Label-free adaptive optics imaging of human retinal macrophage distribution and dynamics.

Microglia are resident central nervous system macrophages and the first responders to neural injury. Until recently, microglia have been studied only in animal models with exogenous or transgenic labeling. While these studies provided a wealth of information on the delicate balance between neuroprotection and neurotoxicity within which these cells operate, extrapolation to human immune function has remained an open question. Here we examine key characteristics of retinal macrophage cells in live human eyes, both healthy and diseased, with the unique capabilities of our adaptive optics-optical coherence tomography approach and owing to their propitious location above the inner limiting membrane (ILM), allowing direct visualization of cells. Our findings indicate that human ILM macrophage cells may be distributed distinctly, age differently, and have different dynamic characteristics than microglia in other animals. For example, we observed a macular pattern that was sparse centrally and peaked peripherally in healthy human eyes. Moreover, human ILM macrophage density decreased with age (∼2% of cells per year). Our results in glaucomatous eyes also indicate that ILM macrophage cells appear to play an early and regionally specific role of nerve fiber layer phagocytosis in areas of active disease. While we investigate ILM macrophage cells distinct from the larger sample of overall retinal microglia, the ability to visualize macrophage cells without fluorescent labeling in the live human eye represents an important advance for both ophthalmology and neuroscience, which may lead to novel disease biomarkers and new avenues of exploration in disease progression.

3-D printed photoreceptor phantoms for evaluating lateral resolution of adaptive optics imaging systems.

With adaptive optics (AO), optical coherence tomography and scanning laser ophthalmoscopy imaging systems can resolve individual photoreceptor cells in living eyes, due to enhanced lateral spatial resolution. However, no standard test method exists for experimentally quantifying this parameter in ophthalmic AO imagers. Here, we present three-dimensional (3-D) printed phantoms, which enable the measurement of lateral resolution in an anatomically relevant manner. We used two-photon polymerization to fabricate two phantoms, which mimic the mosaic of cone photoreceptor outer segments at multiple retinal eccentricities. With these phantoms, we demonstrated that the resolution of two multimodal AO systems is similar to theoretical predictions, with some intriguing speckle effects.

Application of optical coherence tomography and optical path length method for monitoring corneal thickness and refractive index change during corneal cross-linking.

Corneal cross-linking (CXL) using UVA irradiation with a riboflavin photosensitizer has emerged as a new treatment paradigm for corneal ectatic disorders. The thickness threshold for protection of intraocular structures has often been challenged with ongoing developments, and corneal thinning becomes an important safety concern, especially for patients with thin corneas. In this study with an ex vivo bovine eye model, we monitored corneal thinning and corneal refractive index changes using optical coherence tomography (OCT) integrated with an adaptation of the optical path length method. CXL experiments were performed based on the standard protocol that includes removal of the corneal epithelium to facilitate diffusion of riboflavin into the stroma. The corneal stromal thickness and group refractive index were measured by a 1310 nm Fourier-domain OCT imaging system at three critical points of the procedure: immediately after epithelial removal, after 30 min riboflavin instillation, and after 30 min UVA irradiation with continuing instillation. We found that the refractive index of the bovine cornea changed significantly from epithelial removal to riboflavin instillation and UVA irradiation, increasing from 1.377±0.005 (mean±standard deviation) after de-epithelization to 1.387±0.003 after 30 min instillation and 1.388±0.008 after subsequent irradiation. The corneas also underwent a considerable decrease (10%-20%) in stromal thickness with thinning of 95±29 µm (mean±standard deviation) after riboflavin instillation and a further decrease (∼5%) with thinning of 42±19 µm after UVA irradiation. Our study highlights the importance of corneal thickness monitoring during CXL, especially after riboflavin instillation when the decrease is the largest, to avoid delivering endothelial cytotoxic doses. An increase in refractive index heightens the concern for corneal thinning and the need for careful monitoring as a safety precaution.

Revealing speckle obscured living human retinal cells with artificial intelligence assisted adaptive optics optical coherence tomography.

