Motion Contrast, Phase Gradient, and Simultaneous OCT Images Assist in the Interpretation of Dark-Field Images in Eyes with Retinal Pathology.

The cellular-level visualization of retinal microstructures such as blood vessel wall components, not available with other imaging modalities, is provided with unprecedented details by dark-field imaging configurations; however, the interpretation of such images alone is sometimes difficult since multiple structural disturbances may be present in the same time. Particularly in eyes with retinal pathology, microstructures may appear in high-resolution retinal images with a wide range of sizes, sharpnesses, and brightnesses. In this paper we show that motion contrast and phase gradient imaging modalities, as well as the simultaneous acquisition of depth-resolved optical coherence tomography (OCT) images, provide additional insight to help understand the retinal neural and vascular structures seen in dark-field images and may enable improved diagnostic and treatment plans.

The Surviving, Not Thriving, Photoreceptors in Patients with ABCA4 Stargardt Disease.

Stargardt disease (STGD1), associated with biallelic variants in the ABCA4 gene, is the most common heritable macular dystrophy and is currently untreatable. To identify potential treatment targets, we characterized surviving STGD1 photoreceptors. We used clinical data to identify macular regions with surviving STGD1 photoreceptors. We compared the hyperreflective bands in the optical coherence tomographic (OCT) images that correspond to structures in the STGD1 photoreceptor inner segments to those in controls. We used adaptive optics scanning light ophthalmoscopy (AO-SLO) to study the distribution of cones and AO-OCT to evaluate the interface of photoreceptors and retinal pigment epithelium (RPE). We found that the profile of the hyperreflective bands differed dramatically between patients with STGD1 and controls. AO-SLOs showed patches in which cone densities were similar to those in healthy retinas and others in which the cone population was sparse. In regions replete with cones, there was no debris at the photoreceptor-RPE interface. In regions with sparse cones, there was abundant debris. Our results raise the possibility that pharmaceutical means may protect surviving photoreceptors and so mitigate vision loss in patients with STGD1.

Optical coherence tomography-based micro-particle image velocimetry.

We present a new application of optical coherence tomography (OCT), widely used in biomedical imaging, to flow analysis in near-wall hydrodynamics for marine research. This unique capability, called OCT micro-particle image velocimetry, provides a high-resolution view of microscopic flow phenomena and measurement of flow statistics within the first millimeter of a boundary layer. The technique is demonstrated in a small flow cuvette and in a water tunnel.

Cellular-Level Analysis of Retinal Blood Vessel Walls Based on Phase Gradient Images.

Diseases such as diabetes affect the retinal vasculature and the health of the neural retina, leading to vision problems. We describe here an imaging method and analysis procedure that enables characterization of the retinal vessel walls with cellular-level resolution, potentially providing markers for eye diseases. Adaptive optics scanning laser ophthalmoscopy is used with a modified detection scheme to include four simultaneous offset aperture channels. The magnitude of the phase gradient derived from these offset images is used to visualize the structural characteristics of the vessels. The average standard deviation image provides motion contrast and enables segmentation of the vessel lumen. Segmentation of blood vessel walls provides quantitative measures of geometrical characteristics of the vessel walls, including vessel and lumen diameters, wall thickness, and wall-to-lumen ratio. Retinal diseases may affect the structural integrity of the vessel walls, their elasticity, their permeability, and their geometrical characteristics. The ability to measure these changes is valuable for understanding the vascular effects of retinal diseases, monitoring disease progression, and drug testing. In addition, loss of structural integrity of the blood vessel wall may result in microaneurysms, a hallmark lesion of diabetic retinopathy, which may rupture or leak and further create vision impairment. Early identification of such structural abnormalities may open new treatment avenues for disease management and vision preservation. Functional testing of retinal circuitry through high-resolution measurement of vasodilation as a response to controlled light stimulation of the retina (neurovascular coupling) is another application of our method and can provide an unbiased evaluation of one's vision and enable early detection of retinal diseases and monitoring treatment results.

Non-Rigid Registration for High-Resolution Retinal Imaging.

Adaptive optics provides improved resolution in ophthalmic imaging when retinal microstructures need to be identified, counted, and mapped. In general, multiple images are averaged to improve the signal-to-noise ratio or analyzed for temporal dynamics. Image registration by cross-correlation is straightforward for small patches; however, larger images require more sophisticated registration techniques. Strip-based registration has been used successfully for photoreceptor mosaic alignment in small patches; however, if the deformations along strips are not simple displacements, averaging can degrade the final image. We have applied a non-rigid registration technique that improves the quality of processed images for mapping cones over large image patches. In this approach, correction of local deformations compensates for local image stretching, compressing, bending, and twisting due to a number of causes. The main result of this procedure is improved definition of retinal microstructures that can be better identified and segmented. Derived metrics such as cone density, wall-to-lumen ratio, and quantification of structural modification of blood vessel walls have diagnostic value in many retinal diseases, including diabetic retinopathy and age-related macular degeneration, and their improved evaluations may facilitate early diagnostics of retinal diseases.

