Jungtae Rha

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.

Photoreceptor Structure and Function in Patients with Congenital Achromatopsia

PURPOSE To assess photoreceptor structure and function in patients with congenital achromatopsia. METHODS Twelve patients were enrolled. All patients underwent a complete ocular examination, spectral-domain optical coherence tomography (SD-OCT), full-field electroretinographic (ERG), and color vision testing. Macular microperimetry (MP; in four patients) and adaptive optics (AO) imaging (in nine patients) were also performed. Blood was drawn for screening of disease-causing genetic mutations. RESULTS Mean (± SD) age was 30.8 (± 16.6) years. Mean best-corrected visual acuity was 0.85 (± 0.14) logarithm of the minimal angle of resolution (logMAR) units. Seven patients (58.3%) showed either an absent foveal reflex or nonspecific retinal pigment epithelium mottling to mild hypopigmentary changes on fundus examination. Two patients showed an atrophic-appearing macular lesion. On anomaloscopy, only 5 patients matched over the entire range from 0 to 73. SD-OCT examination showed a disruption or loss of the macular inner/outer segments (IS/OS) junction of the photoreceptors in 10 patients (83.3%). Seven of these patients showed an optically empty space at the level of the photoreceptors in the fovea. AO images of the photoreceptor mosaic were highly variable but significantly disrupted from normal. On ERG testing, 10 patients (83.3%) showed evidence of residual cone responses to a single-flash stimulus response. The macular MP testing showed that the overall mean retinal sensitivity was significantly lower than normal (12.0 vs. 16.9 dB, P < 0.0001). CONCLUSIONS The current approach of using high-resolution techniques to assess photoreceptor structure and function in patients with achromatopsia should be useful in guiding selection of patients for future therapeutic trials as well as monitoring therapeutic response in these trials.

Arrested Development: High-Resolution Imaging of Foveal Morphology in Albinism

Albinism, an inherited disorder of melanin biosynthesis, disrupts normal retinal development, with foveal hypoplasia as one of the more commonly associated ocular phenotypes. However the cellular integrity of the fovea in albinism is not well understood - there likely exist important anatomical differences that underlie phenotypic variability within the disease and that also may affect responsiveness to therapeutic intervention. Here, using spectral-domain optical coherence tomography (SD-OCT) and adaptive optics (AO) retinal imaging, we obtained high-resolution images of the foveal region in six individuals with albinism. We provide a quantitative analysis of cone density and outer segment elongation demonstrating that foveal cone specialization is variable in albinism. In addition, our data reveal a continuum of foveal pit morphology, roughly aligning with schematics of normal foveal development based on post-mortem analyses. Different albinism subtypes, genetic mutations, and constitutional pigment background likely play a role in determining the degree of foveal maturation.

Spatial and temporal variation of rod photoreceptor reflectance in the human retina

Using adaptive optics imaging tools to image the living retina, numerous investigators have reported temporal fluctuation in the reflectivity of individual cone photoreceptors. In addition, there is cone-to-cone (spatial) variation in reflectivity. As it has only recently become possible to image the complete rod photoreceptor mosaic in the living human retina, we sought to characterize the reflectivity of individual rods and compare their behavior to that of foveal/parafoveal cones. Across two subjects, we were able to successfully track the reflectance behavior of 1,690 rods and 1,980 cones over 12 hours. Rod and cone photoreceptors showed similar regional and temporal variability in their reflectance profiles, suggesting the presence of a common governing physiological process. Within the rod and cone mosaics, there was no sign of spatial clumping of reflectance profile behavior; that is, the arrangement of cells of a given archetypal reflectance profile within the mosaic was indistinguishable from random. These data demonstrate the ability to track the behavior of rod reflectivity over time. Finally, as these and other reflectance changes may be an indicator of photoreceptor function, a future extension of this method will be to analyze this behavior in patients with rod photoreceptor dysfunction (e.g., retinitis pigmentosa, Usher’s syndrome, and congenital stationary night blindness).

