Phillip Bedggood

Variability in Bleach Kinetics and Amount of Photopigment between Individual Foveal Cones

PURPOSE To study the bleaching dynamics of individual foveal cone photoreceptors using an adaptive optics ophthalmoscope. METHODS After dark adaptation, cones were progressively bleached and imaged by a series of flashes of 545-nm to 570-nm light at 12 Hz. Intensity measurements were made within the foveal avascular zone (FAZ) to avoid confounding signals from the inner retinal blood supply. Over 1300 cones in this region were identified and tracked through the imaging sequences. A single subject was used who demonstrated the necessary steady fixation, wide FAZ, and resolvability of cones close to the foveal center. RESULTS The mean intensity of all cones was well-described by first-order kinetics. Individual cones showed marked differences from the mean, both in rate of bleach and amount of photopigment; there was an inverse correlation between these two parameters. A subset of the cones showed large oscillations in intensity consistent with interference from light scattered within the cone outer segment. These cones also bleached more quickly, implying that rapid bleaching induces greater amounts of scatter. CONCLUSIONS Neighboring cones in the fovea display high variability in their optical properties.

Multiconjugate adaptive optics applied to an anatomically accurate human eye model

Aberrations of both astronomical telescopes and the human eye can be successfully corrected with conventional adaptive optics. This produces diffraction-limited imagery over a limited field of view called the isoplanatic patch. A new technique, known as multiconjugate adaptive optics, has been developed recently in astronomy to increase the size of this patch. The key is to model atmospheric turbulence as several flat, discrete layers. A human eye, however, has several curved, aspheric surfaces and a gradient index lens, complicating the task of correcting aberrations over a wide field of view. Here we utilize a computer model to determine the degree to which this technology may be applied to generate high resolution, wide-field retinal images, and discuss the considerations necessary for optimal use with the eye. The Liou and Brennan schematic eye simulates the aspheric surfaces and gradient index lens of real human eyes. We show that the size of the isoplanatic patch of the human eye is significantly increased through multiconjugate adaptive optics.

Optical imaging of human cone photoreceptors directly following the capture of light.

Capture of light in the photoreceptor outer segment initiates a cascade of chemical events that inhibit neurotransmitter release, ultimately resulting in vision. The massed response of the photoreceptor population can be measured non-invasively by electrical recordings, but responses from individual cells cannot be measured without dissecting the retina. Here we used optical imaging to observe individual human cones in the living eye as they underwent bleaching of photopigment and associated phototransduction. The retina was simultaneously stimulated and observed with high intensity visible light at 1 kHz, using adaptive optics. There was marked variability between individual cones in both photosensitivity and pigment optical density, challenging the conventional assumption that photoreceptors act as identical subunits (coefficient of variation in rate of photoisomerization = 23%). There was also a pronounced inverse correlation between these two parameters (p<10−7); the temporal evolution of image statistics revealed this to be a dynamic relationship, with cone waveguiding efficiency beginning a dramatic increase within 3 ms of light onset. Beginning as early as 2 ms after light onset and including half of cells by ∼7 ms, cone intensity showed reversals characteristic of interference phenomena, with greater delays in reversal corresponding to cones with more photopigment (p<10−3). The timing of these changes is argued to best correspond with either the cessation of dark current, or to related events such as changes in intracellular cGMP. Cone intensity also showed fluctuations of high frequency (332±25 Hz) and low amplitude (3.0±0.85%). Other groups have shown similar fluctuations that were directly evoked by light; if this corresponds to the same phenomenon, we propose that the amplitude of fluctuation may be increased by the use of a bright flash followed by a brief pause, to allow recovery of cone circulating current.

Exploring ocular aberrations with a schematic human eye model.

Purpose. Advances in ophthalmic technologies now offer both the measurement and reduction of ocular aberrations by surgically or otherwise honing refraction in the anterior eye. Ocular aberrations, however, are known to change with a multitude of factors, including field position, accommodation level, and age. Thus, although static correction of aberrations provides some vision improvement, this may be less than expected. Methods. In this article, we use an aspheric, variable-focus, age-dependent, gradient index schematic eye to investigate where ocular aberrations arise in the eye and how these change with field position, accommodation, and age. Optical ray tracing was carried out using optical design software ZEMAX, and Seidel aberration analysis was performed with custom written software in MATLAB. Results and Conclusions. Our modeling is consistent with clinical findings that certain corneal aberrations almost balance those arising from the lens. Our calculations also support the general notion that, by optical sculpting, corneal aberrations can be adjusted to completely balance out those of the lens. This can effectively eliminate the eyes total monochromatic aberrations, but for only one retinal image point at a time. Centered on this point of minimal aberration is a region (the isoplanatic patch) within which the aberrations produce a point spread smaller than some tolerable limit. Also, using available evidence in the literature concerning changes in critical ocular parameters with age and accommodation, our modeling results parallel established clinical findings, and additionally indicate that the major source of aberration change can be attributed to the gradient index distribution in the lens.

Limitations to adaptive optics image quality in rodent eyes

Adaptive optics (AO) retinal image quality of rodent eyes is inferior to that of human eyes, despite the promise of greater numerical aperture. This paradox challenges several assumptions commonly made in AO imaging, assumptions which may be invalidated by the very high power and dioptric thickness of the rodent retina. We used optical modeling to compare the performance of rat and human eyes under conditions that tested the validity of these assumptions. Results showed that AO image quality in the human eye is robust to positioning errors of the AO corrector and to differences in imaging depth and wavelength compared to the wavefront beacon. In contrast, image quality in the rat eye declines sharply with each of these manipulations, especially when imaging off-axis. However, some latitude does exist to offset these manipulations against each other to produce good image quality.

