Foveolar Drusen Decrease Fixation Stability in Pre-Symptomatic AMD.

Purpose: This study aims at linking subtle changes of fixational eye movements (FEM) in controls and in patients with foveal drusen using adaptive optics retinal imaging in order to find anatomo-functional markers for pre-symptomatic age-related macular degeneration (AMD). Methods: We recruited 7 young controls, 4 older controls, and 16 patients with presymptomatic AMD with foveal drusen from the Silversight Cohort. A high-speed research-grade adaptive optics flood illumination ophthalmoscope (AO-FIO) was used for monocular retinal tracking of fixational eye movements. The system allows for sub-arcminute resolution, and high-speed and distortion-free imaging of the foveal area. Foveal drusen position and size were documented using gaze-dependent imaging on a clinical-grade AO-FIO. Results: FEM were measured with high precision (RMS-S2S = 0.0015 degrees on human eyes) and small foveal drusen (median diameter = 60 µm) were detected with high contrast imaging. Microsaccade amplitude, drift diffusion coefficient, and ISOline area (ISOA) were significantly larger for patients with foveal drusen compared with controls. Among the drusen participants, microsaccade amplitude was correlated to drusen eccentricity from the center of the fovea. Conclusions: A novel high-speed high-precision retinal tracking technique allowed for the characterization of FEM at the microscopic level. Foveal drusen altered fixation stability, resulting in compensatory FEM changes. Particularly, drusen at the foveolar level seemed to have a stronger impact on microsaccade amplitudes and ISOA. The unexpected anatomo-functional link between small foveal drusen and fixation stability opens up a new perspective of detecting oculomotor signatures of eye diseases at the presymptomatic stage.

Label-Free Imaging of Inflammation at the Level of Single Cells in the Living Human Eye.

Purpose: Putative microglia were recently detected using adaptive optics ophthalmoscopy in healthy eyes. Here we evaluate the use of nonconfocal adaptive optics scanning light ophthalmoscopy (AOSLO) for quantifying the morphology and motility of presumed microglia and other immune cells in eyes with retinal inflammation from uveitis and healthy eyes. Design: Observational exploratory study. Participants: Twelve participants were imaged, including 8 healthy participants and 4 posterior uveitis patients recruited from the clinic of 1 of the authors (M.H.E.). Methods: The Pittsburgh AOSLO imaging system was used with a custom-designed 7-fiber optical fiber bundle for simultaneous confocal and nonconfocal multioffset detection. The inner retina was imaged at several locations at multiple timepoints in healthy participants and uveitis patients to generate time-lapse images. Main Outcome Measures: Microglia and macrophages were manually segmented from nonconfocal AOSLO images, and their morphological characteristics quantified (including soma size, diameter, and circularity). Cell soma motion was quantified across time for periods of up to 30 minutes and their speeds were calculated by measuring their displacement over time. Results: A spectrum of cell morphologies was detected in healthy eyes from circular amoeboid cells to elongated cells with visible processes, resembling activated and ramified microglia, respectively. Average soma diameter was 16.1 ± 0.9 µm. Cell movement was slow in healthy eyes (0.02 µm/sec on average), but macrophage-like cells moved rapidly in some uveitis patients (up to 3 µm/sec). In an eye with infectious uveitis, many macrophage-like cells were detected; during treatment their quantity and motility decreased as vision improved. Conclusions: In vivo adaptive optics ophthalmoscopy offers promise as a potentially powerful tool for detecting and monitoring inflammation and response to treatment at a cellular level in the living eye. Financial Disclosures: Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article.

Influence of static and dynamic ocular aberrations on full-field optical coherence tomography for in vivo high-resolution retinal imaging.

Under spatially incoherent illumination, time-domain full-field optical coherence tomography (FFOCT) offers the possibility to achieve in vivo retinal imaging at cellular resolution over a wide field of view. Such performance is possible, albeit there is the presence of ocular aberrations even without the use of classical adaptive optics. While the effect of aberrations in FFOCT has been debated these past years, mostly on low-order and static aberrations, we present, for the first time to our knowledge, a method enabling a quantitative study of the effect of statistically representative static and dynamic ocular aberrations on FFOCT image metrics, such as SNR, resolution, and image similarity. While we show that ocular aberrations can decrease FFOCT SNR and resolution by up to 14 dB and fivefold, we take advantage of such quantification to discuss different possible compromises between performance gain and adaptive optics complexity and speed, to optimize both sensor-based and sensorless FFOCT high-resolution retinal imaging.

