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

Yan Zhang, Jungtae Rha, Ravi S. Jonnal, and Donald T. Miller

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Abstract

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 µm. Short bursts of narrow B-scans (100×560 µm) 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 ocular 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 µm (0.54 diopters) in the retina, respectively. The 3D resolution of the B-scans (3.0×3.0×5.7 µm) 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.

©2005 Optical Society of America

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Figures (11)
Fig. 1.
Fig. 1. (A) Concept layout shows the AO system as part of the SD-OCT detection channel. (B) Detailed layout of the AO parallel SD-OCT retina camera. The camera consists of four sub-systems. (1) AO system corrects the ocular aberrations using a 788 nm SLD, Shack-Hartmann wavefront sensor, and Xinetics deformable mirror (short dashed line). (2) Pupil retro-illumination and fixation channels permit alignment of the subject’s eye to the retina camera. (3) Conventional flood-illumination is used to validate focusing in the retina and the physical size of microstructures in the retina (solid line). (4) Parallel SD-OCT acquires single shot B-scan images of the retina (long dashed line). Further details of the camera are given in the text.

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Fig. 2.
Fig. 2. Parallel SD-OCT sensitivity as a function of depth, converted from depth measured in air to tissue by using a refractive index of n=1.38. The four A-scan locations are along the line illumination profile. A-scan location is in microns at the retina, e.g. the 25th A-scan is 25 µm from the central A-scan.

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Fig. 3.
Fig. 3. Total wavefront error over time as measured by the SHWS before and during dynamic correction on one subject. The two traces depict correction with and without the middle five actuators frozen to a fixed correction (the remaining 32 actuators continued to provide dynamic correction). The time at which the actuators are frozen is indicated with no appreciable difference in the traces observed afterward.

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Fig. 4.
Fig. 4. Raw single cone images collected on the subject with the conventional flood-illuminated sub-system as depicted in Fig. 1. The four images represent the sharpest collected at each of two retinal eccentricities (1 and 2.4 deg) with and without AO. Images are 0.67×0.33 deg (200×100 µm) in size and were collected using the 843 nm SLD. Exposure duration was 4 msec, and imaging pupil size was 6 mm.

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Fig. 5.
Fig. 5. (top) Stratus OCT3 and (bottom) scanning SD-OCT B-scans collected in the same subject. Images are centered on the fovea and bisect the superior and inferior retinal fields (Stratus OCT3), and nasal and temporal fields (scanning SD-OCT). B-scans are 4.9 mm (16.3 deg) wide and 0.75 mm in depth. White rectangles depict 100 µm wide by 560 µm deep subsections that are centered at 1 and 2.4 deg eccentricity and imaged with the AO parallel SD-OCT camera. A magnified view of these subsections is shown in Fig. 6 with the corresponding AO parallel SD-OCT B-scans.

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Fig. 6.
Fig. 6. (left two columns) B-scan images acquired with the AO parallel SD-OCT instrument in Fig. 2 with and without AO at 1 and 2.4 deg eccentricity (superior). (right two columns) Stratus OCT3 and scanning SD-OCT B-scans are shown at the same retinal eccentricities (from white rectangular boxes in Fig. 5). All images were acquired on the same subject and are 100 µm wide and 560 µm deep. (bottom) The interface between the inner and outer segments and RPE are enlarged and displayed as amplitude on a linear scale (as opposed to a log scale). Minor thresholding was used to enhance contrast. (far right) Labels for retinal layers are repeated from Fig. 5 and positioned where they should appear in the scanning SD-OCT image. Images without AO are normalized to the corresponding AO images, including the enlarged images. The apparent tilt in the Stratus OCT3 image at 2.4 deg is an eye motion artifact. The Stratus OCT3 images were acquired at 9.6 µm/A-scan and interpolated (bicubic) to 1 µm. The scanning SD-OCT images were acquired at 2 µm/A-scan and interpolated (linear) to 1 µm. The 2 and 9.6 µm sampling intervals are noticeably smaller than their respective lateral resolutions. Depth of focus (dof) is displayed at the left and defined in the text.

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Fig. 7.
Fig. 7. Two collages created by digitally pasting together an alternating sequence of AO parallel SD-OCT images acquired at 1 and 2.4 deg eccentricity. The collages are roughly 3.25 to 3.5 degrees wide. Focus is at the cones. Each set of images was taken from the same short burst videos. The collage at 2.4 deg eccentricity was generated from two images (each 70 A-scans from the central region of a B-scan; dashed white rectangle represents the combined two images and is 140 A-scans) that were repeated about 7 times. The collage at 1 deg eccentricity contains three images (each ~70 A-scans; dashed white rectangle represents 210 A-scans) that were repeated about 5 times. The collages were axially registered to each other by aligning the reflection from the IS/OS segment. Note the enhanced clarity of the major retinal layers compared to that for the highly magnified and narrow AO parallel SD-OCT images in Fig. 6.

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Fig. 8.
Fig. 8. Five-burst B-scan videos collected of the same patch of retina at 2.4 deg eccentricity for three scenarios: (left) focused on the cones and without AO, (middle) focused on the cones and with AO; (right) focus shifted anteriorly 200 µm and with AO. Note that the physical separation between the ILM (upper edge of the topmost bright layer) and anterior side of the photoreceptor outer segments (segmented bright line in lower half) is around 272 µm. Each B-scan is composed of 100 A-scans spaced 1 µm apart. Videos were captured at 500 Hz with 1 msec exposure and run at 4 Hz, which is 125 times slower than the actual acquisition rate. The videos are best viewed in “loop” mode.

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Fig. 9.
Fig. 9. Lateral reflectance profiles through the elongated structures (photoreceptor outer segments) for AO-corrected B-scan images at 2.4 (red) and 1 (blue) deg eccentricities for two short-burst sequences. Frame numbers correspond to the temporal sequence within a single short-burst series where frames 1, 5, and 7 are separated by 8 and 4 msecs, respectively. Profiles are shifted vertically in the plot to facilitate visual comparison. Black arrows indicate the centers of cones whose profiles are stable between frames and insensitive to small retinal motion perpendicular to the slit. Gray arrows indicate the edges of cones and reveal the apparent separation or merging of adjacent cones due to retinal motion perpendicular to the slit. Retinal motion parallel to the slit causes a shift in the entire profile.

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Fig. 10.
Fig. 10. Average power spectra obtained by 1D Fourier transformation (black dashed curves) of the conventional flood illuminated AO images in Fig. 4 after being sampled by the 2.8×100 µm slit and (red and blue curves) of cross sectional slices through the interface between the inner and outer segments, outer plexiform layer, outer nuclear layer, and inner plexiform layer. (left) Power spectra are shown for different depths in the retina at the 2.4 deg eccentricity. (center and right) Power spectra are shown with and without AO at eccentricities of 2.4 and 1 degrees, respectively. All parallel SD-OCT curves are normalized to have the same power at 0 cyc/deg.

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Fig. 11.
Fig. 11. Average profile of 20 contiguous A-scans centered about the brightest region in the B-scan for each of the three imaging scenarios shown in Fig. 8. Images were obtained at 2.4 deg retinal eccentricity.

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Tables (2)

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Table 1. Measured wavefront aberrations across a 6.8 mm pupil for one subject with and without AO compensation. RMS wavefront error is shown for the total aberrations, Zernike defocus and astigmatism, and combined 3rd order and higher.

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Table 2. Average SNR for cones and NFL for three imaging scenarios as described in the text.

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