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1.  In vivo imaging of microscopic structures in the rat retina 
Purpose
The ability to resolve single retinal cells in rodents in vivo has applications in rodent models of the visual system and retinal disease. We have characterized the performance of a fluorescence adaptive optics scanning laser ophthalmoscope (fAOSLO) that provides cellular and subcellular imaging of rat retina in vivo.
Methods
Green fluorescent protein (eGFP) was expressed in retinal ganglion cells of normal Sprague Dawley rats via intravitreal injections of adeno-associated viral vectors. Simultaneous reflectance and fluorescence retinal images were acquired using the fAOSLO. fAOSLO resolution was characterized by comparing in vivo images with subsequent imaging of retinal sections from the same eyes using confocal microscopy.
Results
Retinal capillaries and eGFP-labeled ganglion cell bodies, dendrites, and axons were clearly resolved in vivo with adaptive optics (AO). AO correction reduced the total root mean square wavefront error, on average, from 0.30 μm to 0.05 μm (1.7-mm pupil). The full width at half maximum (FWHM) of the average in vivo line-spread function (LSF) was ∼1.84 μm, approximately 82% greater than the FWHM of the diffraction-limited LSF.
Conclusions
With perfect aberration compensation, the in vivo resolution in the rat eye could be ∼2× greater than that in the human eye due to its large numerical aperture (∼0.43). While the fAOSLO corrects a substantial fraction of the rat eye's aberrations, direct measurements of retinal image quality reveal some blur beyond that expected from diffraction. Nonetheless, subcellular features can be resolved, offering promise for using AO to investigate the rodent eye in vivo with high resolution.
doi:10.1167/iovs.09-3675
PMCID: PMC2873188  PMID: 19578019
2.  In vivo fluorescent imaging of the mouse retina using adaptive optics 
Optics letters  2007;32(6):659-661.
In vivo imaging of the mouse retina using visible and near infrared wavelengths does not achieve diffraction-limited resolution due to wavefront aberrations induced by the eye. Considering the pupil size and axial dimension of the eye, it is expected that unaberrated imaging of the retina would have a transverse resolution of 2 μm. Higher-order aberrations in retinal imaging of human can be compensated for by using adaptive optics. We demonstrate an adaptive optics system for in vivo imaging of fluorescent structures in the retina of a mouse, using a microelectromechanical system membrane mirror and a Shack–Hartmann wavefront sensor that detects fluorescent wavefront.
PMCID: PMC2808135  PMID: 17308593
3.  Woofer-tweeter adaptive optics scanning laser ophthalmoscopic imaging based on Lagrange-multiplier damped least-squares algorithm 
Biomedical Optics Express  2011;2(7):1986-2004.
We implemented a Lagrange-multiplier (LM)-based damped least-squares (DLS) control algorithm in a woofer-tweeter dual deformable-mirror (DM) adaptive optics scanning laser ophthalmoscope (AOSLO). The algorithm uses data from a single Shack-Hartmann wavefront sensor to simultaneously correct large-amplitude low-order aberrations by a woofer DM and small-amplitude higher-order aberrations by a tweeter DM. We measured the in vivo performance of high resolution retinal imaging with the dual DM AOSLO. We compared the simultaneous LM-based DLS dual DM controller with both single DM controller, and a successive dual DM controller. We evaluated performance using both wavefront (RMS) and image quality metrics including brightness and power spectrum. The simultaneous LM-based dual DM AO can consistently provide near diffraction-limited in vivo routine imaging of human retina.
doi:10.1364/BOE.2.001986
PMCID: PMC3130583  PMID: 21750774
(010.1080) Active or adaptive optics; (220.1080) Active or adaptive optics; (170.1790) Confocal microscopy; (330.4460) Ophthalmic optics
4.  Optical properties of the mouse eye 
Biomedical Optics Express  2011;2(4):717-738.
