treatments designed to restore RPE health.

Deletion of the X-linked opsin gene array locus control region (LCR) results in disruption of the cone mosaic

Blue cone monochromacy (BCM) is an X-linked condition in which long- (L) and middle- (M) wavelength-sensitive cone function is absent. Due to the X-linked nature of the condition, female carriers are spared from a full manifestation of the associated defects but can show visual symptoms, including abnormal cone electroretinograms. Here we imaged the cone mosaic in four females carrying an L/M array with deletion of the locus control region, resulting in an absence of L/M opsin gene expression (effectively acting as a cone opsin knockout). On average, they had cone mosaics with reduced density and disrupted organization compared to normal trichromats. This suggests that the absence of opsin in a subset of cones results in their early degeneration, with X-inactivation the likely mechanism underlying phenotypic variability in BCM carriers.

Is visual resolution after adaptive optics correction susceptible to perceptual learning

The visual benefit of correcting high-order aberrations may not be fully realized due to neural mechanisms that compensate for the aberrations of the eye. We examined the extent to which these neural mechanisms might be susceptible to perceptual learning in an adaptive optics (AO)-corrected test of visual resolution. Visual resolution was measured in an adaptive optics scanning laser ophthalmoscope (AOSLO) in 3 conditions: (1) low-order correction (defocus and astigmatism) without AO, (2) 3-mm pupil with AO correction, and (3) 5.81-mm pupil with AO correction. Measurements were made on both eyes in all three conditions before training. Subjects underwent 5 days of monocular training in both AO-corrected conditions and were retested in all three conditions in both eyes after training. The range of minimum angle of resolution (MAR) for each condition was: (1) without AO: 0.53-0.95 arcmin, (2) AO 3-mm pupil: 0.33-0.6 arcmin, and (3) AO 5.81-mm pupil: 0.36-0.56 arcmin. AO correction provided an immediate and significant improvement in visual resolution. There was no significant difference in resolution when correcting aberrations over a 5.81-mm pupil versus a 3-mm pupil. Training on this task provided a minimal improvement in performance. Adaptation to aberrations did not hinder AO correction from providing an immediate visual benefit.

Imaging individual neurons in the retinal ganglion cell layer of the living eye

Significance Retinal ganglion cells are the primary output neurons of the retina that process visual information and transmit it to the brain. We developed a method to reveal these cells in the living eye that does not require the fluorescent labels or high light levels that characterize more invasive methods. The death of these cells causes vision loss in glaucoma, the second leading cause of blindness worldwide. The ability to image these cells in the living eye could accelerate our understanding of their role in normal vision and provide a diagnostic tool for evaluating new therapies for retinal disease. Although imaging of the living retina with adaptive optics scanning light ophthalmoscopy (AOSLO) provides microscopic access to individual cells, such as photoreceptors, retinal pigment epithelial cells, and blood cells in the retinal vasculature, other important cell classes, such as retinal ganglion cells, have proven much more challenging to image. The near transparency of inner retinal cells is advantageous for vision, as light must pass through them to reach the photoreceptors, but it has prevented them from being directly imaged in vivo. Here we show that the individual somas of neurons within the retinal ganglion cell (RGC) layer can be imaged with a modification of confocal AOSLO, in both monkeys and humans. Human images of RGC layer neurons did not match the quality of monkey images for several reasons, including safety concerns that limited the light levels permissible for human imaging. We also show that the same technique applied to the photoreceptor layer can resolve ambiguity about cone survival in age-related macular degeneration. The capability to noninvasively image RGC layer neurons in the living eye may one day allow for a better understanding of diseases, such as glaucoma, and accelerate the development of therapeutic strategies that aim to protect these cells. This method may also prove useful for imaging other structures, such as neurons in the brain.

Closed-loop optical stabilization and digital image registration in adaptive optics scanning light ophthalmoscopy

Eye motion is a major impediment to the efficient acquisition of high resolution retinal images with the adaptive optics (AO) scanning light ophthalmoscope (AOSLO). Here we demonstrate a solution to this problem by implementing both optical stabilization and digital image registration in an AOSLO. We replaced the slow scanning mirror with a two-axis tip/tilt mirror for the dual functions of slow scanning and optical stabilization. Closed-loop optical stabilization reduced the amplitude of eye-movement related-image motion by a factor of 10-15. The residual RMS error after optical stabilization alone was on the order of the size of foveal cones: ~1.66-2.56 μm or ~0.34-0.53 arcmin with typical fixational eye motion for normal observers. The full implementation, with real-time digital image registration, corrected the residual eye motion after optical stabilization with an accuracy of ~0.20-0.25 μm or ~0.04-0.05 arcmin RMS, which to our knowledge is more accurate than any method previously reported.

An adaptive optics imaging system designed for clinical use.