Multimodal high-resolution retinal imaging using a camera-based DMD-integrated adaptive optics flood-illumination ophthalmoscope.

We demonstrate the feasibility of a multimodal adaptive optics flood-illumination ophthalmoscope, able to provide both bright-field and dark-field images (such as phase contrast). The multimodality was made possible by integrating a digital micromirror device (DMD) at the illumination path to project a sequence of complementary high-resolution patterns into the retina. Through a versatile post-processing method that digitally selects backscattered or multiply scattered photons, we were able: (1) to achieve up to four-fold contrast increase of bright-field images when imaging the photoreceptor mosaic and nerve fibers; and (2) to visualize translucent retinal features such as capillaries, red blood cells, vessel walls, ganglion cells, and photoreceptor inner segments through phase contrast.

ABNORMALITIES IN THE RETINAL CAPILLARY PLEXUSES IN COATS DISEASE IN ADULTHOOD ON OPTICAL COHERENCE TOMOGRAPHY ANGIOGRAPHY.

PURPOSE: To describe and quantify the abnormalities of the retinal capillary plexuses using optical coherence tomography angiography in Coats disease. METHODS: Retrospective study. Eleven eyes of 11 patients with Coats disease (9 men and two women aged 32-80 years) compared with nine fellow eyes and 11 healthy control eyes. Horizontal bands of contiguous 3 × 3 mm optical coherence tomography angiograms of the superficial vascular plexus and deep capillary complex were acquired from the optic disk to 6 mm temporal to the fovea, through areas with telangiectasia visible on fluorescein angiography in 9 cases. RESULTS: The vascular density was significantly decreased in both plexuses in eyes with Coats disease compared with normal and fellow eyes within the 6 mm temporal to the fovea (superficial vascular plexus: 21.5 vs. 29.4%, P = 0.00004 and vs. 30.3%, P = 0.00008; deep capillary complex, 16.5 vs. 23.9%, P = 0.00004 and vs. 24.7%, P = 0.00008, respectively). The fractal dimension was also significantly decreased in eyes with Coats disease (superficial vascular plexus: 1.796 vs. 1.848 P = 0.001 and vs. 1.833, P = 0.003; deep capillary complex: 1.762 vs. 1.853, P = 0.003 and vs. 1.838, P = 0.004, respectively). CONCLUSION: Retinal plexuses' vascular density was decreased in Coats disease, including in areas with no visible telangiectasia.

Characterization and Analysis of Retinal Axial Motion at High Spatiotemporal Resolution and Its Implication for Real-Time Correction in Human Retinal Imaging.

High-resolution ophthalmic imaging devices including spectral-domain and full-field optical coherence tomography (SDOCT and FFOCT) are adversely affected by the presence of continuous involuntary retinal axial motion. Here, we thoroughly quantify and characterize retinal axial motion with both high temporal resolution (200,000 A-scans/s) and high axial resolution (4.5 µm), recorded over a typical data acquisition duration of 3 s with an SDOCT device over 14 subjects. We demonstrate that although breath-holding can help decrease large-and-slow drifts, it increases small-and-fast fluctuations, which is not ideal when motion compensation is desired. Finally, by simulating the action of an axial motion stabilization control loop, we show that a loop rate of 1.2 kHz is ideal to achieve 100% robust clinical in-vivo retinal imaging.

Design of a radial multi-offset detection pattern for in vivo phase contrast imaging of the inner retina in humans.

Previous work has shown that multi-offset detection in adaptive optics scanning laser ophthalmoscopy (AOSLO) can be used to image transparent cells such as retinal ganglion cells (RGCs) in monkeys and humans. Though imaging in anesthetized monkeys with high light levels produced high contrast images of RGCs, images from humans failed to reach the same contrast due to several drawbacks in the previous dual-wavelength multi-offset approach. Our aim here was to design and build a multi-offset detection pattern for humans at safe light levels that could reveal transparent cells in the retinal ganglion cell layer with a contrast and acquisition time approaching results only previously obtained in monkeys. Here, we present a new single-wavelength solution that allows for increased light power and eliminates problematic chromatic aberrations. Then, we demonstrate that a radial multi-offset detection pattern with an offset distance of 8-10 Airy Disk Diameter (ADD) is optimal to detect photons multiply scattered in all directions from weakly reflective retinal cells thereby enhancing their contrast. This new setup and image processing pipeline led to improved imaging of inner retinal cells, including the first images of microglia with multi-offset imaging in AOSLO.