The Shack-Hartmann wavefront sensor (SHWS) spots upon which ocular aberration measurements depend have poor quality in mice due to light reflected from multiple retinal layers. We have designed and implemented a SHWS that can favor light from a specific retinal layer and measured monochromatic aberrations in 20 eyes from 10 anesthetized C57BL/6J mice. Using this instrument, we show that mice are myopic, not hyperopic as is frequently reported. We have also measured longitudinal chromatic aberration (LCA) of the mouse eye and found that it follows predictions of the water-filled schematic mouse eye. Results indicate that the optical quality of the mouse eye assessed by measurement of its aberrations is remarkably good, better for retinal imaging than the human eye. The dilated mouse eye has a much larger numerical aperture (NA) than that of the dilated human eye (0.5 NA vs. 0.2 NA), but it has a similar amount of root mean square (RMS) higher order aberrations compared to the dilated human eye. These measurements predict that adaptive optics based on this method of wavefront sensing will provide improvements in retinal image quality and potentially two times higher lateral resolution than that in the human eye.
doi:10.1364/BOE.2.000717
PMCID: PMC3072116  PMID: 21483598
(170.4460) Medical optics and biotechnology: Ophthalmic optics and devices; (330.5370) Vision, color, and visual optics: Physiological optics; (330.4300) Vision system - noninvasive assessment; (110.1080) Active or adaptive optics; (330.7324) Vision, color, and visual optics: Visual optics, comparative animal models
5.  Extracting and compensating dispersion mismatch in ultrahigh-resolution Fourier domain OCT imaging of the retina 
Optics Express  2012;20(23):25357-25368.
We present a numerical approach to extract the dispersion mismatch in ultrahigh-resolution Fourier domain optical coherence tomography (OCT) imaging of the retina. The method draws upon an analogy with a Shack-Hartmann wavefront sensor. By exploiting mathematical similarities between the expressions for aberration in optical imaging and dispersion mismatch in spectral / Fourier domain OCT, Shack-Hartmann principles can be extended from the two-dimensional paraxial wavevector space (or the x-y plane in the spatial domain) to the one-dimensional wavenumber space (or the z-axis in the spatial domain). For OCT imaging of the retina, different retinal layers, such as the retinal nerve fiber layer (RNFL), the photoreceptor inner and outer segment junction (IS/OS), or all the retinal layers near the retinal pigment epithelium (RPE) can be used as point source beacons in the axial direction, analogous to point source beacons used in conventional two-dimensional Shack-Hartman wavefront sensors for aberration characterization. Subtleties regarding speckle phenomena in optical imaging, which affect the Shack-Hartmann wavefront sensor used in adaptive optics, also occur analogously in this application. Using this approach and carefully suppressing speckle, the dispersion mismatch in spectral / Fourier domain OCT retinal imaging can be successfully extracted numerically and used for numerical dispersion compensation to generate sharper, ultrahigh-resolution OCT images.
doi:10.1364/OE.20.025357
PMCID: PMC3601734  PMID: 23187353
(170.3880) Medical and biological imaging; (170.4500) Optical coherence tomography; (170.4470) Ophthalmology; (260.2030) Dispersion
6.  Measuring directionality of the retinal reflection with a Shack-Hartmann wavefront sensor 
Optics express  2009;17(25):23085-23097.
The directional sensitivity of the retina, known as the Stiles-Crawford effect (SCE), originates from the waveguide property of photoreceptors. This effect has been extensively studied in normal and pathologic eyes using highly customized optical instrumentation. Here we investigate a new approach based on a Shack-Hartmann wavefront sensor (SHWS), a technology that has been traditionally employed for measuring wave aberrations (phase) of the eye and is available in clinics. Using a modified research-grade SHWS, we demonstrate in five healthy subjects and at four retinal eccentricities that intensity information can be readily extracted from the SHWS measurement and the spatial distribution of which is consistent with that produced by the optical SCE. The technique is found sufficiently sensitive even at near-infrared wavelengths where the optical SCE is faint. We demonstrate that the optical SCE signal is confined to the core of the SHWS spots with the tails being diffuse and non-directional, suggesting cones fail to recapture light that is multiply scattered in the retina. The high sensitivity of the SHWS to the optical SCE raises concern as to how this effect, intrinsic to the retina, may impact the SHWS measurement of ocular aberrations.
PMCID: PMC3113598  PMID: 20052235
7.  Adaptive-optics optical coherence tomography for high-resolution and high-speed 3D retinal in vivo imaging 
Optics express  2005;13(21):8532-8546.
We have combined Fourier-domain optical coherence tomography (FD-OCT) with a closed-loop adaptive optics (AO) system using a Hartmann-Shack wavefront sensor and a bimorph deformable mirror. The adaptive optics system measures and corrects the wavefront aberration of the human eye for improved lateral resolution (~4 μm) of retinal images, while maintaining the high axial resolution (~6 μm) of stand alone OCT. The AO-OCT instrument enables the three-dimensional (3D) visualization of different retinal structures in vivo with high 3D resolution (4×4×6 μm). Using this system, we have demonstrated the ability to image microscopic blood vessels and the cone photoreceptor mosaic.