Multimodal adaptive optics retinal imager: design and performance.

Optical coherence tomography (OCT) and scanning laser ophthalmoscopy (SLO) are complementary imaging modalities, the combination of which can provide clinicians with a wealth of information to detect retinal diseases, monitor disease progression, or assess new therapies. Adaptive optics (AO) is a tool that enables correction of wavefront distortions from ocular aberrations. We have developed a multimodal adaptive optics system (MAOS) for high-resolution multifunctional use in a variety of research and clinical applications. The system integrates both OCT and SLO imaging channels into an AO beam path. The optics and hardware were designed with specific features for simultaneous SLO/OCT output, for high-fidelity AO correction, for use in humans, primates, and small animals, and for efficient location and orientation of retinal regions of interest. The MAOS system was tested on human subjects and rodents. The design, performance characterization, and initial representative results from the human and animal studies are presented and discussed.

Fiber-based hand-held RCM-OCT probe for noninvasive assessment of skin lesions and therapy guidance.

Noninvasive assessment of skin lesions, especially of basal cell carcinoma (BCC), has benefited more recently from the use of optical imaging techniques such as optical coherence tomography (OCT) and reflectance confocal microscopy (RCM). While RCM provides submicron scale resolution and thus enables identification of skin morphological changes of the skin, with the downside of limited penetration depth, OCT imaging of the same lesion brings the benefit of better resolving its depth of invasion. OCT and RCM can be used either individually or combined within the same instrument for the noninvasive diagnosis of nonmelanoma skin cancers (NMSCs). Their combined use has shown to provide certain benefits such as better characterization of the lesion's margins, both in depth and laterally, as well as improved sensitivity and specificity, as previously demonstrated by our team. In this paper we report a new "fiber-based" implementation of the second-generation RCM-OCT hand-held probe. The fiber-based implementation of both imaging modalities enabled the construction of a smaller footprint/lower weight hand-held probe. Its preliminary evaluation on the skin of healthy volunteers is reported here, demonstrating improved capabilities for resolving sub-cellular structures and image skin morphology with micron-scale resolution to a higher depth than in the previous implementation, while also enabling the construction of angiography maps showing vascular remodeling.

High resolution multimodal clinical ophthalmic imaging system.

We developed a multimodal adaptive optics (AO) retinal imager which is the first to combine high performance AO-corrected scanning laser ophthalmoscopy (SLO) and swept source Fourier domain optical coherence tomography (SSOCT) imaging modes in a single compact clinical prototype platform. Such systems are becoming ever more essential to vision research and are expected to prove their clinical value for diagnosis of retinal diseases, including glaucoma, diabetic retinopathy (DR), age-related macular degeneration (AMD), and retinitis pigmentosa. The SSOCT channel operates at a wavelength of 1 microm for increased penetration and visualization of the choriocapillaris and choroid, sites of major disease activity for DR and wet AMD. This AO system is designed for use in clinical populations; a dual deformable mirror (DM) configuration allows simultaneous low- and high-order aberration correction over a large range of refractions and ocular media quality. The system also includes a wide field (33 deg.) line scanning ophthalmoscope (LSO) for initial screening, target identification, and global orientation, an integrated retinal tracker (RT) to stabilize the SLO, OCT, and LSO imaging fields in the presence of lateral eye motion, and a high-resolution LCD-based fixation target for presentation of visual cues. The system was tested in human subjects without retinal disease for performance optimization and validation. We were able to resolve and quantify cone photoreceptors across the macula to within approximately 0.5 deg (approximately 100-150 microm) of the fovea, image and delineate ten retinal layers, and penetrate to resolve features deep into the choroid. The prototype presented here is the first of a new class of powerful flexible imaging platforms that will provide clinicians and researchers with high-resolution, high performance adaptive optics imaging to help guide therapies, develop new drugs, and improve patient outcomes.

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.

Compact adaptive optics line scanning ophthalmoscope.

We have developed a compact retinal imager that integrates adaptive optics (AO) into a line scanning ophthalmoscope (LSO). The bench-top AO-LSO instrument significantly reduces the size, complexity, and cost of research AO scanning laser ophthalmoscopes (AOSLOs), for the purpose of moving adaptive optics imaging more rapidly into routine clinical use. The AO-LSO produces high resolution retinal images with only one moving part and a significantly reduced instrument footprint and number of optical components. The AO-LSO has a moderate field of view (5.5 deg), which allows montages of the macula or other targets to be obtained more quickly and efficiently. In a preliminary human subjects investigation, photoreceptors could be resolved and counted within approximately 0.5 mm of the fovea. Photoreceptor counts matched closely to previously reported histology. The capillaries surrounding the foveal avascular zone could be resolved, as well as cells flowing within them. Individual nerve fiber bundles could be resolved, especially near the optic nerve head, as well as other structures such as the lamina cribrosa. In addition to instrument design, fabrication, and testing, software algorithms were developed for automated image registration and cone counting.

