Ravi S. Jonnal

Adaptive optics parallel spectral domain optical coherence tomography for imaging the living retina

Although optical coherence tomography (OCT) can axially resolve and detect reflections from individual cells, there are no reports of imaging cells in the living human retina using OCT. To supplement the axial resolution and sensitivity of OCT with the necessary lateral resolution and speed, we developed a novel spectral domain OCT (SD-OCT) camera based on a free-space parallel illumination architecture and equipped with adaptive optics (AO). Conventional flood illumination, also with AO, was integrated into the camera and provided confirmation of the focus position in the retina with an accuracy of +/-10.3 mum. Short bursts of narrow B-scans (100x560 mum) of the living retina were subsequently acquired at 500 Hz during dynamic compensation (up to 14 Hz) that successfully corrected the most significant ocular aberrations across a dilated 6 mm pupil. Camera sensitivity (up to 94 dB) was sufficient for observing reflections from essentially all neural layers of the retina. Signal-to-noise of the detected reflection from the photoreceptor layer was highly sensitive to the level of cular aberrations and defocus with changes of 11.4 and 13.1 dB (single pass) observed when the ocular aberrations (astigmatism, 3rd order and higher) were corrected and when the focus was shifted by 200 mum (0.54 diopters) in the retina, respectively. The 3D resolution of the B-scans (3.0x3.0x5.7 mum) is the highest reported to date in the living human eye and was sufficient to observe the interface between the inner and outer segments of individual photoreceptor cells, resolved in both lateral and axial dimensions. However, high contrast speckle, which is intrinsic to OCT, was present throughout the AO parallel SD-OCT B-scans and obstructed correlating retinal reflections to cell-sized retinal structures.

High-speed volumetric imaging of cone photoreceptors with adaptive optics spectral-domain optical coherence tomography

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 subjects 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.

Adaptive optics flood-illumination camera for high speed retinal imaging.

Current adaptive optics flood-illumination retina cameras operate at low frame rates, acquiring retinal images below seven Hz, which restricts their research and clinical utility. Here we investigate a novel bench top flood-illumination camera that achieves significantly higher frame rates using strobing fiber-coupled superluminescent and laser diodes in conjunction with a scientific-grade CCD. Source strength was sufficient to obviate frame averaging, even for exposures as short as 1/3 msec. Continuous frame rates of 10, 30, and 60 Hz were achieved for imaging 1.8,0.8, and 0.4 deg retinal patches, respectively. Short-burst imaging up to 500 Hz was also achieved by temporarily storing sequences of images on the CCD. High frame rates, short exposure durations (1 msec), and correction of the most significant aberrations of the eye were found necessary for individuating retinal blood cells and directly measuring cellular flow in capillaries. Cone videos of dark adapted eyes showed a surprisingly rapid fluctuation (~1 Hz) in the reflectance of single cones. As further demonstration of the value of the camera, we evaluated the tradeoff between exposure duration and image blur associated with retina motion.

Measuring retinal contributions to the optical Stiles-Crawford effect with optical coherence tomography

The directional component of the retinal reflection, i.e., the optical Stiles-Crawford effect (SCE), is well established to result from the waveguiding property of photoreceptors. Considerable uncertainty, however, remains as to which retinal reflections are waveguided and thus contribute. To this end we have developed a retina camera based on spectral-domain optical coherence tomography (SD-OCT) that axially resolves (approximately 5 microm) these reflections and permits a direct investigation of the SCE origin at near infrared wavelengths. Reflections from the photoreceptor inner/outer segments junction (IS/OS) and near the posterior tip of the outer segments (PTOS) were found highly sensitive to beam entry position in the pupil with a considerable decrease in brightness occurring with an increase in aperture eccentricity. Reflections from the retinal pigment epithelium (RPE) were largely insensitive. The average directionality (rho(oct) value) at 2 degree eccentricity across the four subjects for the IS/OS, PTOS, and RPE were 0.120, 0.270, and 0.016 mm(-2), respectively. The directionality for the IS/OS approached typical psychophysical SCE measurements, while that for the PTOS approached conventional optical SCE measurements. Precise measurement of the optical SCE was found to require significant A-scan averaging.

Imaging cone photoreceptors in three dimensions and in time using ultrahigh resolution optical coherence tomography with adaptive optics

Cone photoreceptors in the living human eye have recently been imaged with micron-scale resolution in all three spatial dimensions using adaptive optics optical coherence tomography. While these advances have allowed non-invasive study of the three-dimensional structure of living human cones, studies of their function and physiology are still hampered by the difficulties to monitor the same cells over time. The purpose of this study is to demonstrate the feasibility of cone monitoring using ultrahigh-resolution adaptive optics optical coherence tomography. Critical to this is incorporation of a high speed CMOS camera (125 KHz) and a novel feature-based, image registration/dewarping algorithm for reducing the deleterious effects of eye motion on volume images. Volume movies were acquired on three healthy subjects at retinal eccentricities from 0.5° to 6°. Image registration/dewarping reduced motion artifacts in the movies from 15 μm to 1.3 μm root mean square, the latter sufficient for identifying and tracking cones. Cone row-to-row spacing and outer segment lengths were consistent with that reported in the literature. Cone length analysis demonstrates that UHR-AO-OCT is sufficiently sensitive to measure real length differences between cones in the same 0.5° retinal patch, and requires no more than five measurements of OS length to achieve 95% confidence. We know of no other imaging modality that can monitor foveal or parafoveal cones over time with comparable resolution in all three dimensions.