PMCID: PMC2605068  PMID: 19096728
8.  Morphology and Topography of Retinal Pericytes in the Living Mouse Retina Using In Vivo Adaptive Optics Imaging and Ex Vivo Characterization 
Purpose.
To noninvasively image retinal pericytes in the living eye and characterize NG2-positive cell topography and morphology in the adult mouse retina.
Methods.
Transgenic mice expressing fluorescent pericytes (NG2, DsRed) were imaged using a two-channel, adaptive optics scanning laser ophthalmoscope (AOSLO). One channel imaged vascular perfusion with near infrared light. A second channel simultaneously imaged fluorescent retinal pericytes. Mice were also imaged using wide-field ophthalmoscopy. To confirm in vivo imaging, five eyes were enucleated and imaged in flat mount with conventional fluorescent microscopy. Cell topography was quantified relative to the optic disc.
Results.
We observed strong DsRed fluorescence from NG2-positive cells. AOSLO revealed fluorescent vascular mural cells enveloping all vessels in the living retina. Cells were stellate on larger venules, and showed banded morphology on arterioles. NG2-positive cells indicative of pericytes were found on the smallest capillaries of the retinal circulation. Wide-field SLO enabled quick assessment of NG2-positive distribution, but provided insufficient resolution for cell counts. Ex vivo microscopy showed relatively even topography of NG2-positive capillary pericytes at eccentricities more than 0.3 mm from the optic disc (515 ± 94 cells/mm2 of retinal area).
Conclusions.
We provide the first high-resolution images of retinal pericytes in the living animal. Subcellular resolution enabled morphological identification of NG2-positive cells on capillaries showing classic features and topography of retinal pericytes. This report provides foundational basis for future studies that will track and quantify pericyte topography, morphology, and function in the living retina over time, especially in the progression of microvascular disease.
We provide the first high-resolution images of retinal pericytes in the living animal. Adaptive optics ophthalmoscopy provided subcellular resolution allowing for identification of fluorescent NG2-positive cells on capillaries, venules, and arterioles in vivo. Ex vivo imaging confirmed in vivo data.
doi:10.1167/iovs.13-12581
PMCID: PMC3869420  PMID: 24150762
diabetic retinopathy; neurovascular coupling; adaptive optics; capillaries; microvascular network
9.  Wavefront sensorless adaptive optics ophthalmoscopy in the human eye 
Optics express  2011;19(15):14160-14171.
Wavefront sensor noise and fidelity place a fundamental limit on achievable image quality in current adaptive optics ophthalmoscopes. Additionally, the wavefront sensor ‘beacon’ can interfere with visual experiments. We demonstrate real-time (25 Hz), wavefront sensorless adaptive optics imaging in the living human eye with image quality rivaling that of wavefront sensor based control in the same system. A stochastic parallel gradient descent algorithm directly optimized the mean intensity in retinal image frames acquired with a confocal adaptive optics scanning laser ophthalmoscope (AOSLO). When imaging through natural, undilated pupils, both control methods resulted in comparable mean image intensities. However, when imaging through dilated pupils, image intensity was generally higher following wavefront sensor-based control. Despite the typically reduced intensity, image contrast was higher, on average, with sensorless control. Wavefront sensorless control is a viable option for imaging the living human eye and future refinements of this technique may result in even greater optical gains.
PMCID: PMC3178895  PMID: 21934779
10.  Wavefront sensorless adaptive optics ophthalmoscopy in the human eye 
Optics Express  2011;19(15):14160-14171.