BACKGROUND: In vivo imaging of the human retina using adaptive optics optical coherence tomography (AO-OCT) has transformed medical imaging by enabling visualization of 3D retinal structures at cellular-scale resolution, including the retinal pigment epithelial (RPE) cells, which are essential for maintaining visual function. However, because noise inherent to the imaging process (e.g., speckle) makes it difficult to visualize RPE cells from a single volume acquisition, a large number of 3D volumes are typically averaged to improve contrast, substantially increasing the acquisition duration and reducing the overall imaging throughput. METHODS: Here, we introduce parallel discriminator generative adversarial network (P-GAN), an artificial intelligence (AI) method designed to recover speckle-obscured cellular features from a single AO-OCT volume, circumventing the need for acquiring a large number of volumes for averaging. The combination of two parallel discriminators in P-GAN provides additional feedback to the generator to more faithfully recover both local and global cellular structures. Imaging data from 8 eyes of 7 participants were used in this study. RESULTS: We show that P-GAN not only improves RPE cell contrast by 3.5-fold, but also improves the end-to-end time required to visualize RPE cells by 99-fold, thereby enabling large-scale imaging of cells in the living human eye. RPE cell spacing measured across a large set of AI recovered images from 3 participants were in agreement with expected normative ranges. CONCLUSIONS: The results demonstrate the potential of AI assisted imaging in overcoming a key limitation of RPE imaging and making it more accessible in a routine clinical setting.

The retinal pigment epithelium (RPE) is a single layer of cells within the eye that is crucial for vision. These cells are unhealthy in many eye diseases, and this can result in vision problems, including blindness. Imaging RPE cells in living human eyes is time consuming and difficult with the current technology. Our method substantially speeds up the process of RPE imaging by incorporating artificial intelligence. This enables larger areas of the eye to be imaged more efficiently. Our method could potentially be used in the future during routine eye tests. This could lead to earlier detection and treatment of eye diseases, and the prevention of some causes of blindness.

Optical coherence tomography-based micro-particle image velocimetry.

We present a new application of optical coherence tomography (OCT), widely used in biomedical imaging, to flow analysis in near-wall hydrodynamics for marine research. This unique capability, called OCT micro-particle image velocimetry, provides a high-resolution view of microscopic flow phenomena and measurement of flow statistics within the first millimeter of a boundary layer. The technique is demonstrated in a small flow cuvette and in a water tunnel.

Introduction to the Feature Issue on Adaptive Optics for Biomedical Applications.

The guest editors introduce a feature issue commemorating the 25th anniversary of adaptive optics in biomedical research.

Improved in vivo optical coherence tomography imaging of animal peripheral nerves using a prism nerve holder.

Significance: Modern optical volumetric imaging modalities, such as optical coherence tomography (OCT), provide enormous information about the structure, function, and physiology of living tissue. Although optical imaging achieves lateral resolution on the order of the wavelength of light used, and OCT achieves axial resolution on a similar micron scale, tissue optical properties, particularly high scattering and absorption, limit light penetration to only a few millimeters. In addition, in vivo imaging modalities are susceptible to significant motion artifacts due to cardiac and respiratory function. These effects limit access to artifact-free optical measurements during peripheral neurosurgery to only a portion of the exposed nerve without further modification to the procedure. Aim: We aim to improve in vivo OCT imaging during peripheral neurosurgery in small and large animals by increasing the amount of visualized nerve volume as well as suppressing motion of the imaged area. Approach: We designed a nerve holder with embedded mirror prisms for peripheral nerve volumetric imaging as well as a specific beam steering strategy to acquire prism and direct view volumes in one session with minimal motion artifacts. Results: The axially imaged volumes from mirror prisms increased the OCT signal intensity by > 22 dB over a 1.25-mm imaging depth in tissue-mimicking phantoms. We then demonstrated the new imaging capabilities in visualizing peripheral nerves from direct and side views in living rats and minipigs using a polarization-sensitive OCT system. Prism views have shown nerve fascicles and vasculature from the bottom half of the imaged nerve which was not visible in direct view. Conclusions: We demonstrated improved OCT imaging during neurosurgery in small and large animals by combining the use of a prism nerve holder with a specifically designed beam scanning protocol. Our strategy can be applied to existing OCT imaging systems with minimal hardware modification, increasing the nerve tissue volume visualized. Enhanced imaging depth techniques may lead to a greater adoption of structural and functional optical biomarkers in preclinical and clinical medicine.

Cellular-Level Visualization of Retinal Pathology in Multiple Sclerosis With Adaptive Optics.

