Adaptive optics SLO/OCT for 3D imaging of human photoreceptors in vivo

We present a new instrument that is capable of imaging human photoreceptors in three dimensions. To achieve high lateral resolution, the system incorporates an adaptive optics system. The high axial resolution is achieved through the implementation of optical coherence tomography (OCT). The instrument records simultaneously both, scanning laser ophthalmoscope (SLO) and OCT en-face images, with a pixel to pixel correspondence. The information provided by the SLO is used to correct for transverse eye motion in post-processing. In order to correct for axial eye motion, the instrument is equipped with a high speed axial eye tracker. In vivo images of foveal cones as well as images recorded at an eccentricity from the fovea showing cones and rods are presented.OCIS codes: (170.3890) Medical optics instrumentation, (110.1080) Active or adaptive optics, (170.4470) Ophthalmology, (330.5310) Vision - photoreceptors, (110.4500) Optical coherence tomography     

Adaptive optics optical coherence tomography with dynamic retinal tracking

Adaptive optics optical coherence tomography (AO-OCT) is a highly sensitive and noninvasive method for three dimensional imaging of the microscopic retina. Like all in vivo retinal imaging techniques, however, it suffers the effects of involuntary eye movements that occur even under normal fixation. In this study we investigated dynamic retinal tracking to measure and correct eye motion at KHz rates for AO-OCT imaging. A customized retina tracking module was integrated into the sample arm of the 2nd-generation Indiana AO-OCT system and images were acquired on three subjects. Analyses were developed based on temporal amplitude and spatial power spectra in conjunction with strip-wise registration to independently measure AO-OCT tracking performance. After optimization of the tracker parameters, the system was found to correct eye movements up to 100 Hz and reduce residual motion to 10 µm root mean square. Between session precision was 33 µm. Performance was limited by tracker-generated noise at high temporal frequencies.OCIS codes: (110.1080) Active or adaptive optics, (170.4500) Optical coherence tomography, (120.3890) Medical optics instrumentation, (170.0110) Imaging systems, (170.4470) Ophthalmology, (330.5310) Vision - photoreceptors     

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.OCIS codes: (110.1080) Active or adaptive optics, (120.3890) Medical optics instrumentation, (170.3880) Medical and biological imaging, (170.4470) Ophthalmology, (330.2210) Vision - eye movements     

Phase-sensitive imaging of the outer retina using optical coherence tomography and adaptive optics

The cone photoreceptor's outer segment (OS) experiences changes in optical path length, both in response to visible stimuli and as a matter of its daily course of renewal and shedding. These changes are of interest, to quantify function in healthy cells and assess dysfunction in diseased ones. While optical coherence tomography (OCT), combined with adaptive optics (AO), has permitted unprecedented three-dimensional resolution in the living retina, it has not generally been able to measure these OS dynamics, whose scale is smaller than OCT's axial resolution of a few microns. A possible solution is to take advantage of the phase information encoded in the OCT signal. Phase-sensitive implementations of spectral-domain optical coherence tomography (SD-OCT) have been demonstrated, capable of resolving sample axial displacements much smaller than the imaging wavelength, but these have been limited to ex vivo samples. In this paper we present a novel technique for retrieving phase information from OCT volumes of the outer retina. The key component of our technique is quantification of phase differences within the retina. We provide a quantitative analysis of such phase information and show that-when combined with appropriate methods for filtering and unwrapping-it can improve the sensitivity to OS length change by more than an order of magnitude, down to 45 nm, slightly thicker than a single OS disc. We further show that phase sensitivity drops off with retinal eccentricity, and that the best location for phase imaging is close to the fovea. We apply the technique to the measurement of sub-resolution changes in the OS over matters of hours. Using custom software for registration and tracking, these microscopic changes are monitored in hundreds of cones over time. In two subjects, the OS was found to have average elongation rates of 150 nm/hr, values which agree with our previous findings.     