Wavefront sensor noise and fidelity place a fundamental limit on achievable image quality in current adaptive optics ophthalmoscopes. Additionally, the wavefront sensor ‘beacon’ can interfere with visual experiments. We demonstrate real-time (25 Hz), wavefront sensorless adaptive optics imaging in the living human eye with image quality rivaling that of wavefront sensor based control in the same system. A stochastic parallel gradient descent algorithm directly optimized the mean intensity in retinal image frames acquired with a confocal adaptive optics scanning laser ophthalmoscope (AOSLO). When imaging through natural, undilated pupils, both control methods resulted in comparable mean image intensities. However, when imaging through dilated pupils, image intensity was generally higher following wavefront sensor-based control. Despite the typically reduced intensity, image contrast was higher, on average, with sensorless control. Wavefront sensorless control is a viable option for imaging the living human eye and future refinements of this technique may result in even greater optical gains.
doi:10.1364/OE.19.014160
PMCID: PMC3178895  PMID: 21934779
(000.3860) Mathematical methods in physics; (110.1080) Active or Adaptive optics; (330.4460) Ophthalmic optics and devices
11.  Intersubject Variability of Foveal Cone Photoreceptor Density in Relation to Eye Length 
The smallest foveal cones can now be visualized using adaptive optics scanning laser ophthalmoscopy. Cone density is not affected by eye length inside the foveola.
Purpose.
Adaptive optics scanning laser ophthalmoscopy (AOSLO) under optimized wavefront correction allows for routine imaging of foveal cone photoreceptors. The intersubject variability of foveal cone density was measured and its relation to eye length evaluated.
Methods.
AOSLO was used to image 18 healthy eyes with axial lengths from 22.86 to 28.31 mm. Ocular biometry and an eye model were used to estimate the retinal magnification factor. Individual cones in the AOSLO images were labeled, and the locations were used to generate topographic maps representing the spatial distribution of density. Representative retinal (cones/mm2) and angular (cones/deg2) cone densities at specific eccentricities were calculated from these maps.
Results.
The entire foveal cone mosaic was resolved in four eyes, whereas the cones within 0.03 mm eccentricity remained unresolved in most eyes. The preferred retinal locus deviated significantly (P < 0.001) from the point of peak cone density for all except one individual. A significant decrease in retinal density (P < 0.05) with increasing axial length was observed at 0.30 mm eccentricity but not closer. Longer, more myopic eyes generally had higher angular density near the foveal center than the shorter eyes, but by 1°, this difference was nullified by retinal expansion, and so angular densities across all eyes were similar.
Conclusions.
The AOSLO can resolve the smallest foveal cones in certain eyes. Although myopia causes retinal stretching in the fovea, its effect within the foveola is confounded by factors other than cone density that have high levels of intersubject variability.
doi:10.1167/iovs.10-5499
PMCID: PMC3055782  PMID: 20688730
12.  Adaptive optics photoacoustic microscopy 
Optics Express  2010;18(21):21770-21776.
We have developed an adaptive optics photoacoustic microscope (AO-PAM) for high-resolution imaging of biological tissues, especially the retina. To demonstrate the feasibility of AO-PAM we first designed the AO system to correct the wavefront errors of the illuminating light of PAM. The aberrations of the optical system delivering the illuminating light to the sample in PAM was corrected with a close-loop AO system consisting of a 141-element MEMS-based deformable mirror (DM) and a Shack-Hartmann (SH) wavefront sensor operating at 15 Hz. The photoacoustic signal induced by the illuminating laser beam was detected by a custom-built needle ultrasonic transducer. When the wavefront errors were corrected by the AO system, the lateral resolution of PAM was measured to be better than 2.5 μm using a low NA objective lens. We tested the system on imaging ex vivo ocular samples, e.g., the ciliary body and retinal pigment epithelium (RPE) of a pig eye. The AO-PAM images showed significant quality improvement. For the first time we were able to resolve single RPE cells with PAM.
PMCID: PMC3289054  PMID: 20941077
13.  Adaptive optics photoacoustic microscopy 
Optics Express  2010;18(21):21770-21776.
We have developed an adaptive optics photoacoustic microscope (AO-PAM) for high-resolution imaging of biological tissues, especially the retina. To demonstrate the feasibility of AO-PAM we first designed the AO system to correct the wavefront errors of the illuminating light of PAM. The aberrations of the optical system delivering the illuminating light to the sample in PAM was corrected with a close-loop AO system consisting of a 141-element MEMS-based deformable mirror (DM) and a Shack-Hartmann (SH) wavefront sensor operating at 15 Hz. The photoacoustic signal induced by the illuminating laser beam was detected by a custom-built needle ultrasonic transducer. When the wavefront errors were corrected by the AO system, the lateral resolution of PAM was measured to be better than 2.5 µm using a low NA objective lens. We tested the system on imaging ex vivo ocular samples, e.g., the ciliary body and retinal pigment epithelium (RPE) of a pig eye. The AO-PAM images showed significant quality improvement. For the first time we were able to resolve single RPE cells with PAM.
doi:10.1364/OE.18.021770
PMCID: PMC3289054  PMID: 20941077
(110.1085) Adaptive imaging; (110.5120) Photoacoustic imaging; (110.0180) Microscopy
14.  Live imaging using adaptive optics with fluorescent protein guide-stars 
Optics Express  2012;20(14):15969-15982.
