Christina Schwarz

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.

Impact on stereo-acuity of two presbyopia correction approaches: monovision and small aperture inlay

Some of the different currently applied approaches that correct presbyopia may reduce stereovision. In this work, stereo-acuity was measured for two methods: (1) monovision and (2) small aperture inlay in one eye. When performing the experiment, a prototype of a binocular adaptive optics vision analyzer was employed. The system allowed simultaneous measurement and manipulation of the optics in both eyes of a subject. The apparatus incorporated two programmable spatial light modulators: one phase-only device using liquid crystal on silicon technology for wavefront manipulation and one intensity modulator for controlling the exit pupils. The prototype was also equipped with a stimulus generator for creating retinal disparity based on two micro-displays. The three-needle test was programmed for characterizing stereo-acuity. Subjects underwent a two-alternative forced-choice test. The following cases were tested for the stimulus placed at distance: (a) natural vision; (b) 1.5 D monovision; (c) 0.75 D monovision; (d) natural vision and small pupil; (e) 0.75 D monovision and small pupil. In all cases the standard pupil diameter was 4 mm and the small pupil diameter was 1.6 mm. The use of a small aperture significantly reduced the negative impact of monovision on stereopsis. The results of the experiment suggest that combining micro-monovision with a small aperture, which is currently being implemented as a corneal inlay, can yield values of stereoacuity close to those attained under normal binocular vision.

Binocular visual acuity for the correction of spherical aberration in polychromatic and monochromatic light.

Correction of spherical (SA) and longitudinal chromatic aberrations (LCA) significantly improves monocular visual acuity (VA). In this work, the visual effect of SA correction in polychromatic and monochromatic light on binocular visual performance is investigated. A liquid crystal based binocular adaptive optics visual analyzer capable of operating in polychromatic light is employed in this study. Binocular VA improves when SA is corrected and LCA effects are reduced separately and in combination, resulting in the highest value for SA correction in monochromatic light. However, the binocular summation ratio is highest for the baseline condition of uncorrected SA in polychromatic light. Although SA correction in monochromatic light has a greater impact monocularly than binocularly, bilateral correction of both SA and LCA may further improve binocular spatial visual acuity which may support the use of aspheric-achromatic ophthalmic devices, in particular, intraocular lenses (IOLs).

Binocular adaptive optics vision analyzer with full control over the complex pupil functions.

We present a binocular adaptive optics vision analyzer fully capable of controlling both amplitude and phase of the two complex pupil functions in each eye of the subject. A special feature of the instrument is its comparatively simple setup. A single reflective liquid crystal on silicon spatial light modulator working in pure phase modulation generates the phase profiles for both pupils simultaneously. In addition, another liquid crystal spatial light modulator working in transmission operates in pure intensity modulation to produce a large variety of pupil masks for each eye. Subjects perform visual tasks through any predefined variations of the complex pupil function for both eyes. As an example of the system efficiency, we recorded images of the stimuli through the system as they were projected at the subjects retina. This instrument proves to be extremely versatile for designing and testing novel ophthalmic elements and simulating visual outcomes, as well as for further research of binocular vision.

Comparison of binocular through-focus visual acuity with monovision and a small aperture inlay

Corneal small aperture inlays provide extended depth of focus as a solution to presbyopia. As this procedure is becoming more popular, it is interesting to compare its performance with traditional approaches, such as monovision. Here, binocular visual acuity was measured as a function of object vergence in three subjects by using a binocular adaptive optics vision analyzer. Visual acuity was measured at two luminance levels (photopic and mesopic) under several optical conditions: 1) natural vision (4 mm pupils, best corrected distance vision), 2) pure-defocus monovision ( + 1.25 D add in the nondominant eye), 3) small aperture monovision (1.6 mm pupil in the nondominant eye), and 4) combined small aperture and defocus monovision (1.6 mm pupil and a + 0.75 D add in the nondominant eye). Visual simulations of a small aperture corneal inlay suggest that the device extends DOF as effectively as traditional monovision in photopic light, in both cases at the cost of binocular summation. However, individual factors, such as aperture centration or sensitivity to mesopic conditions should be considered to assure adequate visual outcomes.