Purpose: To apply adaptive optics-optical coherence tomography (AO-OCT) to quantify multiple sclerosis (MS)-induced changes in axonal bundles in the macular nerve fiber layer, ganglion cell somas, and macrophage-like cells at the vitreomacular interface. Methods: We used AO-OCT imaging in a pilot study of MS participants (n = 10), including those without and with a history of optic neuritis (ON, n = 4), and healthy volunteers (HV, n = 9) to reveal pathologic changes to inner retinal cells and structures affected by MS. Results: We found that nerve fiber layer axonal bundles had 38% lower volume in MS participants (1.5 × 10-3 mm3) compared to HVs (2.4 × 10-3 mm3; P < 0.001). Retinal ganglion cell (RGC) density was 51% lower in MS participants (12.3 cells/mm2 × 1000) compared to HVs (25.0 cells/mm2 × 1000; P < 0.001). Spatial differences across the macula were observed in RGC density. RGC diameter was 15% higher in MS participants (11.7 µm) compared to HVs (10.1 µm; P < 0.001). A nonsignificant trend of higher density of macrophage-like cells in MS eyes was also observed. For all AO-OCT measures, outcomes were worse for MS participants with a history of ON compared to MS participants without a history of ON. AO-OCT measures were associated with key visual and physical disabilities in the MS cohort. Conclusions: Our findings demonstrate the utility of AO-OCT for highly sensitive and specific detection of neurodegenerative changes in MS. Moreover, the results shed light on the mechanisms that underpin specific neuronal pathology that occurs when MS attacks the retina. The new findings support the further development of AO-based biomarkers for MS.

Deep learning-enabled volumetric cone photoreceptor segmentation in adaptive optics optical coherence tomography images of normal and diseased eyes.

Objective quantification of photoreceptor cell morphology, such as cell diameter and outer segment length, is crucial for early, accurate, and sensitive diagnosis and prognosis of retinal neurodegenerative diseases. Adaptive optics optical coherence tomography (AO-OCT) provides three-dimensional (3-D) visualization of photoreceptor cells in the living human eye. The current gold standard for extracting cell morphology from AO-OCT images involves the tedious process of 2-D manual marking. To automate this process and extend to 3-D analysis of the volumetric data, we propose a comprehensive deep learning framework to segment individual cone cells in AO-OCT scans. Our automated method achieved human-level performance in assessing cone photoreceptors of healthy and diseased participants captured with three different AO-OCT systems representing two different types of point scanning OCT: spectral domain and swept source.

Development of polarization-sensitive optical coherence tomography imaging platform and metrics to quantify electrostimulation-induced peripheral nerve injury in vivo in a small animal model.

Significance: Neuromodulation devices are rapidly evolving for the treatment of neurological diseases and conditions. Injury from implantation or long-term use without obvious functional losses is often only detectable through terminal histology. New technologies are needed that assess the peripheral nervous system (PNS) under normal and diseased or injured conditions. Aim: We aim to demonstrate an imaging and stimulation platform that can elucidate the biological mechanisms and impacts of neurostimulation in the PNS and apply it to the sciatic nerve to extract imaging metrics indicating electrical overstimulation. Approach: A sciatic nerve injury model in a 15-rat cohort was observed using a newly developed imaging and stimulation platform that can detect electrical overstimulation effects with polarization-sensitive optical coherence tomography. The sciatic nerve was electrically stimulated using a custom-developed nerve holder with embedded electrodes for 1 h, followed by a 1-h recovery period, delivered at above-threshold Shannon model k -values in experimental groups: sham control (SC, n = 5 , 0.0 mA / 0 Hz ), stimulation level 1 (SL1, n = 5 , 3.4 mA / 50 Hz , and k = 2.57 ), and stimulation level 2 (SL2, n = 5 , 6.8 mA / 100 Hz , and k = 3.17 ). Results: The stimulation and imaging system successfully captured study data across the cohort. When compared to a SC after a 1-week recovery, the fascicle closest to the stimulation lead showed an average change of + 4 % / - 309 % (SL1/SL2) in phase retardation and - 79 % / - 148 % in optical attenuation relative to SC. Analysis of immunohistochemistry (IHC) shows a + 1 % / - 36 % difference in myelin pixel counts and - 13 % / + 29 % difference in axon pixel counts, and an overall increase in cell nuclei pixel count of + 20 % / + 35 % . These metrics were consistent with IHC and hematoxylin/eosin tissue section analysis. Conclusions: The poststimulation changes observed in our study are manifestations of nerve injury and repair, specifically degeneration and angiogenesis. Optical imaging metrics quantify these processes and may help evaluate the safety and efficacy of neuromodulation devices.