Spatially and temporally dependent optical aberrations induced by the inhomogeneous refractive index of live samples limit the resolution of live dynamic imaging. We introduce an adaptive optical microscope with a direct wavefront sensing method using a Shack-Hartmann wavefront sensor and fluorescent protein guide-stars for live imaging. The results of imaging Drosophila embryos demonstrate its ability to correct aberrations and achieve near diffraction limited images of medial sections of large Drosophila embryos. GFP-polo labeled centrosomes can be observed clearly after correction but cannot be observed before correction. Four dimensional time lapse images are achieved with the correction of dynamic aberrations. These studies also demonstrate that the GFP-tagged centrosome proteins, Polo and Cnn, serve as excellent biological guide-stars for adaptive optics based microscopy.
doi:10.1364/OE.20.015969
PMCID: PMC3601654  PMID: 22772285
(110.1080) Active or adaptive optics; (010.7350) Wave-front sensing; (180.2520) Fluorescence microscopy; (180.6900) Three-dimensional microscopy; (170.3880) Medical and biological imaging
15.  Closed loop adaptive optics for microscopy without a wavefront sensor 
Proceedings of SPIE  2010;7570:10.1117/12.840943.
A three-dimensional wide-field image of a small fluorescent bead contains more than enough information to accurately calculate the wavefront in the microscope objective back pupil plane using the phase retrieval technique. The phase-retrieved wavefront can then be used to set a deformable mirror to correct the point-spread function (PSF) of the microscope without the use of a wavefront sensor. This technique will be useful for aligning the deformable mirror in a widefield microscope with adaptive optics and could potentially be used to correct aberrations in samples where small fluorescent beads or other point sources are used as reference beacons. Another advantage is the high resolution of the retrieved wavefont as compared with current Shack-Hartmann wavefront sensors. Here we demonstrate effective correction of the PSF in 3 iterations. Starting from a severely aberrated system, we achieve a Strehl ratio of 0.78 and a greater than 10-fold increase in maximum intensity.
doi:10.1117/12.840943
PMCID: PMC3877333  PMID: 24392198
Adaptive Optics; Microscopy; Biomedical Imaging
16.  Adaptive optics scanning laser ophthalmoscope with integrated wide-field retinal imaging and tracking 
We have developed a new, unified implementation of the adaptive optics scanning laser ophthalmoscope (AOSLO) incorporating a wide-field line-scanning ophthalmoscope (LSO) and a closed-loop optical retinal tracker. AOSLO raster scans are deflected by the integrated tracking mirrors so that direct AOSLO stabilization is automatic during tracking. The wide-field imager and large-spherical-mirror optical interface design, as well as a large-stroke deformable mirror (DM), enable the AOSLO image field to be corrected at any retinal coordinates of interest in a field of >25 deg. AO performance was assessed by imaging individuals with a range of refractive errors. In most subjects, image contrast was measurable at spatial frequencies close to the diffraction limit. Closed-loop optical (hardware) tracking performance was assessed by comparing sequential image series with and without stabilization. Though usually better than 10 μm rms, or 0.03 deg, tracking does not yet stabilize to single cone precision but significantly improves average image quality and increases the number of frames that can be successfully aligned by software-based post-processing methods. The new optical interface allows the high-resolution imaging field to be placed anywhere within the wide field without requiring the subject to re-fixate, enabling easier retinal navigation and faster, more efficient AOSLO montage capture and stitching.
PMCID: PMC3071649  PMID: 21045887
17.  Visual Function and Cortical Organization in Carriers of Blue Cone Monochromacy 
PLoS ONE  2013;8(2):e57956.
