Weihua Gao

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.

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.

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.

Imaging Retinal Capillaries Using Ultrahigh-Resolution Optical Coherence Tomography and Adaptive Optics

PURPOSE Ultrahigh-resolution optical coherence tomography (UHR-OCT) with adaptive optics (AO) provides micrometer-scale 3D resolution that is attractive for imaging the retinal microvasculature. Such imaging may be useful for early detection of pathologic changes as in diabetic retinopathy. Here the authors investigate this potential for detecting individual capillaries in healthy subjects. METHODS UHR-AO-OCT volumes centered on the fovea were acquired from seven subjects (age range, 25-61 years) with three preselected with no foveal avascular zone (FAZ). Images were compared with entoptic diagrams using the capillaries at the rim of the FAZ. Methods of comparison were testing for the presence of a FAZ, noting distinct features in the capillary pattern, and measuring the size of the FAZ. Additional analysis included measurements of capillary diameter and depth range with retinal eccentricity. RESULTS UHR-AO-OCT results are consistent with entoptic observations for all three methods of comparison. FAZ diameters measured by UHR-AO-OCT and entoptic imaging are strongly correlated (R(2) = 0.86). Average capillary diameter near the FAZ rim is 5.1 (4.6) ± 1.4 μm, with the value in parentheses accounting for axial image blur. This is consistent with histology (average, ~4.7 μm). Depth range of the capillaries increases monotonically with eccentricity (0°-1.25°) and is larger and more variable for subjects without FAZ. CONCLUSIONS UHR-AO-OCT permits observation of many of the capillaries proximal to the FAZ, including those of average size based on published histology. This supports the view that the vast majority of capillaries in the retina are likely detectable with UHR-AO-OCT.

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.

Measuring directionality of the retinal reflection with a Shack-Hartmann wavefront sensor

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.

3D imaging of cone photoreceptors over extended time periods using optical coherence tomography with adaptive optics

Optical coherence tomography with adaptive optics (AO-OCT) is a highly sensitive, noninvasive method for 3D imaging of the microscopic retina. The purpose of this study is to advance AO-OCT technology by enabling repeated imaging of cone photoreceptors over extended periods of time (days). This sort of longitudinal imaging permits monitoring of 3D cone dynamics in both normal and diseased eyes, in particular the physiological processes of disc renewal and phagocytosis, which are disrupted by retinal diseases such as age related macular degeneration and retinitis pigmentosa. For this study, the existing AO-OCT system at Indiana underwent several major hardware and software improvements to optimize system performance for 4D cone imaging. First, ultrahigh speed imaging was realized using a Basler Sprint camera. Second, a light source with adjustable spectrum was realized by integration of an Integral laser (Femto Lasers, λc=800nm, ▵λ=160nm) and spectral filters in the source arm. For cone imaging, we used a bandpass filter with λc=809nm and ▵λ=81nm (2.6 μm nominal axial resolution in tissue, and 167 KHz A-line rate using 1,408 px), which reduced the impact of eye motion compared to previous AO-OCT implementations. Third, eye motion artifacts were further reduced by custom ImageJ plugins that registered (axially and laterally) the volume videos. In two subjects, cone photoreceptors were imaged and tracked over a ten day period and their reflectance and outer segment (OS) lengths measured. High-speed imaging and image registration/dewarping were found to reduce eye motion to a fraction of a cone width (1 μm root mean square). The pattern of reflections in the cones was found to change dramatically and occurred on a spatial scale well below the resolution of clinical instruments. Normalized reflectance of connecting cilia (CC) and OS posterior tip (PT) of an exemplary cone was 54±4, 47±4, 48±6, 50±5, 56±1% and 46±4, 53±4, 52±6, 50±5, 44±1% for days #1,3,6,8,10 respectively. OS length of the same cone was 28.9, 26.4, 26.4, 30.6, and 28.1 ìm for days #1,3,6,8,10 respectively. It is plausible these changes are an optical correlate of the natural process of OS renewal and shedding.

Imaging retinal nerve fiber bundles at ultrahigh-speed and ultrahigh-resolution using OCT with adaptive optics

Ultrahigh speed line scan detectors based on CMOS technology have been recently demonstrated in ultrahigh resolution spectral-domain optical coherence tomography (UHR-SD-OCT) for retinal imaging. While successful, fundamental tradeoffs exist been image acquisition time, image sampling density, and sensitivity, all of which impact the extent of motion artifacts, visualization of fine spatial detail, and detection of faint reflections. Here we investigate these tradeoffs for imaging retinal nerve fiber bundles (RNFBs) using UHR-SD-OCT with adaptive optics (AO). Volume scans of 3°x3° and 1.5°x1.5° were acquired at retinal locations of 3° nasal and 6° superior to the fovea on a healthy subject. Dynamic AO compensation across a 6 mm pupil provided near-diffraction-limited performance. The acquisition rates were 22.5k lines/s and 125k lines/s with A-lines spaced at 0.9 μm and 1.8 μm and B-scans at 1.8 μm and 9 μm. Focus was optimized for visualizing the retinal nerve fiber bundles (RNFBs). En face projection and crosssectional views of the RNFBs were extracted from the volumes and compared to images acquired with established conventional CCD-based line-scan camera. The projection view was found highly sensitive to eye motion artifacts, yet could only be partially compensated with coarser sampling, since fine sampling was necessary to observe the microscopic features in the RNFBs. For the cross-sectional view, speckle noise rather than eye motion artifacts limited bundle clarity. The highest B-scan density (1.8 μm spacing) coupled with B-scan averaging proved the best combination. Regardless of view, the higher line rate provided better RNFB clarity.