Multimodal adaptive optics retinal imager: design and performance.

Optical coherence tomography (OCT) and scanning laser ophthalmoscopy (SLO) are complementary imaging modalities, the combination of which can provide clinicians with a wealth of information to detect retinal diseases, monitor disease progression, or assess new therapies. Adaptive optics (AO) is a tool that enables correction of wavefront distortions from ocular aberrations. We have developed a multimodal adaptive optics system (MAOS) for high-resolution multifunctional use in a variety of research and clinical applications. The system integrates both OCT and SLO imaging channels into an AO beam path. The optics and hardware were designed with specific features for simultaneous SLO/OCT output, for high-fidelity AO correction, for use in humans, primates, and small animals, and for efficient location and orientation of retinal regions of interest. The MAOS system was tested on human subjects and rodents. The design, performance characterization, and initial representative results from the human and animal studies are presented and discussed.

In vivo imaging of photoreceptor disruption associated with age-related macular degeneration: A pilot study.

BACKGROUND AND OBJECTIVE: Age-related macular degeneration is one of the leading causes of vision loss in the developed world. As the disease progresses, the central part of the retina, called the macula, is compromised leading to a disruption of both structure and visual function. In this study, we investigate the disruption of macular photoreceptor cells in vivo as a function of disease stage in patients with the dry form of age-related macular degeneration AMD. MATERIALS AND METHODS: An investigational confocal Adaptive Optics Scanning Laser Ophthalmoscope (AO-SLO) was used to obtain high resolution images of the macular photoreceptor mosaic in patients previously diagnosed with AMD. Four patients were selected as representative cases, comprising each of the four clinical stages of AMD progression. RESULTS: AO-SLO imaging revealed slight disruption in the photoreceptor mosaic in early stage AMD due to focal drusen formation and identified several small drusen deposits that were not observed with standard clinical imaging techniques. An increase in photoreceptor disruption was visualized within the macula in direct correlation with the stage of AMD progression leading to a decrease in visual acuity. Large coalescent drusen and areas of geographic atrophy in advanced stage dry AMD exhibited a significant decrease in visible photoreceptor density. Significant decrease in photoreceptor counts (∼35-50%) were observed when comparing earlier stages of AMD progression (Categories I and II) to later stages of the disease (Categories III and IV). CONCLUSIONS: This study demonstrates the capabilities of adaptive optics retinal imaging to monitor disruption of individual photoreceptor cells as a function of disease progression yielding valuable diagnostic findings in early stage AMD beyond what can be learned about the health of photoreceptors using conventional retinal imaging techniques. Lasers Surg. Med. 44: 603-610, 2012. © 2012 Wiley Periodicals, Inc.

Ultrahigh-speed multimodal adaptive optics system for microscopic structural and functional imaging of the human retina.

We describe the design and performance of a multimodal and multifunctional adaptive optics (AO) system that combines scanning laser ophthalmoscopy (SLO) and optical coherence tomography (OCT) for simultaneous retinal imaging at 13.4 Hz. The high-speed AO-OCT channel uses a 3.4 MHz Fourier-domain mode-locked (FDML) swept source. The system achieves exquisite resolution and sensitivity for pan-macular and transretinal visualization of retinal cells and structures while providing a functional assessment of the cone photoreceptors. The ultra-high speed also enables wide-field scans for clinical usability and angiography for vascular visualization. The FDA FDML-AO system is a powerful platform for studying various retinal and neurological diseases for vision science research, retina physiology investigation, and biomarker development.

Widespread subclinical cellular changes revealed across a neural-epithelial-vascular complex in choroideremia using adaptive optics.