Carriers of blue cone monochromacy have fewer cone photoreceptors than normal. Here we examine how this disruption at the level of the retina affects visual function and cortical organization in these individuals. Visual resolution and contrast sensitivity was measured at the preferred retinal locus of fixation and visual resolution was tested at two eccentric locations (2.5° and 8°) with spectacle correction only. Adaptive optics corrected resolution acuity and cone spacing were simultaneously measured at several locations within the central fovea with adaptive optics scanning laser ophthalmoscopy (AOSLO). Fixation stability was assessed by extracting eye motion data from AOSLO videos. Retinotopic mapping using fMRI was carried out to estimate the area of early cortical regions, including that of the foveal confluence. Without adaptive optics correction, BCM carriers appeared to have normal visual function, with normal contrast sensitivity and visual resolution, but with AO-correction, visual resolution was significantly worse than normal. This resolution deficit is not explained by cone loss alone and is suggestive of an associated loss of retinal ganglion cells. However, despite evidence suggesting a reduction in the number of retinal ganglion cells, retinotopic mapping showed no reduction in the cortical area of the foveal confluence. These results suggest that ganglion cell density may not govern the foveal overrepresentation in the cortex. We propose that it is not the number of afferents, but rather the content of the information relayed to the cortex from the retina across the visual field that governs cortical magnification, as under normal viewing conditions this information is similar in both BCM carriers and normal controls.
doi:10.1371/journal.pone.0057956
PMCID: PMC3585243  PMID: 23469117
18.  Ocular wavefront aberrations in patients with macular diseases 
Retina (Philadelphia, Pa.)  2009;29(9):1356-1363.
Background
There have been reports that by compensating for the ocular aberrations using adaptive optical systems it may be possible to improve the resolution of clinical retinal imaging systems beyond what is now possible. In order to develop such system to observe eyes with retinal disease, understanding of the ocular wavefront aberrations in individuals with retinal disease is required.
Methods
82 eyes of 66 patients with macular disease (epiretinal membrane, macular edema, macular hole etc.) and 85 eyes of 51 patients without retinal disease were studied. Using a ray-tracing wavefront device, each eye was scanned at both small and large pupil apertures and Zernike coefficients up to 6th order were acquired.
Results
In phakic eyes, 3rd order root mean square errors (RMS) in macular disease group were statistically greater than control, an average of 12% for 5mm and 31% for 3mm scan diameters (p<0.021). In pseudophakic eyes, there also was an elevation of 3rd order RMS, on average 57% for 5mm and 51% for 3mm scan diameters (p<0.031).
Conclusion
Higher order wavefront aberrations in eyes with macular disease were greater than in control eyes without disease. Our study suggests that such aberrations may result from irregular or multiple reflecting retinal surfaces. Modifications in wavefront sensor technology will be needed to accurately determine wavefront aberration and allow correction using adaptive optics in eyes with macular irregularities.
doi:10.1097/IAE.0b013e3181a5e657
PMCID: PMC3711882  PMID: 19574950
Macular disease; Ray tracing; Retinal imaging; Wavefront aberration
19.  Comparison of adaptive optics scanning light ophthalmoscopic fluorescein angiography and offset pinhole imaging 
Biomedical Optics Express  2014;5(4):1173-1189.
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.
doi:10.1364/BOE.5.001173
PMCID: PMC3985984  PMID: 24761299
(110.1080) Active or adaptive optics; (290.4210) Multiple scattering; (170.4460) Ophthalmic optics and devices; (170.4470) Ophthalmology
20.  Bringing the Visible Universe into Focus with Robo-AO 
The angular resolution of ground-based optical telescopes is limited by the degrading effects of the turbulent atmosphere. In the absence of an atmosphere, the angular resolution of a typical telescope is limited only by diffraction, i.e., the wavelength of interest, λ, divided by the size of its primary mirror's aperture, D. For example, the Hubble Space Telescope (HST), with a 2.4-m primary mirror, has an angular resolution at visible wavelengths of ~0.04 arc seconds. The atmosphere is composed of air at slightly different temperatures, and therefore different indices of refraction, constantly mixing. Light waves are bent as they pass through the inhomogeneous atmosphere. When a telescope on the ground focuses these light waves, instantaneous images appear fragmented, changing as a function of time. As a result, long-exposure images acquired using ground-based telescopes - even telescopes with four times the diameter of HST - appear blurry and have an angular resolution of roughly 0.5 to 1.5 arc seconds at best.
Astronomical adaptive-optics systems compensate for the effects of atmospheric turbulence. First, the shape of the incoming non-planar wave is determined using measurements of a nearby bright star by a wavefront sensor. Next, an element in the optical system, such as a deformable mirror, is commanded to correct the shape of the incoming light wave. Additional corrections are made at a rate sufficient to keep up with the dynamically changing atmosphere through which the telescope looks, ultimately producing diffraction-limited images.