Analysis of contrast and motion signals generated by human blood constituents in capillary flow.

The flow of individual corpuscles through retinal capillaries may now be observed noninvasively by using adaptive optics (AO). To explore their imaging properties, we imaged retinal capillary flow in two healthy subjects at 593 nm with a flood-based AO ophthalmoscope, at a variety of retinal locations and levels of defocus. The image intensity of red cells and plasma depends upon capillary depth relative to focus: red cells appear brighter than background, and plasma darker, for capillaries posterior to focus. The reverse is true for capillaries anterior to focus. Contrast reversals were obtained over 0.05 D (∼14 μm), which are well within the typical undulations in depth of retinal capillaries. We relate these observations to phase-contrast defocusing microscopy. This defocusing effect confounds flow measurements, which rely on correlation of image intensity between successive locations along the same capillary, a requirement made further difficult by high physiological variability in flow. Peak correlation was maintained >0.25 over a distance of 22±15 μm (roughly the spacing between red cells) and over a duration of 154±49 ms (roughly eight times the temporal period between red cells). We provide a 2D correlogram approach that significantly improves robustness in the face of optical and physiological variability, compared to the traditional spatiotemporal plot, without requiring additional data.

Evidence of Flicker-Induced Functional Hyperaemia in the Smallest Vessels of the Human Retinal Blood Supply.

Regional changes in blood flow are initiated within neural tissue to help fuel local differences in neural activity. Classically, this response was thought to arise only in larger arterioles and venules. However, recently, it has been proposed that a) the smallest vessels of the circulation make a comparable contribution, and b) the response should be localised intermittently along such vessels, due to the known distribution of contractile mural cells. To assess these hypotheses in human neural tissue in vivo, we imaged the retinal microvasculature (diameters 3–28 μm) non-invasively, using adaptive optics, before and after delivery of focal (360 μm) patches of flickering visible light. Our results demonstrated a definite average response in 35% of all vessel segments analysed. In these responding vessels, the magnitude of proportional dilation (mean ± SEM for pre-capillary arterioles 13 ± 5%, capillaries 31 ± 8%, and post-capillary venules 10 ± 3%) is generally far greater than the magnitudes we and others have measured in the larger retinal vessels, supporting proposition a) above. The dilations observed in venules were unexpected based on previous animal work, and may be attributed either to differences in stimulus or species. Response heterogeneity across the network was high; responses were also heterogeneous along individual vessels (45% of vessel segments showed demonstrable locality in their response). These observations support proposition b) above. We also observed a definite average constriction across 7% of vessel segments (mean ± SEM constriction for capillaries -16 ± 3.2%, and post-capillary venules -18 ± 12%), which paints a picture of dynamic redistribution of flow throughout the smallest vessel networks in the retina in response to local, stimulus-driven metabolic demand.

Quantitative spatial and temporal analysis of fluorescein angiography dynamics in the eye.

Purpose We describe a novel approach to analyze fluorescein angiography to investigate fluorescein flow dynamics in the rat posterior retina as well as identify abnormal areas following laser photocoagulation. Methods Experiments were undertaken in adult Long Evans rats. Using a rodent retinal camera, videos were acquired at 30 frames per second for 30 seconds following intravenous introduction of sodium fluorescein in a group of control animals (n = 14). Videos were image registered and analyzed using principle components analysis across all pixels in the field. This returns fluorescence intensity profiles from which, the half-rise (time to 50% brightness), half-fall (time for 50% decay) back to an offset (plateau level of fluorescence). We applied this analysis to video fluorescein angiography data collected 30 minutes following laser photocoagulation in a separate group of rats (n = 7). Results Pixel-by-pixel analysis of video angiography clearly delineates differences in the temporal profiles of arteries, veins and capillaries in the posterior retina. We find no difference in half-rise, half-fall or offset amongst the four quadrants (inferior, nasal, superior, temporal). We also found little difference with eccentricity. By expressing the parameters at each pixel as a function of the number of standard deviation from the average of the entire field, we could clearly identify the spatial extent of the laser injury. Conclusions This simple registration and analysis provides a way to monitor the size of vascular injury, to highlight areas of subtle vascular leakage and to quantify vascular dynamics not possible using current fluorescein angiography approaches. This can be applied in both laboratory and clinical settings for in vivo dynamic fluorescent imaging of vasculature.

Comparison of sorting algorithms to increase the range of Hartmann-Shack aberrometry

Recently many software-based approaches have been suggested for improving the range and accuracy of Hartmann-Shack aberrometry. We compare the performance of four representative algorithms, with a focus on aberrometry for the human eye. Algorithms vary in complexity from the simplistic traditional approach to iterative spline extrapolation based on prior spot measurements. Range is assessed for a variety of aberration types in isolation using computer modeling, and also for complex wavefront shapes using a real adaptive optics system. The effects of common sources of error for ocular wavefront sensing are explored. The results show that the simplest possible iterative algorithm produces comparable range and robustness compared to the more complicated algorithms, while keeping processing time minimal to afford real-time analysis.

Contrast-based sensorless adaptive optics for retinal imaging.

Conventional adaptive optics ophthalmoscopes use wavefront sensing methods to characterize ocular aberrations for real-time correction. However, there are important situations in which the wavefront sensing step is susceptible to difficulties that affect the accuracy of the correction. To circumvent these, wavefront sensorless adaptive optics (or non-wavefront sensing AO; NS-AO) imaging has recently been developed and has been applied to point-scanning based retinal imaging modalities. In this study we show, for the first time, contrast-based NS-AO ophthalmoscopy for full-frame in vivo imaging of human and animal eyes. We suggest a robust image quality metric that could be used for any imaging modality, and test its performance against other metrics using (physical) model eyes.