Visible-light Optical Coherence Tomography Research Articles

Longitudinal imaging of vitreal hyperreflective foci in mice with acute optic nerve damage using visible-light optical coherence tomography.

Hyperreflective foci (HRFs) appear in optical coherence tomography (OCT) images of the retina and vitreous of patients with various ocular diseases. HRFs are hypothesized to be immune cells that appear in response to ischemia or tissue damage. To accurately identify HRFs and establish their clinical significance, it is necessary to replicate the detection of similar patterns in vivo in a small animal model. We combined visible-light OCT with temporal speckle averaging (TSA) to visualize and track vitreal HRFs (VHRFs) densities for three days after an optic nerve crush (ONC) injury. Resulting vis-OCT images revealed that VHRF density significantly increased approximately 10-fold at 12 h after ONC and returned to baseline three days after ONC. Additional immunohistochemistry results confirmed these VHRFs as inflammatory cells induced from optic nerve damage.

Current Status and Future Perspectives of Optic Nerve Imaging in Glaucoma.

Being the primary site of degeneration, the optic nerve has always been the focus of structural glaucoma assessment. The technical advancements, mainly of optical coherence tomography (OCT), now allow for a very precise quantification of the optic nerve head and peripapillary retina morphology. By far the most commonly used structural optic nerve parameter is the thickness of the parapapillary retinal nerve fiber, which has great clinical utility but also suffers from significant limitations, mainly in advanced glaucoma. Emerging novel imaging technologies, such as OCT angiography, polarization-sensitive or visible-light OCT and adaptive optics, offer new biomarkers that have the potential to significantly improve structural glaucoma diagnostics. Another great potential lies in the processing of the data already available. Artificial intelligence does not only help increase the reliability of current biomarkers but can also integrate data from various imaging modalities and other clinical measures to increase diagnostic accuracy. And it can, in a more efficient way, draw information from available datasets, such as an OCT scan, compared to the current concept of biomarkers, which only use a fraction of the whole dataset.

Comparative In Vivo Imaging of Retinal Structures in Tree Shrews, Humans, and Mice.

Rodent models, such as mice and rats, are commonly used to examine retinal ganglion cell damage in eye diseases. However, as nocturnal animals, rodent retinal structures differ from primates, imposing significant limitations in studying retinal pathology. Tree shrews (Tupaia belangeri) are small, diurnal paraprimates that exhibit superior visual acuity and color vision compared with mice. Like humans, tree shrews have a dense retinal nerve fiber layer (RNFL) and a thick ganglion cell layer (GCL), making them a valuable model for investigating optic neuropathies. In this study, we applied high-resolution visible-light optical coherence tomography to characterize the tree shrew retinal structure in vivo and compare it with that of humans and mice. We quantitatively characterize the tree shrew's retinal layer structure in vivo, specifically examining the sublayer structures within the inner plexiform layer (IPL) for the first time. Next, we conducted a comparative analysis of retinal layer structures among tree shrews, mice, and humans. We then validated our in vivo findings in the tree shrew inner retina using ex vivo confocal microscopy. The in vivo and ex vivo analyses of the shrew retina build the foundation for future work to accurately track and quantify the retinal structural changes in the IPL, GCL, and RNFL during the development and progression of human optic diseases.

A dual-channel visible light optical coherence tomography system enables wide-field, full-range, and shot-noise limited human retinal imaging

Visible light optical coherence tomography (VIS-OCT) is an emerging ophthalmic imaging method featuring ultrahigh depth resolution, retinal microvascular oximetry, and distinct scattering contrast in the visible spectral range. The clinical utility of VIS-OCT is hampered by the fundamental trade-off between the imaging depth range and axial resolution, which are determined by the spectral resolution and bandwidth, respectively. To address this trade-off, here we developed a dual-channel VIS-OCT system with three major advancements including the first linear-in-K VIS-OCT spectrometer to decrease the roll-off, reference pathlength modulation to expand the imaging depth range, and per-A-line noise cancellation to remove excess noise, Due to these unique designs, this system achieves 7.2 dB roll-off over the full 1.74 mm depth range (water) with shot-noise limited performance. The system uniquely enables >60° wide-field imaging which would allow simultaneous imaging of the peripheral retina and optic nerve head, as well as ultrahigh 1.3 µm depth resolution (water). Benefiting from the additional near-infrared (NIR) channel of the dual-channel design, this system is compatible with Doppler OCT and OCT angiography (OCTA). The comprehensive structure-function measurement enabled by this dual-channel VIS-OCT system is an advance towards adoption of VIS-OCT in clinical applications.

Visible-Light Optical Coherence Tomography Fibergraphy of the Tree Shrew Retinal Ganglion Cell Axon Bundles.

We seek to develop techniques for high-resolution imaging of the tree shrew retina for visualizing and parameterizing retinal ganglion cell (RGC) axon bundles in vivo. We applied visible-light optical coherence tomography fibergraphy (vis-OCTF) and temporal speckle averaging (TSA) to visualize individual RGC axon bundles in the tree shrew retina. For the first time, we quantified individual RGC bundle width, height, and cross-sectional area and applied vis-OCT angiography (vis-OCTA) to visualize the retinal microvasculature in tree shrews. Throughout the retina, as the distance from the optic nerve head (ONH) increased from 0.5 mm to 2.5 mm, bundle width increased by 30%, height decreased by 67%, and cross-sectional area decreased by 36%. We also showed that axon bundles become vertically elongated as they converge toward the ONH. Ex vivo confocal microscopy of retinal flat-mounts immunostained with Tuj1 confirmed our in vivo vis-OCTF findings.