Handheld simultaneous scanning laser ophthalmoscopy and optical coherence tomography system

Scanning laser ophthalmoscopy (SLO) and optical coherence tomography (OCT) are widely used retinal imaging modalities that can assist in the diagnosis of retinal pathologies. The combination of SLO and OCT provides a more comprehensive imaging system and a method to register OCT images to produce motion corrected retinal volumes. While high quality, bench-top SLO-OCT systems have been discussed in the literature and are available commercially, there are currently no handheld designs. We describe the first design and fabrication of a handheld SLO/spectral domain OCT probe. SLO and OCT images were acquired simultaneously with a combined power under the ANSI limit. High signal-to-noise ratio SLO and OCT images were acquired simultaneously from a normal subject with visible motion artifacts. Fully automated motion estimation methods were performed in post-processing to correct for the inter- and intra-frame motion in SLO images and their concurrently acquired OCT volumes. The resulting set of reconstructed SLO images and the OCT volume were without visible motion artifacts. At a reduced field of view, the SLO resolved parafoveal cones without adaptive optics at a retinal eccentricity of 11° in subjects with good ocular optics. This system may be especially useful for imaging young children and subjects with less stable fixation.OCIS codes: (170.4460) Ophthalmic optics and devices, (080.3620) Lens system design, (170.0110) Imaging systems, (170.5755) Retina scanning, (170.4470) Ophthalmology, (110.4500) Optical coherence tomography, (110.4153) Motion estimation and optical flow     

Understanding the changes of cone reflectance in adaptive optics flood illumination retinal images over three years

Although there is increasing interest in the investigation of cone reflectance variability, little is understood about its characteristics over long time scales. Cone detection and its automation is now becoming a fundamental step in the assessment and monitoring of the health of the retina and in the understanding of the photoreceptor physiology. In this work we provide an insight into the cone reflectance variability over time scales ranging from minutes to three years on the same eye, and for large areas of the retina (≥ 2.0 × 2.0 degrees) at two different retinal eccentricities using a commercial adaptive optics (AO) flood illumination retinal camera. We observed that the difference in reflectance observed in the cones increases with the time separation between the data acquisitions and this may have a negative impact on algorithms attempting to track cones over time. In addition, we determined that displacements of the light source within 0.35 mm of the pupil center, which is the farthest location from the pupil center used by operators of the AO camera to acquire high-quality images of the cone mosaic in clinical studies, does not significantly affect the cone detection and density estimation.OCIS codes: (110.1080) Active or adaptive optics, (330.7331) Visual optics, receptor optics, (170.3880) Medical and biological imaging     

Modal content of living human cone photoreceptors

Decades of experimental and theoretical investigations have established that photoreceptors capture light based on the principles of optical waveguiding. Yet considerable uncertainty remains, even for the most basic prediction as to whether photoreceptors support more than a single waveguide mode. To test for modal behavior in human cone photoreceptors in the near infrared, we took advantage of adaptive-optics optical coherence tomography (AO-OCT, λ_c = 785 nm) to noninvasively image in three dimensions the reflectance profile of cones. Modal content of reflections generated at the cone inner segment and outer segment junction (IS/OS) and cone outer segment tip (COST) was examined over a range of cone diameters in 1,802 cones from 0.6° to 10° retinal eccentricity. Second moment analysis in conjunction with theoretical predictions indicate cone IS and OS have optical properties consistent of waveguides, which depend on segment diameter and refractive index. Cone IS was found to support a single mode near the fovea (≤3°) and multiple modes further away (>4°). In contrast, no evidence of multiple modes was found in the cone OSs. The IS/OS and COST reflections share a common optical aperture, are most circular near the fovea, show no orientation preference, and are temporally stable. We tested mode predictions of a conventional step-index fiber model and found that in order to fit our AO-OCT results required a lower estimate of the IS refractive index and introduction of an IS focusing/tapering effect.OCIS codes: (010.1080) Active or adaptive optics, (110.4500) Optical coherence tomography, (330.5310) Vision - photoreceptors, (330.4300) Vision system - noninvasive assessment, (330.7331) Visual optics, receptor optics     