Cone photoreceptor mosaic disruption associated with Cys203Arg mutation in the M-cone opsin

Missense mutations in the cone opsins have been identified as a relatively common cause of red/green color vision defects, with the most frequent mutation being the substitution of arginine for cysteine at position 203 (C203R). When the corresponding cysteine is mutated in rhodopsin, it disrupts proper folding of the pigment, causing severe, early onset retinitis pigmentosa. While the C203R mutation has been associated with loss of cone function in color vision deficiency, it is not known what happens to cones expressing this mutant opsin. Here, we used high-resolution retinal imaging to examine the cone mosaic in two individuals with genes encoding a middle-wavelength sensitive (M) pigment with the C203R mutation. We found a significant reduction in cone density compared to normal and color-deficient controls, accompanying disruption in the cone mosaic in both individuals, and thinning of the outer nuclear layer. The C203R mosaics were different from that produced by another mutation (LIAVA) previously shown to disrupt the cone mosaic. Comparison of these mosaics provides insight into the timing and degree of cone disruption and has implications for the prospects for restoration of vision loss associated with various cone opsin mutations.

Variable optical activation of human cone photoreceptors visualized using a short coherence light source

It has been shown that after a visible stimulus, optical oscillations of nearly all cone photoreceptors can be observed using long coherence length light and in a few cones using short coherence length light. Here, we show that after exposure to a visible stimulus, a short coherence length imaging source reveals light-evoked oscillation signals in a large number of cones. More than 80% of cones in a given retinal area are activated (modulation in the reflectance signal) after stimulation, and the pattern of their activation can be subjectively classified into one of four categories. The application of light-evoked signal detection techniques for in vivo retinal imaging may prove useful for assessing the functional status of cones in normal and diseased retinae.

Retinal Imaging Using Commercial Broadband Optical Coherence Tomography

Aims To examine the practical improvement in image quality afforded by a broadband light source in a clinical setting and to define image quality metrics for future use in evaluating spectral domain optical coherence tomography (SD-OCT) images. Methods A commercially available SD-OCT system, configured with a standard source as well as an external broadband light source, was used to acquire 4 mm horizontal line scans of the right eye of 10 normal subjects. Scans were averaged to reduce speckling and multiple retinal layers were analysed in the resulting images. Results For all layers there was a significant improvement in the mean local contrast (average improvement by a factor of 1.66) when using the broadband light source. Intersession variability was shown not to be a major contributing factor to the observed improvement in image quality obtained with the broadband light source. We report the first observation of sublamination within the inner plexiform layer visible with SD-OCT. Conclusion The practical improvement with the broadband light source was significant, although it remains to be seen what the utility will be for diagnostic pathology. The approach presented here serves as a model for a more quantitative analysis of SD-OCT images, allowing for more meaningful comparisons between subjects, clinics and SD-OCT systems.

High-Resolution Images of Retinal Structure in Patients with Choroideremia

PURPOSE To study retinal structure in choroideremia patients and carriers using high-resolution imaging techniques. METHODS Subjects from four families (six female carriers and five affected males) with choroideremia (CHM) were characterized with best-corrected visual acuity (BCVA), kinetic and static perimetry, full-field electroretinography, and fundus autofluorescence (FAF). High-resolution macular images were obtained with adaptive optics scanning laser ophthalmoscopy (AOSLO) and spectral domain optical coherence tomography (SD-OCT). Coding regions of the CHM gene were sequenced. RESULTS Molecular analysis of the CHM gene identified a deletion of exons 9 to 15 in family A, a splice site mutation at position 79+1 of exon 1 in family B, deletion of exons 6 to 8 in family C, and a substitution at position 106 causing a premature stop in family D. BCVA ranged from 20/16 to 20/63 in carriers and from 20/25 to 5/63 in affected males. FAF showed abnormalities in all subjects. SD-OCT showed outer retinal layer loss, outer retinal tubulations at the margin of outer retinal loss, and inner retinal microcysts. Patchy cone loss was present in two symptomatic carriers. In two affected males, cone mosaics were disrupted with increased cone spacing near the fovea but more normal cone spacing near the edge of atrophy. CONCLUSIONS High-resolution retinal images in CHM carriers and affected males demonstrated RPE and photoreceptor cell degeneration. As both RPE and photoreceptor cells were affected, these cell types may degenerate simultaneously in CHM. These findings provide insight into the effect of CHM mutations on macular retinal structure, with implications for the development of treatments for CHM. (ClinicalTrials.gov number, NCT00254605.).