The fidelity of the wavefront sensor measurement is based upon how well the incoming light is spatially and temporally sampled1. Finer sampling requires brighter reference objects. While the brightest stars can serve as reference objects for imaging targets from several to tens of arc seconds away in the best conditions, most interesting astronomical targets do not have sufficiently bright stars nearby. One solution is to focus a high-power laser beam in the direction of the astronomical target to create an artificial reference of known shape, also known as a 'laser guide star'. The Robo-AO laser adaptive optics system2,3 employs a 10-W ultraviolet laser focused at a distance of 10 km to generate a laser guide star. Wavefront sensor measurements of the laser guide star drive the adaptive optics correction resulting in diffraction-limited images that have an angular resolution of ~0.1 arc seconds on a 1.5-m telescope.
doi:10.3791/50021
PMCID: PMC3622497  PMID: 23426078
Physics; Issue 72; Astronomy; Mechanical Engineering; Astrophysics; Optics; Adaptive optics; lasers; wavefront sensing; robotics; stars; galaxies; imaging; supernova; telescopes
21.  Adaptive optics with pupil tracking for high resolution retinal imaging 
Biomedical Optics Express  2012;3(2):225-239.
Adaptive optics, when integrated into retinal imaging systems, compensates for rapidly changing ocular aberrations in real time and results in improved high resolution images that reveal the photoreceptor mosaic. Imaging the retina at high resolution has numerous potential medical applications, and yet for the development of commercial products that can be used in the clinic, the complexity and high cost of the present research systems have to be addressed. We present a new method to control the deformable mirror in real time based on pupil tracking measurements which uses the default camera for the alignment of the eye in the retinal imaging system and requires no extra cost or hardware. We also present the first experiments done with a compact adaptive optics flood illumination fundus camera where it was possible to compensate for the higher order aberrations of a moving model eye and in vivo in real time based on pupil tracking measurements, without the real time contribution of a wavefront sensor. As an outcome of this research, we showed that pupil tracking can be effectively used as a low cost and practical adaptive optics tool for high resolution retinal imaging because eye movements constitute an important part of the ocular wavefront dynamics.
doi:10.1364/BOE.3.000225
PMCID: PMC3269841  PMID: 22312577
(110.1080) Active or adaptive optics; (100.4999) Pattern recognition, target tracking; (170.4460) Ophthalmic optics and devices; (170.3890) Medical optics instrumentation
22.  Extracting Wavefront Error From Shack-Hartmann Images Using Spatial Demodulation 
PURPOSE:
To determine whether the spatial demodulation processing of Shack-Hartmann images is suitable for extracting wavefront gradients for ocular wavefront sensors.
METHODS:
We developed a custom software program to implement the spatial demodulation technique. To test the algorithm’s performance, we generated simulated spot images and obtained an eye examination image. We generated a collection of simulated aberrated spot images corresponding to: astigmatic wavefront (-5.00 -2.00 × 17), highly aberrated defocus (±20.00 diopters [D]), high-resolution defocus (-0.01 D), and third-order aberrations (trefoil and coma). The eye examination image and its measured Zernike coefficients were obtained from a Shack-Hartmann ocular aberrations system. We evaluated the output from the algorithm in terms of comparing the results to the known Zernike coefficients (for the simulated images) or the previously measured Zernike coefficients (for the eye examination image).
RESULTS:
The spatial demodulation algorithm was able to correctly recover the aberrations to better than 1/100 (0.01) D for the simulated spot images. The processing of the eye examination image yielded results within approximately 1/4 (0.25) D to the values provided by the Shack-Hartmann system.
CONCLUSIONS:
From the set of simulated images and the eye examination image used to test the spatial demodulation technique, it appears that the method is suitable for application in ocular wavefront aberrations Shack-Hartmann systems. The method appears capable of accurately processing high levels of aberrations (±20.00 D) as well as providing high resolution as evidenced by finding the -0.01 D defocus. The method may be especially well suited for processing highly aberrated wavefronts.
PMCID: PMC1851688  PMID: 17124895
23.  Adaptive Optics Scanning Laser Ophthalmoscope-Based Microperimetry 
Optometry and Vision Science  2012;89(5):563-574.