MEMS segmented-based adaptive optics scanning laser ophthalmoscope

The performance of a MEMS (micro-electro-mechanical-system) segmented deformable mirror was evaluated in an adaptive optics (AO) scanning laser ophthalmoscope. The tested AO mirror (Iris AO, Inc, Berkeley, CA) is composed of 37 hexagonal segments that allow piston/tip/tilt motion up to 5 μm stroke and ±5 mrad angle over a 3.5 mm optical aperture. The control system that implements the closed-loop operation employs a 1:1 matched 37-lenslet Shack-Hartmann wavefront sensor whose measurements are used to apply modal corrections to the deformable mirror. After a preliminary evaluation of the AO mirror optical performance, retinal images from 4 normal subjects over a 0.9°x0.9° field size were acquired through a 6.4 mm ocular pupil, showing resolved retinal features at the cellular level. Cone photoreceptors were observed as close as 0.25 degrees from the foveal center. In general, the quality of these images is comparable to that obtained using deformable mirrors based on different technologies.     

Wavefront sensorless adaptive optics optical coherence tomography for in vivo retinal imaging in mice

We present wavefront sensorless adaptive optics (WSAO) Fourier domain optical coherence tomography (FD-OCT) for in vivo small animal retinal imaging. WSAO is attractive especially for mouse retinal imaging because it simplifies optical design and eliminates the need for wavefront sensing, which is difficult in the small animal eye. GPU accelerated processing of the OCT data permitted real-time extraction of image quality metrics (intensity) for arbitrarily selected retinal layers to be optimized. Modal control of a commercially available segmented deformable mirror (IrisAO Inc.) provided rapid convergence using a sequential search algorithm. Image quality improvements with WSAO OCT are presented for both pigmented and albino mouse retinal data, acquired in vivo.OCIS codes: (170.4460) Ophthalmic optics and devices, (110.1080) Active or adaptive optics, (110.4500) Optical coherence tomography     

Noninvasive imaging of the human rod photoreceptor mosaic using a confocal adaptive optics scanning ophthalmoscope

The rod photoreceptors are implicated in a number of devastating retinal diseases. However, routine imaging of these cells has remained elusive, even with the advent of adaptive optics imaging. Here, we present the first in vivo images of the contiguous rod photoreceptor mosaic in nine healthy human subjects. The images were collected with three different confocal adaptive optics scanning ophthalmoscopes at two different institutions, using 680 and 775 nm superluminescent diodes for illumination. Estimates of photoreceptor density and rod:cone ratios in the 5°-15° retinal eccentricity range are consistent with histological findings, confirming our ability to resolve the rod mosaic by averaging multiple registered images, without the need for additional image processing. In one subject, we were able to identify the emergence of the first rods at approximately 190 μm from the foveal center, in agreement with previous histological studies. The rod and cone photoreceptor mosaics appear in focus at different retinal depths, with the rod mosaic best focus (i.e., brightest and sharpest) being at least 10 μm shallower than the cones at retinal eccentricities larger than 8°. This study represents an important step in bringing high-resolution imaging to bear on the study of rod disorders.     

Imaging of retinal vasculature using adaptive optics SLO/OCT

We use our previously developed adaptive optics (AO) scanning laser ophthalmoscope (SLO)/ optical coherence tomography (OCT) instrument to investigate its capability for imaging retinal vasculature. The system records SLO and OCT images simultaneously with a pixel to pixel correspondence which allows a direct comparison between those imaging modalities. Different field of views ranging from 0.8°x0.8° up to 4°x4° are supported by the instrument. In addition a dynamic focus scheme was developed for the AO-SLO/OCT system in order to maintain the high transverse resolution throughout imaging depth. The active axial eye tracking that is implemented in the OCT channel allows time resolved measurements of the retinal vasculature in the en-face imaging plane. Vessel walls and structures that we believe correspond to individual erythrocytes could be visualized with the system.OCIS codes: (170.3890) Medical optics instrumentation, (110.1080) Active or adaptive optics, (170.4470) Ophthalmology, (330.5310) Vision - photoreceptors, (110.4500) Optical coherence tomography     

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.     