Choroideremia is an X-linked, blinding retinal degeneration with progressive loss of photoreceptors, retinal pigment epithelial (RPE) cells, and choriocapillaris. To study the extent to which these layers are disrupted in affected males and female carriers, we performed multimodal adaptive optics imaging to better visualize the in vivo pathogenesis of choroideremia in the living human eye. We demonstrate the presence of subclinical, widespread enlarged RPE cells present in all subjects imaged. In the fovea, the last area to be affected in choroideremia, we found greater disruption to the RPE than to either the photoreceptor or choriocapillaris layers. The unexpected finding of patches of photoreceptors that were fluorescently-labeled, but structurally and functionally normal, suggests that the RPE blood barrier function may be altered in choroideremia. Finally, we introduce a strategy for detecting enlarged cells using conventional ophthalmic imaging instrumentation. These findings establish that there is subclinical polymegathism of RPE cells in choroideremia.

A Novel Fiber-Optic Confocal Laser Caliper Approach for Non-Contact Optical Characterization of Silk Fibroin Thin Films Created by Riboflavin Photo-Crosslinking.

Silk fibroin with its attractive combination of advanced properties is promising for regenerative treatments of corneal disorders. Novel photonics approach is used to characterize the thickness and refractive index of silk fibroin thin films photo-crosslinked with a natural photosensitizer Riboflavin.

Quantification of Retinal Ganglion Cell Morphology in Human Glaucomatous Eyes.

Purpose: To characterize retinal ganglion cell morphological changes in patients with primary open-angle glaucoma associated with hemifield defect (HD) using adaptive optics-optical coherence tomography (AO-OCT). Methods: Six patients with early to moderate primary open-angle glaucoma with an average age of 58 years associated with HD and six age-matched healthy controls with an average age of 61 years were included. All participants underwent in vivo retinal ganglion cell (RGC) imaging at six primary locations across the macula with AO-OCT. Ganglion cell layer (GCL) somas were manually counted, and morphological parameters of GCL soma density, size, and symmetry were calculated. RGC cellular characteristics were correlated with functional visual field measurements. Results: GCL soma density was 12,799 ± 7747 cells/mm2, 9370 ± 5572 cells/mm2, and 2134 ± 1494 cells/mm2 at 3°, 6°, and 12°, respectively, in glaucoma patients compared with 25,058 ± 4649 cells/mm2, 15,551 ± 2301 cells/mm2, and 3891 ± 1105 cells/mm2 (P < 0.05 for all locations) at the corresponding retinal locations in healthy participants. Mean soma diameter was significantly larger in glaucoma patients (14.20 ± 2.30 µm) compared with the health controls (12.32 ± 1.94 µm, P < 0.05 for all locations); symmetry was 0.36 ± 0.32 and 0.86 ± 0.13 in glaucoma and control cohorts, respectively. Conclusions: Glaucoma patients had lower GCL soma density and symmetry, greater soma size, and increased variation of GCL soma reflectance compared with age-matched control subjects. The morphological changes corresponded with HD, and the cellular level structural loss correlated with visual function loss in glaucoma. AO-based morphological parameters could be potential sensitive biomarkers for glaucoma.

Cell - Vessel Mismatch in Glaucoma: Correlation of Ganglion Cell Layer Soma and Capillary Densities.

Purpose: The purpose of this study was to characterize the relationship between retinal ganglion cell layer (GCL) soma density and capillary density in glaucomatous eyes. Methods: Six glaucoma subjects with known hemifield defects and 6 age-matched controls were imaged with adaptive optics - optical coherence tomography (AO-OCT) at 6 locations: 3 degrees, 6 degrees, and 12 degrees temporal to the fovea above and below the midline. GCL soma density and capillary density were measured at each location. Coefficients of determination (pseudo R2) and slopes between GCL soma and capillary density were determined from mixed-effects regressions and were compared between glaucoma and control subjects, between more and less affected hemifield in subjects with glaucoma, and between subjects with early and moderate glaucoma, both in a local, bivariate model and then a global, multivariable model controlling for eccentricity and soma size. Results: The global correlation between GCL soma and capillary density was stronger in control versus subjects with glaucoma (R2 = 0.59 vs. 0.22), less versus more affected hemifields (R2 = 0.55 vs. 0.01), and subjects with early versus moderate glaucoma subjects (R2 = 0.44 vs. 0.18). When controlling for eccentricity and soma size, we noted an inverse soma-capillary density local relationship in subjects with glaucoma (-388 ± 190 cells/mm2 per 1% change in capillary density, P = 0.046) and more affected hemifields (-602 ± 257 cells/mm2 per 1% change in capillary density, P = 0.03). Conclusions: An inverted soma-capillary density local relationship in areas affected by glaucoma potentially explains weaker global correlations observed between GCL soma and capillary density, suggesting cell-vessel mismatch is associated with the disease.