Purpose
To develop and test the application of an adaptive optics scanning laser ophthalmoscope (AOSLO) with eye tracking for high-resolution microperimetric testing.
Methods
An AOSLO was used to conduct simultaneous high-resolution retinal imaging and visual function testing in six normal subjects. Visual sensitivity was measured at test locations between the fovea and 5.0° eccentricity via an increment threshold approach using a 40-trial, yes-no adaptive Bayesian staircase procedure (QUEST). A high-speed eye tracking algorithm enabled real-time video stabilization and the delivery of diffraction-limited Goldmann I-sized stimuli (diameter = 6.5 arcmin = ~32 μm; λ = 680 nm) to targeted retinal loci for 200 msec. Test locations were selected either manually by the examiner or automatically using Fourier-based image registration. Cone spacing was assessed at each test location and sensitivity was plotted against retinal eccentricity. Finally, a 4.2 arcminute stimulus was used to probe the angioscotoma associated with a blood vessel located at 2.5° eccentricity.
Results
Visual sensitivity decreases with eccentricity at a rate of −1.32 dB per degree (R2 = 0.60). The vertical and horizontal errors of the targeted stimulus delivery algorithm averaged 0.81 and 0.89 arcminutes (~4 microns), respectively. Based on a pre-determined exclusion criterion, the stimulus was successfully delivered to its targeted location on 90.1% of all trials. Automated recovery of test locations afforded the repeat testing of the same set of cones over a period of three months. Thresholds measured over a parafoveal blood vessel were 1.96 times higher (p<0.05; one-tailed t-test) than those measured in directly adjacent retina.
Conclusions
AOSLO-based microperimetry has the potential to test visual sensitivity with fine retinotopic precision. Automated recovery of previously-tested locations allows these measures to be tracked longitudinally. This approach can be implemented by researchers interested in establishing the functional correlates of photoreceptor mosaic structure in patients with retinal disease.
doi:10.1097/OPX.0b013e3182512b98
PMCID: PMC3348404  PMID: 22446720
adaptive optics; microperimetry; scanning laser ophthalmoscopy
24.  High-speed volumetric imaging of cone photoreceptors with adaptive optics spectral-domain optical coherence tomography 
Optics express  2006;14(10):4380-4394.
We report the first observations of the three-dimensional morphology of cone photoreceptors in the living human retina. Images were acquired with a high-speed adaptive optics (AO) spectral-domain optical coherence tomography (SD-OCT) camera. The AO system consisted of a Shack-Hartmann wavefront sensor and bimorph mirror (AOptix) that measured and corrected the ocular and system aberrations at a closed-loop rate of 12 Hz. The bimorph mirror was positioned between the XY mechanical scanners and the subject’s eye. The SD-OCT system consisted of a superluminescent diode and a 512 pixel line scan charge-coupled device (CCD) that acquired 75,000 A-scans/s. This rate is more than two times faster than that previously reported. Retinal motion artifacts were minimized by quickly acquiring small volume images of the retina with and without AO compensation. Camera sensitivity was sufficient to detect reflections from all major retinal layers. The regular distribution of bright spots observed within C-scans at the inner segment / outer segment (IS/OS) junctions and at the posterior tips of the OS were found to be highly correlated with one another and with the expected cone spacing. No correlation was found between the posterior tips of the OS and the other retinal layers examined, including the retinal pigment epithelium.
doi:10.1364/OE.14.004380
PMCID: PMC2605071  PMID: 19096730
25.  Wavefront-aberration sorting and correction for a dual-deformable-mirror adaptive-optics system 
Optics letters  2008;33(22):2602-2604.
Many next-generation adaptive optics (AO) systems for vision will have two deformable mirrors (DMs) instead of one: a high-stroke, low-resolution mirror (the woofer) and a low-stroke, high-resolution mirror (the tweeter). We developed a zonal wavefront-control algorithm and validated it using simulations. Rather than separating the woofer and tweeter corrections into two independent control processes or using a modal decomposition, the algorithm we proposed uses wavefront slope measurements from a single Shack–Hartmann wavefront sensor to generate control signals for both deformable mirrors within a single zonal control. A Lagrange multiplier is chosen to integrate two DMs into a single-DM wavefront control, and a damped least-squares control is employed to suppress the correlation between the two DMs.
PMCID: PMC2738988  PMID: 19015681

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