Night Myopia Studied with an Adaptive Optics Visual Analyzer

Purpose Eyes with distant objects in focus in daylight are thought to become myopic in dim light. This phenomenon, often called “night myopia” has been studied extensively for several decades. However, despite its general acceptance, its magnitude and causes are still controversial. A series of experiments were performed to understand night myopia in greater detail. Methods We used an adaptive optics instrument operating in invisible infrared light to elucidate the actual magnitude of night myopia and its main causes. The experimental setup allowed the manipulation of the eyes aberrations (and particularly spherical aberration) as well as the use of monochromatic and polychromatic stimuli. Eight subjects with normal vision monocularly determined their best focus position subjectively for a Maltese cross stimulus at different levels of luminance, from the baseline condition of 20 cd/m2 to the lowest luminance of 22×10−6 cd/m2. While subjects performed the focusing tasks, their eyes defocus and aberrations were continuously measured with the 1050-nm Hartmann-Shack sensor incorporated in the adaptive optics instrument. The experiment was repeated for a variety of controlled conditions incorporating specific aberrations of the eye and chromatic content of the stimuli. Results We found large inter-subject variability and an average of −0.8 D myopic shift for low light conditions. The main cause responsible for night myopia was the accommodation shift occurring at low light levels. Other factors, traditionally suggested to explain night myopia, such as chromatic and spherical aberrations, have a much smaller effect in this mechanism. Conclusions An adaptive optics visual analyzer was applied to study the phenomenon of night myopia. We found that the defocus shift occurring in dim light is mainly due to accommodation errors.

In Vivo Two-Photon Fluorescence Kinetics of Primate Rods and Cones.

Purpose The retinoid cycle maintains vision by regenerating bleached visual pigment through metabolic events, the kinetics of which have been difficult to characterize in vivo. Two-photon fluorescence excitation has been used previously to track autofluorescence directly from retinoids and pyridines in the visual cycle in mouse and frog retinas, but the mechanisms of the retinoid cycle are not well understood in primates. Methods We developed a two-photon fluorescence adaptive optics scanning light ophthalmoscope dedicated to in vivo imaging in anesthetized macaques. Using pulsed light at 730 nm, two-photon fluorescence was captured from rods and cones during light and dark adaptation through the eyes pupil. Results The fluorescence from rods and cones increased with light exposure but at different rates. During dark adaptation, autofluorescence declined, with cone autofluorescence decreasing approximately 4 times faster than from rods. Rates of autofluorescence decrease in rods and cones were approximately 4 times faster than their respective rates of photopigment regeneration. Also, subsets of sparsely distributed cones were less fluorescent than their neighbors immediately following bleach at 565 nm and they were comparable with the S cone mosaic in density and distribution. Conclusions Although other molecules could be contributing, we posit that these fluorescence changes are mediated by products of the retinoid cycle. In vivo two-photon ophthalmoscopy provides a way to monitor noninvasively stages of the retinoid cycle that were previously inaccessible in the living primate eye. This can be used to assess objectively photoreceptor function in normal and diseased retinas.

Wavefront measurements of phase plates combining a point-diffraction interferometer and a Hartmann-Shack sensor

A dual setup composed of a point diffraction interferometer (PDI) and a Hartmann-Shack (HS) wavefront sensor was built to compare the estimates of wavefront aberrations provided by the two different and complementary techniques when applied to different phase plates. Results show that under the same experimental and fitting conditions both techniques provide similar information concerning the wavefront aberration map. When taking into account all Zernike terms up to 6th order, the maximum difference in root-mean-square wavefront error was 0.08 microm, and this reduced up to 0.03 microm when excluding lower-order terms. The effects of the pupil size and the order of the Zernike expansion used to reconstruct the wavefront were evaluated. The combination of the two techniques can accurately measure complicated phase profiles, combining the robustness of the HS and the higher resolution and dynamic range of the PDI.

Binocular visual performance with aberration correction as a function of light level

The extent to which monocular visual performance of subjects with normal amounts of ocular aberrations can be improved with adaptive optics (AO) depends on both the pupil diameter and the luminance for visual testing. Here, the benefit of correction of higher order aberrations for binocular visual performance was assessed over a range of luminances for natural light-adapted pupil sizes with a binocular AO visual simulator. Results show that binocular aberration correction benefits for visual acuity and contrast sensitivity increase with decreasing luminances. Also, the advantage of binocular over monocular viewing increases when visual acuity becomes worse. The findings suggest that binocular summation mitigates poor visual performance under low luminance conditions.

Relationship between wave aberrations and histological features in ex vivo porcine crystalline lenses

Wave aberrations of isolated ex vivo porcine crystalline lenses were measured by using a point-diffraction interferometer. This method allowed us to gain greater insight into the detailed aberration structure of eye lenses showing systematic presence of some dominant aberrations. In order of significance, astigmatism together with spherical aberration, coma, and trefoil are the main aberrations present in all lenses. We found a high correlation between the axis of both astigmatism and trefoil with the Y-shaped suture planes of the lens, revealing a subtle relationship between the induced aberrations and the histological features.