Dissecting the microvascular contributions to diffuse correlation spectroscopy measurements of cerebral hemodynamics using optical coherence tomography angiography.

Significance: Diffuse correlation spectroscopy (DCS) is an emerging noninvasive, diffuse optical modality that purportedly enables direct measurements of microvasculature blood flow. Functional optical coherence tomography angiography (OCT-A) can resolve blood flow in vessels as fine as capillaries and thus has the capability to validate key attributes of the DCS signal. Aim: To characterize activity in cortical vasculature within the spatial volume that is probed by DCS and to identify populations of blood vessels that are most representative of the DCS signals. Approach: We performed simultaneous measurements of somatosensory-evoked cerebral blood flow in mice in vivo using both DCS and OCT-A. Results: We resolved sensory-evoked blood flow in the somatosensory cortex with both modalities. Vessels with diameters smaller than 10 µ m featured higher peak flow rates during the initial poststimulus positive increase in flow, whereas larger vessels exhibited considerably larger magnitude of the subsequent undershoot. The simultaneously recorded DCS waveforms correlated most highly with flow in the smallest vessels, yet featured a more prominent undershoot. Conclusions: Our direct, multiscale, multimodal cross-validation measurements of functional blood flow support the assertion that the DCS signal preferentially represents flow in microvasculature. The significantly greater undershoot in DCS, however, suggests a more spatially complex relationship to flow in cortical vasculature during functional activation.

Weakly supervised individual ganglion cell segmentation from adaptive optics OCT images for glaucomatous damage assessment.

Cell-level quantitative features of retinal ganglion cells (GCs) are potentially important biomarkers for improved diagnosis and treatment monitoring of neurodegenerative diseases such as glaucoma, Parkinson's disease, and Alzheimer's disease. Yet, due to limited resolution, individual GCs cannot be visualized by commonly used ophthalmic imaging systems, including optical coherence tomography (OCT), and assessment is limited to gross layer thickness analysis. Adaptive optics OCT (AO-OCT) enables in vivo imaging of individual retinal GCs. We present an automated segmentation of GC layer (GCL) somas from AO-OCT volumes based on weakly supervised deep learning (named WeakGCSeg), which effectively utilizes weak annotations in the training process. Experimental results show that WeakGCSeg is on par with or superior to human experts and is superior to other state-of-the-art networks. The automated quantitative features of individual GCLs show an increase in structure-function correlation in glaucoma subjects compared to using thickness measures from OCT images. Our results suggest that by automatic quantification of GC morphology, WeakGCSeg can potentially alleviate a major bottleneck in using AO-OCT for vision research.

Integrating adaptive optics-SLO and OCT for multimodal visualization of the human retinal pigment epithelial mosaic.

In vivo imaging of human retinal pigment epithelial (RPE) cells has been demonstrated through multiple adaptive optics (AO)-based modalities. However, whether consistent and complete information regarding the cellular structure of the RPE mosaic is obtained across these modalities remains uncertain due to limited comparisons performed in the same eye. Here, an imaging platform combining multimodal AO-scanning light ophthalmoscopy (AO-SLO) with AO-optical coherence tomography (AO-OCT) is developed to make a side-by-side comparison of the same RPE cells imaged across four modalities: AO-darkfield, AO-enhanced indocyanine green (AO-ICG), AO-infrared autofluorescence (AO-IRAF), and AO-OCT. Co-registered images were acquired in five subjects, including one patient with choroideremia. Multimodal imaging provided multiple perspectives of the RPE mosaic that were used to explore variations in RPE cell contrast in a subject-, location-, and even cell-dependent manner. Estimated cell-to-cell spacing and density were found to be consistent both across modalities and with normative data. Multimodal images from a patient with choroideremia illustrate the benefit of using multiple modalities to infer the cellular structure of the RPE mosaic in an affected eye, in which disruptions to the RPE mosaic may locally alter the signal strength, visibility of individual RPE cells, or even source of contrast in unpredictable ways.