Adaptive optics retinal imaging in the living mouse eye

Correction of the eye's monochromatic aberrations using adaptive optics (AO) can improve the resolution of in vivo mouse retinal images [Biss et al., Opt. Lett. 32(6), 659 (2007) and Alt et al., Proc. SPIE 7550, 755019 (2010)], but previous attempts have been limited by poor spot quality in the Shack-Hartmann wavefront sensor (SHWS). Recent advances in mouse eye wavefront sensing using an adjustable focus beacon with an annular beam profile have improved the wavefront sensor spot quality [Geng et al., Biomed. Opt. Express 2(4), 717 (2011)], and we have incorporated them into a fluorescence adaptive optics scanning laser ophthalmoscope (AOSLO). The performance of the instrument was tested on the living mouse eye, and images of multiple retinal structures, including the photoreceptor mosaic, nerve fiber bundles, fine capillaries and fluorescently labeled ganglion cells were obtained. The in vivo transverse and axial resolutions of the fluorescence channel of the AOSLO were estimated from the full width half maximum (FWHM) of the line and point spread functions (LSF and PSF), and were found to be better than 0.79 μm ± 0.03 μm (STD)(45% wider than the diffraction limit) and 10.8 μm ± 0.7 μm (STD)(two times the diffraction limit), respectively. The axial positional accuracy was estimated to be 0.36 μm. This resolution and positional accuracy has allowed us to classify many ganglion cell types, such as bistratified ganglion cells, in vivo.     

Improved visualization of outer retinal morphology with aberration cancelling reflective optical design for adaptive optics - optical coherence tomography

We present an aberration cancelling optical design for a reflective adaptive optics - optical coherence tomography (AO-OCT) retinal imaging system. The optical performance of this instrument is compared to our previous multimodal AO-OCT/AO-SLO retinal imaging system. The feasibility of new instrumentation for improved visualization of microscopic retinal structures is discussed. Examples of images acquired with this new AO-OCT instrument are presented.OCIS codes: (110.4500) Optical coherence tomography, (010.1080) Active or adaptive optics, (220.1000) Aberration compensation, (170.0110) Imaging systems, (170.4470) Ophthalmology, (120.3890) Medical optics instrumentation     

Characterizing the point spread function of retinal OCT devices with a model eye-based phantom

We have designed, fabricated, and tested a nanoparticle-embedded phantom (NEP) incorporated into a model eye in order to characterize the point spread function (PSF) of retinal optical coherence tomography (OCT) devices in three dimensions under realistic imaging conditions. The NEP comprises a sparse distribution of highly backscattering silica-gold nanoshells embedded in a transparent UV-curing epoxy. The commercially-available model eye replicates the key optical structures and focusing power of the human eye. We imaged the model eye-NEP combination with a research-grade spectral domain OCT system designed for in vivo retinal imaging and quantified the lateral and axial PSF dimensions across the field of view in the OCT images. We also imaged the model eye-NEP in a clinical OCT system. Subtle features in the PSF and its dimensions were consistent with independent measurements of lateral and axial resolution. This model eye-based phantom can provide retinal OCT device developers and users a means to rapidly, objectively, and consistently assess the PSF, a fundamental imaging performance metric.     

Accurately motion-corrected Lissajous OCT with multi-type image registration

Passive motion correction methods for optical coherence tomography (OCT) use image registration to estimate eye movements. To improve motion correction, a multi-image cross-correlation that employs spatial features in different image types is introduced. Lateral motion correction using en face OCT and OCT-A projections on Lissajous-scanned OCT data is applied. Motion correction using OCT-A projection of whole depth and OCT amplitude, OCT logarithmic intensity, and OCT maximum intensity projections were evaluated in retinal imaging with 76 patients. The proposed method was compared with motion correction using OCT-A projection of whole depth. The comparison shows improvements in the image quality of motion-corrected superficial OCT-A images and image registration.     