Partial-field illumination ophthalmoscope: improving the contrast of a camera-based retinal imager.

Effective and accurate in vivo diagnosis of retinal pathologies requires high performance imaging devices, combining a large field of view and the ability to discriminate the ballistic signal from the diffuse background in order to provide a highly contrasted image of the retinal structures. Here, we have implemented the partial-field illumination ophthalmoscope, a patterned illumination modality, integrated to a high pixel rate adaptive optics full-field microscope. This non-invasive technique enables us to mitigate the low signal-to-noise ratio, intrinsic of full-field ophthalmoscopes, by partially illuminating the retina with complementary patterns to reconstruct a wide-field image. This new, to the best of our knowledge, modality provides an image contrast spanning from the full-field to the confocal contrast, depending on the pattern size. As a result, it offers various trade-offs in terms of contrast and acquisition speed, guiding the users towards the most efficient system for a particular clinical application.

Manifestation of aberrations in full-field optical coherence tomography.

We report on a theoretical model for image formation in full-field optical coherence tomography (FFOCT). Because the spatial incoherence of the illumination acts as a virtual confocal pinhole in FFOCT, its imaging performance is equivalent to a scanning time-gated coherent confocal microscope. In agreement with optical experiments enabling a precise control of aberrations, FFOCT is shown to have nearly twice the resolution of standard imaging at moderate aberration level. Beyond a rigorous study on the sensitivity of FFOCT with respect to aberrations, this theoretical model paves the way towards an optimized design of adaptive optics and computational tools for high-resolution and deep imaging of biological tissues.

Strip-based digital image registration for distortion minimization and robust eye motion measurement from scanned ophthalmic imaging systems.

Retinal image-based eye motion measurement from scanned ophthalmic imaging systems, such as scanning laser ophthalmoscopy, has allowed for precise real-time eye tracking at sub-micron resolution. However, the constraints of real-time tracking result in a high error tolerance that is detrimental for some eye motion measurement and imaging applications. We show here that eye motion can be extracted from image sequences when these constraints are lifted, and all data is available at the time of registration. Our approach identifies and discards distorted frames, detects coarse motion to generate a synthetic reference frame and then uses it for fine scale motion tracking with improved sensitivity over a larger area. We demonstrate its application here to tracking scanning laser ophthalmoscopy (TSLO) and adaptive optics scanning light ophthalmoscopy (AOSLO), and show that it can successfully capture most of the eye motion across each image sequence, leaving only between 0.1-3.4% of non-blink frames untracked, while simultaneously minimizing image distortions induced from eye motion. These improvements will facilitate precise measurement of fixational eye movements (FEMs) in TSLO and longitudinal tracking of individual cells in AOSLO.

Spatial-frequency-based image reconstruction to improve image contrast in multi-offset adaptive optics ophthalmoscopy.

Off-axis detection methods in adaptive optics (AO) ophthalmoscopy can enhance image contrast of translucent retinal structures such as cone inner segments and retinal ganglion cells. Here, we propose a 2D optical model showing that the phase contrast produced by these methods depends on the offset orientation. While one axis provides an asymmetric light distribution, hence high phase contrast, the perpendicular axis provides a symmetric one, thus substantially lower contrast. We support this model with in vivo human data acquired with a multi-offset AO scanning light ophthalmoscope. Then, using this finding, we provide a post-processing method, named spatial-frequency-based image reconstruction, to optimally combine images from different off-axis detector orientations, significantly increasing the structural cellular contrast of in vivo human retinal neurons such as cone inner segment, putative rods, and retinal ganglion cells.

Adaptive-glasses time-domain FFOCT for wide-field high-resolution retinal imaging with increased SNR.

