Human Retinal Pigment Epithelium: In Vivo Cell Morphometry, Multispectral Autofluorescence, and Relationship to Cone Mosaic.

Purpose: To characterize in vivo morphometry and multispectral autofluorescence of the retinal pigment epithelial (RPE) cell mosaic and its relationship to cone cell topography across the macula. Methods: RPE cell morphometrics were computed in regularly spaced regions of interest (ROIs) from contiguous short-wavelength autofluorescence (SWAF) and photoreceptor reflectance images collected across the macula in one eye of 10 normal participants (23-65 years) by using adaptive optics scanning light ophthalmoscopy (AOSLO). Infrared autofluorescence (IRAF) images of the RPE were collected with AOSLO in seven normal participants (22-65 years), with participant overlap, and compared to SWAF quantitatively and qualitatively. Results: RPE cell statistics could be analyzed in 84% of SWAF ROIs. RPE cell density consistently decreased with eccentricity from the fovea (participant mean ± SD: 6026 ± 1590 cells/mm2 at fovea; 4552 ± 1370 cells/mm2 and 3757 ± 1290 cells/mm2 at 3.5 mm temporally and nasally, respectively). Mean cone-to-RPE cell ratio decreased rapidly from 16.6 at the foveal center to <5 by 1 mm. IRAF revealed cells in six of seven participants, in agreement with SWAF RPE cell size and location. Differences in cell fluorescent structure, contrast, and visibility beneath vasculature were observed between modalities. Conclusions: Improvements in AOSLO autofluorescence imaging permit efficient visualization of RPE cells with safe light exposures, allowing individual characterization of RPE cell morphometry that is variable between participants. The normative dataset and analysis of RPE cell IRAF and SWAF herein are essential for understanding microscopic characteristics of cell fluorescence and may assist in interpreting disease progression in RPE cells.

Imaging individual neurons in the retinal ganglion cell layer of the living eye.

Although imaging of the living retina with adaptive optics scanning light ophthalmoscopy (AOSLO) provides microscopic access to individual cells, such as photoreceptors, retinal pigment epithelial cells, and blood cells in the retinal vasculature, other important cell classes, such as retinal ganglion cells, have proven much more challenging to image. The near transparency of inner retinal cells is advantageous for vision, as light must pass through them to reach the photoreceptors, but it has prevented them from being directly imaged in vivo. Here we show that the individual somas of neurons within the retinal ganglion cell (RGC) layer can be imaged with a modification of confocal AOSLO, in both monkeys and humans. Human images of RGC layer neurons did not match the quality of monkey images for several reasons, including safety concerns that limited the light levels permissible for human imaging. We also show that the same technique applied to the photoreceptor layer can resolve ambiguity about cone survival in age-related macular degeneration. The capability to noninvasively image RGC layer neurons in the living eye may one day allow for a better understanding of diseases, such as glaucoma, and accelerate the development of therapeutic strategies that aim to protect these cells. This method may also prove useful for imaging other structures, such as neurons in the brain.

Erratum: An adaptive optics imaging system designed for clinical use: publisher's note.

This publisher's note amends the author list and Acknowledgments of a recent publication [Biomed. Opt. Express6, 2120 (2015)].[This corrects the article on p. 2120 in vol. 6, PMID: 26114033.].

An adaptive optics imaging system designed for clinical use.

Here we demonstrate a new imaging system that addresses several major problems limiting the clinical utility of conventional adaptive optics scanning light ophthalmoscopy (AOSLO), including its small field of view (FOV), reliance on patient fixation for targeting imaging, and substantial post-processing time. We previously showed an efficient image based eye tracking method for real-time optical stabilization and image registration in AOSLO. However, in patients with poor fixation, eye motion causes the FOV to drift substantially, causing this approach to fail. We solve that problem here by tracking eye motion at multiple spatial scales simultaneously by optically and electronically integrating a wide FOV SLO (WFSLO) with an AOSLO. This multi-scale approach, implemented with fast tip/tilt mirrors, has a large stabilization range of ± 5.6°. Our method consists of three stages implemented in parallel: 1) coarse optical stabilization driven by a WFSLO image, 2) fine optical stabilization driven by an AOSLO image, and 3) sub-pixel digital registration of the AOSLO image. We evaluated system performance in normal eyes and diseased eyes with poor fixation. Residual image motion with incremental compensation after each stage was: 1) ~2-3 arc minutes, (arcmin) 2) ~0.5-0.8 arcmin and, 3) ~0.05-0.07 arcmin, for normal eyes. Performance in eyes with poor fixation was: 1) ~3-5 arcmin, 2) ~0.7-1.1 arcmin and 3) ~0.07-0.14 arcmin. We demonstrate that this system is capable of reducing image motion by a factor of ~400, on average. This new optical design provides additional benefits for clinical imaging, including a steering subsystem for AOSLO that can be guided by the WFSLO to target specific regions of interest such as retinal pathology and real-time averaging of registered images to eliminate image post-processing.