Retinal imaging with polarization-sensitive optical coherence tomography and adaptive optics.

Various layers of the retina are well known to alter the polarization state of light. Such changes in polarization may be a sensitive indicator of tissue structure and function, and as such have gained increased clinical attention. Here we demonstrate a polarization-sensitive optical coherence tomography (PS-OCT) system that incorporates adaptive optics (AO) in the sample arm and a single line scan camera in the detection arm. We quantify the benefit of AO for PS-OCT in terms of signal-to-noise, lateral resolution, and speckle size. Double pass phase retardation per unit depth values ranging from 0.25 degrees/microm to 0.65 degrees/microm were found in the birefringent nerve fiber layer at 6 degrees eccentricity, superior to the fovea, with the highest values being noticeably higher than previously reported with PS-OCT around the optic nerve head. Moreover, fast axis orientation and degree of polarization uniformity measurements made with AO-PS-OCT demonstrate polarization scrambling in the retinal pigment epithelium at the highest resolution reported to date.

Automated algorithm for breast tissue differentiation in optical coherence tomography.

An automated algorithm for differentiating breast tissue types based on optical coherence tomography (OCT) data is presented. Eight parameters are derived from the OCT reflectivity profiles and their means and covariance matrices are calculated for each tissue type from a training set (48 samples) selected based on histological examination. A quadratic discrimination score is then used to assess the samples from a validation set. The algorithm results for a set of 89 breast tissue samples were correlated with the histological findings, yielding specificity and sensitivity of 0.88. If further perfected to work in real time and yield even higher sensitivity and specificity, this algorithm would be a valuable tool for biopsy guidance and could significantly increase procedure reliability by reducing both the number of nondiagnostic aspirates and the number of false negatives.

Three-dimensional pointwise comparison of human retinal optical property at 845 and 1060 nm using optical frequency domain imaging.

To compare the optical properties of the human retina, 3-D volumetric images of the same eye are acquired with two nearly identical optical coherence tomography (OCT) systems at center wavelengths of 845 and 1060 nm using optical frequency domain imaging (OFDI). To characterize the contrast of individual tissue layers in the retina at these two wavelengths, the 3-D volumetric data sets are carefully spatially matched. The relative scattering intensities from different layers such as the nerve fiber, photoreceptor, pigment epithelium, and choroid are measured and a quantitative comparison is presented. OCT retinal imaging at 1060 nm is found to have a significantly better depth penetration but a reduced contrast between the retinal nerve fiber, the ganglion cell, and the inner plexiform layers compared to the OCT retinal imaging at 845 nm.

Visualizing the vasculature of the entire human eye posterior hemisphere without a contrast agent.

The platform described here combines the non-invasive measurement of the retina/choroid structure and ocular blood flow based on optical coherence tomography (OCT) and wide-field semi-quantitative global flow visualization using line-scanning Doppler flowmetry (LSDF). The combination of these two imaging modalities within the same platform enables comprehensive assessment of blood flow in the retina and choroid in animals and human subjects for diagnostic purposes. Ultra-widefield vasculature visualization is demonstrated here for the first time without injecting additional contrast agents and based only on the motion of particles within the vasculature.

Dual-beam Fourier domain optical Doppler tomography of zebrafish.

We have developed a dual-beam Fourier domain optical Doppler tomography (FD-ODT) system to image zebrafish (Danio rerio) larvae. Two beams incident on the zebrafish with a fixed angular separation allow absolute blood flow velocity measurement to be made regardless of vessel orientation in a sagittal plane along which the heart and most of the major vasculature lie. Two spectrometers simultaneously acquire spectra from two interferometers with a typical (maximum) line rate of 18 (28) kHz. The system was calibrated using diluted milk and microspheres and a 0.5-mm thick flow cell. The average deviation from the set velocity from 1.4 to 34.6 mm/s was 4.1%. Three-dimensional structural raster videos were acquired of an entire fish, and through the head, heart, and upper tail of the fish. Coarse features that were resolved include the telencephalon, retina, both heart chambers (atrium and ventricle), branchial arches, and notochord. Other fine structures within these organs were also resolved. Zebrafish are an important tool for high-throughput screening of new pharmacological agents. The ability to generate high-resolution three-dimensional structural videos and accurately measure absolute flow rates in major vessels with FD-ODT provides researchers with additional metrics by which the efficacy of new drugs can be assessed.