The highest three-dimensional (3D) resolution possible in in vivo retinal imaging is achieved by combining optical coherence tomography (OCT) and adaptive optics. However, this combination brings important limitations, such as small field-of-view and complex, cumbersome systems, preventing so far the translation of this technology from the research lab to clinics. In this Letter, we mitigate these limitations by combining our compact time-domain full-field OCT (FFOCT) with a multi-actuator adaptive lens positioned just in front of the eye, in a technique we call the adaptive-glasses wavefront sensorless approach. Through this approach, we demonstrate that ocular aberrations can be corrected, increasing the FFOCT signal-to-noise ratio (SNR) and enabling imaging of different retinal layers with a 3D cellular resolution over a 5∘×5∘ field-of-view, without apparent anisoplanatism.

Coherence gate shaping for wide field high-resolution in vivo retinal imaging with full-field OCT.

Allying high-resolution with a large field-of-view (FOV) is of great importance in the fields of biology and medicine, but it is particularly challenging when imaging non-flat living samples such as the human retina. Indeed, high-resolution is normally achieved with adaptive optics (AO) and scanning methods, which considerably reduce the useful FOV and increase the system complexity. An alternative technique is time-domain full-field optical coherence tomography (FF-OCT), which has already shown its potential for in-vivo high-resolution retinal imaging. Here, we introduce coherence gate shaping for FF-OCT, to optically shape the coherence gate geometry to match the sample curvature, thus achieving a larger FOV than previously possible. Using this instrument, we obtained high-resolution images of living human photoreceptors close to the foveal center without AO and with a 1 mm × 1 mm FOV in a single shot. This novel advance enables the extraction of photoreceptor-based biomarkers with ease and spatiotemporal monitoring of individual photoreceptors. We compare our findings with AO-assisted ophthalmoscopes, highlighting the potential of FF-OCT, as a compact system, to become a routine clinical imaging technique.

Optical Incoherence Tomography: a method to generate tomographic retinal cross-sections with non-interferometric adaptive optics ophthalmoscopes.

We present Optical Incoherence Tomography (OIT): a completely digital method to generate tomographic retinal cross-sections from en-face through-focus image stacks acquired by non-interferometric imaging systems, such as en-face adaptive optics (AO)-ophthalmoscopes. We demonstrate that OIT can be applied to different imaging modalities using back-scattered light, including systems without inherent optical sectioning and, for the first time, multiply-scattered light, revealing a distinctive cross-sectional view of the retina. The axial dimension of OIT cross-sections is given in terms of focus position rather than optical path, as in OCT. We explore this property to guide focus position in cases where the user is "blind" focusing, allowing precise plane selection for en-face imaging of retinal pigment epithelium, the vascular plexuses and translucent retinal neurons, such as photoreceptor inner segments and retinal ganglion cells, using respectively autofluorescence, motion contrast and split detection techniques.

Retinal Capillary Plexus Pattern and Density from Fovea to Periphery Measured in Healthy Eyes with Swept-Source Optical Coherence Tomography Angiography.

Optical coherence tomography angiography is evolving towards wider fields of view. As single widefield acquisitions have a lower resolution, preventing an accurate segmentation of vascular plexuses in the periphery, we examined the retinal vascularisation from the macula to the periphery in all retinal quadrants, using 3 × 3-mm volume scans, to obtain montages with sufficient image resolution up to 11 mm from the foveal centre. Images were qualitatively and quantitatively analysed, using C- and B-scan approaches to calculate the capillary density (CD) and the interplexus distance (IPD). Three vascular plexuses (i.e., superficial vascular plexus: SVP, intermediate capillary plexus: ICP, and deep capillary plexus: DCP) were observed up to the mid-periphery in all sectors. The CD of the SVP decreased from about 5 mm of eccentricity, along with ganglion cell density decrease. The CD of the ICP progressively decreased from the fovea towards the periphery, along with the retinal thinning and then vanished from 8 to 9 mm of eccentricity, becoming undetectable beyond. This ICP disappearance resulted in an increased IPD between the SVP and the DCP in an area known to be frequently affected by capillary drop-out in diabetic retinopathy. The DCP only showed a slightly decreased CD towards the retinal periphery.

High-resolution in-vivo human retinal imaging using full-field OCT with optical stabilization of axial motion.