Here we demonstrate a new imaging system that addresses several major problems limiting the clinical utility of conventional adaptive optics scanning light ophthalmoscopy (AOSLO), including its small field of view (FOV), reliance on patient fixation for targeting imaging, and substantial post-processing time. We previously showed an efficient image based eye tracking method for real-time optical stabilization and image registration in AOSLO. However, in patients with poor fixation, eye motion causes the FOV to drift substantially, causing this approach to fail. We solve that problem here by tracking eye motion at multiple spatial scales simultaneously by optically and electronically integrating a wide FOV SLO (WFSLO) with an AOSLO. This multi-scale approach, implemented with fast tip/tilt mirrors, has a large stabilization range of ± 5.6°. Our method consists of three stages implemented in parallel: 1) coarse optical stabilization driven by a WFSLO image, 2) fine optical stabilization driven by an AOSLO image, and 3) sub-pixel digital registration of the AOSLO image. We evaluated system performance in normal eyes and diseased eyes with poor fixation. Residual image motion with incremental compensation after each stage was: 1) ~2-3 arc minutes, (arcmin) 2) ~0.5-0.8 arcmin and, 3) ~0.05-0.07 arcmin, for normal eyes. Performance in eyes with poor fixation was: 1) ~3-5 arcmin, 2) ~0.7-1.1 arcmin and 3) ~0.07-0.14 arcmin. We demonstrate that this system is capable of reducing image motion by a factor of ~400, on average. This new optical design provides additional benefits for clinical imaging, including a steering subsystem for AOSLO that can be guided by the WFSLO to target specific regions of interest such as retinal pathology and real-time averaging of registered images to eliminate image post-processing.

Cone and Rod Loss in Stargardt Disease Revealed by Adaptive Optics Scanning Light Ophthalmoscopy

IMPORTANCE Stargardt disease (STGD1) is characterized by macular atrophy and flecks in the retinal pigment epithelium. The causative ABCA4 gene encodes a protein localizing to photoreceptor outer segments. The pathologic steps by which ABCA4 mutations lead to clinically detectable retinal pigment epithelium changes remain unclear. We investigated early STGD1 using adaptive optics scanning light ophthalmoscopy. OBSERVATIONS Adaptive optics scanning light ophthalmoscopy imaging of 2 brothers with early STGD1 and their unaffected parents was compared with conventional imaging. Cone and rod spacing were increased in both patients (P < .001) with a dark cone appearance. No foveal cones were detected in the older brother. In the younger brother, foveal cones were enlarged with low density (peak cone density, 48.3 × 103 cones/mm2). The ratio of cone to rod spacing was increased in both patients, with greater divergence from normal approaching the foveal center, indicating that cone loss predominates centrally and rod loss increases peripherally. Both parents had normal photoreceptor mosaics. Genetic testing revealed 3 disease-causing mutations. CONCLUSIONS AND RELEVANCE This study provides in vivo images of rods and cones in STGD1. Although the primary clinical features of STGD1 are retinal pigment epithelial lesions, adaptive optics scanning light ophthalmoscopy reveals increased cone and rod spacing in areas that appear normal in conventional images, suggesting that photoreceptor loss precedes clinically detectable retinal pigment epithelial disease in STGD1.

Long-Term Reduction in Infrared Autofluorescence Caused by Infrared Light Below the Maximum Permissible Exposure

PURPOSE Many retinal imaging instruments use infrared wavelengths to reduce the risk of light damage. However, we have discovered that exposure to infrared illumination causes a long-lasting reduction in infrared autofluorescence (IRAF). We have characterized the dependence of this effect on radiant exposure and investigated its origin. METHODS A scanning laser ophthalmoscope was used to obtain IRAF images from two macaques before and after exposure to 790-nm light (15-450 J/cm(2)). Exposures were performed with either raster-scanning or uniform illumination. Infrared autofluorescence images also were obtained in two humans exposed to 790-nm light in a separate study. Humans were assessed with direct ophthalmoscopy, Goldmann visual fields, multifocal ERG, and photopic microperimetry to determine whether these measures revealed any effects in the exposed locations. RESULTS A significant decrease in IRAF after exposure to infrared light was seen in both monkeys and humans. In monkeys, the magnitude of this reduction increased with retinal radiant exposure. Partial recovery was seen at 1 month, with full recovery within 21 months. Consistent with a photochemical origin, IRAF decreases caused by either raster-scanning or uniform illumination were not significantly different. We were unable to detect any effect of the light exposure with any measure other than IRAF imaging. We cannot exclude the possibility that changes could be detected with more sensitive tests or longer follow-up. CONCLUSIONS This long-lasting effect of infrared illumination in both humans and monkeys occurs at exposure levels four to five times below current safety limits. The photochemical basis for this phenomenon remains unknown.