Polarization-sensitive spectral-domain optical coherence tomography using a single line scan camera.

Polarization-sensitive optical coherence tomography can be used to measure the birefringence of biological tissue such as the human retina. Previous measurements with a time-domain polarization-sensitive optical coherence tomography system revealed that the birefringence of the human retinal nerve fiber layer is not constant, but varies as a function of location around the optic nerve head. Here we present a spectral-domain polarization-sensitive optical coherence tomography system that uses a spectrometer configuration with a single line scan camera and a Wollaston prism in the detection arm. Since only one camera has to be synchronized with other components in the system, the design is simplified considerably. This system is 60 times faster than a time-domain polarization-sensitive optical coherence tomography system. Data was acquired using concentric circular scans around the optic nerve head of a young healthy volunteer and the acquisition time for 12 circular scans was reduced from 72 s to 1.2 s. The acquired data sets demonstrate variations in retinal thickness and double pass phase retardation per unit depth that were similar to data from the same volunteer taken with a time-domain polarization-sensitive system. The double pass phase retardation per unit depth of the retinal nerve fiber layer varied between 0.18 and 0.40 degrees/mum, equivalent to a birefringence of 2.2 * 10(-4) and 4.8 * 10(-4) respectively, measured at 840 nm.

Three dimensional tracking for volumetric spectral-domain optical coherence tomography.

We present a three-dimensional (3D) tracker for a clinical ophthalmic spectral domain optical coherence tomography (SD-OCT) system that combines depth-tracking with lateral tracking, providing a stabilized reference frame for 3D data recording and post acquisition analysis. The depth-tracking system is implemented through a real-time dynamic feedback mechanism to compensate for motion artifact in the axial direction. Active monitoring of the retina and adapting the reference arm of the interferometer allowed the whole thickness of the retina to be stabilized to within +/-100 mum. We achieve a relatively constant SNR from image to image by stabilizing the image of the retina with respect to the depth dependent sensitivity of SD-OCT. The depth tracking range of our system is 5.2 mm in air and the depth is adjusted every frame.nhancement in the stability of the images with the depth-tracking algorithm is demonstrated on a healthy volunteer.

Autocalibration of spectral-domain optical coherence tomography spectrometers for in vivo quantitative retinal nerve fiber layer birefringence determination.

Accurate wavelength assignment of each spectral element for spectral-domain optical coherence tomography (SD-OCT) and optical frequency domain imaging (OFDI) is required for proper construction of biological tissue cross-sectional images. This becomes more critical for functional extensions of these techniques, especially in polarization-sensitive optical coherence tomography (PS-OCT), where incorrect wavelength assignment between the two orthogonal polarization channels leads to polarization artifacts. We present an autocalibration method for wavelength assignment that does not require separate calibration measurements and that can be applied directly on actual data. Removal of the birefringence artifact is demonstrated in a PS-OCT system with picometer accuracy in the relative wavelength assignment, resulting in a residual phase error of 0.25 deg/100 microm. We also demonstrate, for the first time, a quantitative birefringence map of an in vivo human retinal nerve fiber layer.

In vivo optical frequency domain imaging of human retina and choroid.

Optical frequency domain imaging (OFDI) using swept laser sources is an emerging second-generation method for optical coherence tomography (OCT). Despite the widespread use of conventional OCT for retinal disease diagnostics, until now imaging the posterior eye segment with OFDI has not been possible. Here we report the development of a highperformance swept laser at 1050 nm and an ophthalmic OFDI system that offers an A-line rate of 18.8 kHz, sensitivity of >92 dB over a depth range of 2.4 mm with an optical exposure level of 550 muW, and deep penetration into the choroid. Using these new technologies, we demonstrate comprehensive human retina, optic disc, and choroid imaging in vivo. This advance enables us to view choroidal vasculature in vivo without intravenous injection of fluorescent dyes and may provide a useful tool for evaluating choroidal as well as retinal diseases.

Pulsed illumination spectral-domain optical coherence tomography for human retinal imaging.

We present pulsed illumination spectral-domain optical coherence tomography (SD-OCT) for in vivo human retinal imaging. We analyze the signal-to-noise (SNR) for continuous wave (CW) and pulsed illumination SD-OCT. The lateral beam scan motion is responsible for a SNR drop due to lateral scanning induced interference fringe washout. Pulsed illumination can reduce the SNR drop by shorter sample illumination time during the integration time of a camera. First, we demonstrate the SNR benefit of pulsed illumination over CW as function of lateral scan speed for a paper sample. Finally, we show better SNR in retinal images of a normal subject with pulsed illumination SD-OCT over CW at high lateral scanning speed.