Time-domain full-field OCT (FF-OCT) represents an imaging modality capable of recording high-speed en-face sections of a sample at a given depth. One of the biggest challenges to transfer this technique to image in-vivo human retina is the presence of continuous involuntary head and eye axial motion during image acquisition. In this paper, we demonstrate a solution to this problem by implementing an optical stabilization in an FF-OCT system. This was made possible by combining an FF-OCT system, an SD-OCT system, and a high-speed voice-coil translation stage. B-scans generated by the SD-OCT were used to measure the retina axial position and to drive the position of the high-speed voice coil translation stage, where the FF-OCT reference arm is mounted. Closed-loop optical stabilization reduced the RMS error by a factor of 7, significantly increasing the FF-OCT image acquisition efficiency. By these means, we demonstrate the capacity of the FF-OCT to resolve cone mosaic as close as 1.5 o from the fovea center with high consistency and without using adaptive optics.

Near infrared adaptive optics flood illumination retinal angiography.

Image-based angiography is a well-adapted technique to characterize vasculature, and has been used in retinal neurovascular studies. Because the microvasculature is of particular interest, being the site of exchange between blood and tissue, a high spatio-temporal resolution is required, implying the use of adaptive optics ophthalmoscopes with a high frame rate. Creating the opportunity for decoupled stimulation and imaging of the retina makes the use of near infrared (NIR) imaging light desirable, while the need for a large field of view and a lack of distortion implies the use of a flood illumination-based setup. However, flood-illumination NIR video sequences of erythrocytes, or red blood cells (RBC), have a limited contrast compared to scanning systems and visible light. As a result, they cannot be processed via existing image-based angiography methods. We have therefore developed a new computational method relying on a spatio-temporal filtering of the sequence to isolate blood flow from noise in low-contrast sequences. Applying this computational approach enabled us to perform angiography with an adaptive optics flood illumination ophthalmoscope (AO-FIO) using NIR light, both in bright-field and dark-field modalities. Finally, we demonstrate the capabilities of our system to differentiate blood flow velocity on a retinal capillary network in vivo.

Higher adaptive optics loop rate enhances axial resolution in nonconfocal ophthalmoscopes.

In this Letter, we propose a way to better understand the impact of dynamic ocular aberrations in the axial resolution of nonconfocal adaptive optics (AO) ophthalmoscopes via a simulation of the 3D PSF in the retina for various AO-loop rates. We then use optical incoherence tomography, a method enabling the generation of tomographic retinal cross sections in incoherent imaging systems, to evaluate the benefits of a fast AO-loop rate on axial resolution and, consequently, on AO-corrected retinal image quality. We used the PARIS AO flood-illumination ophthalmoscope for this study, where retinal images from different focal planes at an AO-loop rate of 10 and 50 Hz were acquired.

High loop rate adaptive optics flood illumination ophthalmoscope with structured illumination capability.

The design and performance of an adaptive optics flood illumination ophthalmoscope (AO-FIO) platform, based on eye motion and dynamic aberrations experimental analysis, are described. The system incorporates a custom-built real-time controller, enabling up to 70 Hz loop rate without jitter, and an AO-corrected illumination capable of projecting high-resolution features in the retina. Wide-field (2.7°×5.4°) and distortionless images from vessel walls, capillaries, and the lamina cribrosa are obtained with an enhanced contrast and signal-to-noise ratio, thanks to careful control of AO parameters. The high spatial and temporal resolution (image acquisition up to 200 Hz) performance achieved by this platform enables the visualization of vessel deformation and blood flow. This system opens up the prospect of a return to favor of flood illumination adaptive optics systems provided that its high pixel rate and structured illumination capabilities are exploited.

Fixational eye movement: a negligible source of dynamic aberration.

To evaluate the contribution of fixational eye movements to dynamic aberration, 50 healthy eyes were examined with an original custom-built Shack-Hartmann aberrometer, running at a temporal frequency of 236Hz, with 22 lenslets across a 5mm pupil, synchronized with a 236Hz pupil tracker. A comparison of the dynamic behavior of the first 21 Zernike modes (starting from defocus) with and without digital pupil stabilization, on a 3.4s sequence between blinks, showed that the contribution of fixational eye movements to dynamic aberration is negligible. Therefore we highlighted the fact that a pupil tracker coupled to an Adaptive Optics Ophthalmoscope is not essential to achieve diffraction-limited resolution.