Volumetric retinal imaging with ultrahigh-resolution spectral-domain optical coherence tomography and adaptive optics using two broadband light sources

Ultrabroadband sources, such as multiplexed superluminescent diodes (SLDs) and femtosecond lasers, have been successfully employed in adaptive optics optical coherence tomography (AO-OCT) systems for ultrahigh resolution retinal imaging. The large cost differential of these sources, however, motivates the need for a performance comparison. Here, we compare the performance of a Femtolasers Integral Ti:Sapphire laser and a Superlum BroadLighter T840, using the same AO-OCT system and the same subject. In addition, we investigate the capability of our instrument equipped with the Integral to capture volume images of the fovea and adjacent regions on a second subject using the AO to control focus in the retina and custom and freeware image registration software to reduce eye motion artifacts. Monochromatic ocular aberrations were corrected with a woofer-tweeter AO system. Coherence lengths of the Integral and BroadLighter were measured in vivo at 3.2 microm and 3.3 microm, respectively. The difference in dynamic range was 5 dB, close to the expected variability of the experiment. Individual cone photoreceptors, retinal capillaries and nerve fiber bundles were distinguished in all three dimensions with both sources. The acquired retinal volumes are provided for viewing in OSA ISP, allowing the reader to data mine at the microscope level.

The Cellular Origins of the Outer Retinal Bands in Optical Coherence Tomography Images

PURPOSE To test the recently proposed hypothesis that the second outer retinal band, observed in clinical OCT images, originates from the inner segment ellipsoid, by measuring: (1) the thickness of this band within single cone photoreceptors, and (2) its respective distance from the putative external limiting membrane (band 1) and cone outer segment tips (band 3). METHODS Adaptive optics-optical coherence tomography images were acquired from four subjects without known retinal disease. Images were obtained at foveal (2°) and perifoveal (5°) locations. Cone photoreceptors (n = 9593) were identified and segmented in three dimensions using custom software. Features corresponding to bands 1, 2, and 3 were automatically identified. The thickness of band 2 was assessed in each cell by fitting the longitudinal reflectance profile of the band with a Gaussian function. Distances between bands 1 and 2, and between 2 and 3, respectively, were also measured in each cell. Two independent calibration techniques were employed to determine the depth scale (physical length per pixel) of the imaging system. RESULTS When resolved within single cells, the thickness of band 2 is a factor of three to four times narrower than in corresponding clinical OCT images. The distribution of band 2 thickness across subjects and eccentricities had a modal value of 4.7 μm, with 48% of the cones falling between 4.1 and 5.2 μm. No significant differences were found between cells in the fovea and perifovea. The distance separating bands 1 and 2 was found to be larger than the distance between bands 2 and 3, across subjects and eccentricities, with a significantly larger difference at 5° than 2°. CONCLUSIONS On the basis of these findings, we suggest that ascription of the outer retinal band 2 to the inner segment ellipsoid is unjustified, because the ellipsoid is both too thick and proximally located to produce the band.

Imaging retinal nerve fiber bundles using optical coherence tomography with adaptive optics.

Early detection of axonal tissue loss in retinal nerve fiber layer (RNFL) is critical for effective treatment and management of diseases such as glaucoma. This study aims to evaluate the capability of ultrahigh-resolution optical coherence tomography with adaptive optics (UHR-AO-OCT) for imaging the RNFL axonal bundles (RNFBs) with 3×3×3μm(3) resolution in the eye. We used a research-grade UHR-AO-OCT system to acquire 3°×3° volumes in four normal subjects and one subject with an arcuate retinal nerve fiber layer defect (n=5; 29-62years). Cross section (B-scans) and en face (C-scan) slices extracted from the volumes were used to assess visibility and size distribution of individual RNFBs. In one subject, we reimaged the same RNFBs twice over a 7month interval and compared bundle width and thickness between the two imaging sessions. Lastly we compared images of an arcuate RNFL defect acquired with UHR-AO-OCT and commercial OCT (Heidelberg Spectralis). Individual RNFBs were distinguishable in all subjects at 3° retinal eccentricity in both cross-sectional and en face views (width: 30-50μm, thickness: 10-15μm). At 6° retinal eccentricity, RNFBs were distinguishable in three of the five subjects in both views (width: 30-45μm, thickness: 20-40μm). Width and thickness RNFB measurements taken 7months apart were strongly correlated (p<0.0005). Mean difference and standard deviation of the differences between the two measurement sessions were -0.1±4.0μm (width) and 0.3±1.5μm (thickness). UHR-AO-OCT outperformed commercial OCT in terms of clarity of the microscopic retina. To our knowledge, these are the first measurements of RNFB cross section reported in the living human eye.

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.

Coherence gating and adaptive optics in the eye

An en face coherence gated camera equipped with adaptive optics (AO) has been constructed for imaging single cells in the living human retina. The high axial resolution of coherence gating combined with the high transverse resolution of AO provides a powerful imaging tool whose image quality can surpass either methodology performing alone. The AO system relies on a 37-actuator Xinetics mirror and a Shack-Hartmann wavefront sensor that executes up to 22 corrections per second. The coherence gate is realized with a free-space Michelson interferometer that employs a scientific-grade 12-bit CCD array for recording 2-D retinal interferograms. Images were collected of microstructures the size of single cells in the in vivo retina. Early results suggest that a coherence gated adaptive optics camera should substantially improve our ability to detect single cells in the retina over the current state-of-the-art AO retina cameras, including conventional flood illuminated and confocal scanning laser ophthalmoscopes. To our knowledge, this is the first effort to combine coherence gating and adaptive optics.