Calibration-free sinusoidal rectification and uniform retinal irradiance in scanning light ophthalmoscopy.

Sinusoidal rectification (i.e., desinusoiding) is necessary for scanning imaging systems and is typically achieved by calculating a rectification transform from a calibration image such as a regular grid. This approach is susceptible to error due to electronic or mechanical instability that can alter the phase of the imaging window with respect to the calibration transform. Here, we show a calibration-free rectification method implemented from live video of a scanning light ophthalmoscope (SLO) with or without adaptive optics (AO). This approach, which capitalizes on positional differences in the images obtained in the forward and backward scan directions, dynamically keeps the imaging window in phase with the motion of the sinusoidal resonant scanner, preventing errors from signal drift over time. A benefit of this approach is that it allows the light power across the field-of-view (FOV) to be modulated inversely to achieve uniform irradiance on the retina, a feature desirable for functional imaging methods and light safety in SLOs.

Closed-loop optical stabilization and digital image registration in adaptive optics scanning light ophthalmoscopy.

Eye motion is a major impediment to the efficient acquisition of high resolution retinal images with the adaptive optics (AO) scanning light ophthalmoscope (AOSLO). Here we demonstrate a solution to this problem by implementing both optical stabilization and digital image registration in an AOSLO. We replaced the slow scanning mirror with a two-axis tip/tilt mirror for the dual functions of slow scanning and optical stabilization. Closed-loop optical stabilization reduced the amplitude of eye-movement related-image motion by a factor of 10-15. The residual RMS error after optical stabilization alone was on the order of the size of foveal cones: ~1.66-2.56 µm or ~0.34-0.53 arcmin with typical fixational eye motion for normal observers. The full implementation, with real-time digital image registration, corrected the residual eye motion after optical stabilization with an accuracy of ~0.20-0.25 µm or ~0.04-0.05 arcmin RMS, which to our knowledge is more accurate than any method previously reported.

The source of moving particles in parafoveal capillaries detected by adaptive optics scanning laser ophthalmoscopy.

PURPOSE: To investigate the source of moving particles in parafoveal capillaries detected by adaptive optics scanning laser ophthalmoscopy (AO-SLO). METHODS: AO-SLO videos were acquired from the parafoveal areas of eyes of healthy subjects. The gray-scale values inside and outside the moving particles were measured and compared. Thereafter, successive frames of the captured videos were analyzed under higher magnification to detect changes in the gray values of bright spots inside the capillaries, before and during passage of the particles. Simultaneously, changes in the gray values of areas without the bright spots were measured for comparison. Then, the authors analyzed the packing arrangements of the bright spots in the particles, and measured the particle velocity using spatiotemporal images of the target capillary. RESULTS: There were no significant differences in the gray values between the moving particles and the cone mosaic outside the parafoveal capillaries adjacent to the particles. The gray value of the bright spots in the dark shadow of the vessels increased when the particles passed through, while the dark areas without bright spots remained dark. There were no significant differences in the packing arrangements between the bright spots and surrounding cone mosaic. Further, the concordance rate of packing arrangements of bright spots between two consecutive moving particles in the capillary was 95.8%. The mean particle velocity was 1.34 ± 0.42 mm/s. CONCLUSIONS: The particles moving in the capillaries are suggested to be reflections of photoreceptor aggregates that pass through circulating transparent objects such as leukocytes or plasma gaps.