Motion artifact and speckle noise reduction in polarization sensitive optical coherence tomography by retinal tracking

We present a novel polarization sensitive optical coherence tomography (PS-OCT) system with an integrated retinal tracker. The tracking operates at up to 60 Hz, correcting PS-OCT scanning positions during the acquisition to avoid artifacts caused by eye motion. To demonstrate the practical performance of the system, we imaged several healthy volunteers and patients with AMD both with B-scan repetitions for frame averaging and with 3D raster scans. Under large retinal motions with up to 1 mm amplitude at 0.5 ~a few Hz frequency range, motion artifact suppression in the PS-OCT images as well as standard deviation noise reduction in the frame averaged retardation images are presented.OCIS codes: (170.0170) Medical optics and biotechnology, (170.0110) Imaging systems, (170.2655) Functional monitoring and imaging, (170.4470) Ophthalmology, (170.4500) Optical coherence tomography     

Variability in parafoveal cone mosaic in normal trichromatic individuals

Parafoveal function is important for daily visual tasks such as reading. Here the variability in cone density along the four cardinal meridians in parafoveal regions of the retina was investigated in vivo using an adaptive optics fundus camera. Ten healthy normal trichromatic individuals were included in the study. There were significant differences in cone density between individuals at all four tested eccentricities (0.5, 1, 2 and 3°) and meridians. Cone density ranged from 34,900 to 63,000 cones/mm(2) at 1° horizontally, and from 31,600 to 60,700 at 1° vertically. The results were consistent with those of Curcio et al. (1990), although between-individual variability is greater than previously reported in the parafovea from 1 to 3.2°.     

Dual band dual focus optical coherence tomography for imaging the whole eye segment

We developed an improved dual band dual focus spectral domain optical coherence tomography (SD-OCT) for in vivo 2D/3D imaging of the whole eye segment, including the whole anterior segment and retina. The system featured two OCT channels with two different bands centered at 840 nm and 1050 nm, which were designed to image the retina and the anterior segments of the eye, respectively. By combing the two probe light beams for co-axial scanning and separating them for focusing at different segments of the eye with a combination of three dichroic mirrors, we not only minimized the loss of the backscattered light from the sample but also improved the imaging depth, scan range and resolution. The full resolved complex (FRC) method was applied to double the imaging depth for the whole anterior segment imaging, with which an imaging depth of 36.71 mm in air was achieved. We demonstrated that this system was capable of measuring the dynamic changes of ocular dimensions, including the asphericity of the cornea and lens, during accommodation.OCIS codes: (110.4500) Optical coherence tomography, (330.7322) Visual optics, accommodation, (170.3880) Medical and biological imaging, (170.4460) Ophthalmic optics and devices, (170.4470) Ophthalmology, (170.4500) Optical coherence tomography     

Optimizing 3D retinal vasculature imaging in diabetic retinopathy using registration and averaging of OCT-A

High resolution visualization of optical coherence tomography (OCT) and OCT angiography (OCT-A) data is required to fully take advantage of the imaging modality’s three-dimensional nature. However, artifacts induced by patient motion often degrade OCT-A data quality. This is especially true for patients with deteriorated focal vision, such as those with diabetic retinopathy (DR). We propose a novel methodology for software-based OCT-A motion correction achieved through serial acquisition, volumetric registration, and averaging. Motion artifacts are removed via a multi-step 3D registration process, and visibility is significantly enhanced through volumetric averaging. We demonstrate that this method permits clear 3D visualization of retinal pathologies and their surrounding features, 3D visualization of inner retinal capillary connections, as well as reliable visualization of the choriocapillaris layer.