Deep learning network for parallel self-denoising and segmentation in visible light optical coherence tomography of the human retina.

Visible light optical coherence tomography (VIS-OCT) of the human retina is an emerging imaging modality that uses shorter wavelengths in visible light range than conventional near-infrared (NIR) light. It provides one-micron level axial resolution to better separate stratified retinal layers, as well as microvascular oximetry. However, due to the practical limitation of laser safety and comfort, the permissible illumination power is much lower than NIR OCT, which can be challenging to obtain high-quality VIS-OCT images and subsequent image analysis. Therefore, improving VIS-OCT image quality by denoising is an essential step in the overall workflow in VIS-OCT clinical applications. In this paper, we provide the first VIS-OCT retinal image dataset from normal eyes, including retinal layer annotation and "noisy-clean" image pairs. We propose an efficient co-learning deep learning framework for parallel self-denoising and segmentation simultaneously. Both tasks synergize within the same network and improve each other's performance. The significant improvement of segmentation (2% higher Dice coefficient compared to segmentation-only process) for ganglion cell layer (GCL), inner plexiform layer (IPL) and inner nuclear layer (INL) is observed when available annotation drops to 25%, suggesting an annotation-efficient training. We also showed that the denoising model trained on our dataset generalizes well for a different scanning protocol.

Tracking the dynamics of thrombus formation in a blood vessel-on-chip with visible-light optical coherence tomography.

Thrombus formation is a physiological response to damage in a blood vessel that relies on a complex interplay of platelets, coagulation factors, immune cells, and the vessel wall. The dynamics of thrombus formation are essential for a deeper understanding of many disease processes, like bleeding, wound healing, and thrombosis. However, monitoring thrombus formation is challenging due to the limited imaging options available to analyze flowing blood. In this work, we use a visible-light optical coherence tomography (vis-OCT) system to monitor the dynamic process of the formation of thrombi in a microfluidic blood vessel-on-chip (VoC) device. Inside the VoC, thrombi form in a channel lined with a monolayer of endothelial cells and perfused by human whole blood. We show that the correlation of the vis-OCT signal can be utilized as a marker for thrombus formation. By thresholding the correlation during thrombus formation, we track and quantify the growth of the thrombi over time. We validate our results with fluorescence microscopic imaging of fibrin and platelet markers at the end of the blood perfusion assay. In conclusion, we demonstrate that the correlation of the vis-OCT signal can be used to visualize both the spatial and temporal behavior of the thrombus formation in flowing human whole blood.

Imaging human macular pigments with visible light optical coherence tomography and superluminescent diodes.

We demonstrate superluminescent diodes (SLDs) for visible light optical coherence tomography (OCT) of the human retina. SLDs are less costly than supercontinuum sources and have lower intrinsic excess noise, enabling imaging closer to the shot noise limit. While single SLDs are not broadband, they provide power concentrated at specific wavelengths relevant to retinal function. As a new, to the best of our knowledge, application, we image human macular pigments (MPs), which are thought to both aid vision and protect against advanced age-related macular degeneration. Using the unique depth-resolved capabilities of OCT, we localize MPs in depth to Henle's fibers beneath the foveal pit in the living human retina. Our approach reduces the cost of visible light OCT to nearly that of near-infrared (NIR) OCT while also providing information about clinically relevant MPs which cannot be measured in the NIR.

A Generic and Effective System Dispersion Compensation Method: Development and Validation in Visible-Light OCT

Compared with optical coherence tomography (OCT) in the near-infrared domain, the visible-light OCT (vis-OCT) system affords a higher axial resolution for discerning subtle pathological changes associated with early diseases. However, the significant material dispersion at the visible-light range leads to a severe problem for dispersion management in vis-OCT systems, which results in a compromised axial resolution. While dispersion compensators (such as prism pairs) are commonly used, a digital method is still highly desirable and has been widely used to compensate for the residual dispersion imbalance between the reference and sample arms in an OCT system. In this paper, we develop a generic approach to effectively compensate for the system dispersion, especially the higher-order dispersion in the vis-OCT system, by using a single arbitrary measurement of the mirror-reflection (SAMMR) method and its resulting phase information. Compared with the previous methods, including the method based on the Taylor series iterative fitting and differential method, the proposed method does not need to extract the dispersion coefficients or use the metric functions and affords a better performance for axial resolution and the signal-to-noise ratio in vis-OCT systems. Its effectiveness is further validated in an OCT system operating in the near-infrared domain.

Optical Coherence Tomography and Optical Coherence Tomography Angiography: Essential Tools for Detecting Glaucoma and Disease Progression.

Early diagnosis and detection of disease progression are critical to successful therapeutic intervention in glaucoma, the leading cause of irreversible blindness worldwide. Optical coherence tomography (OCT) is a non-invasive imaging technique that allows objective quantification in vivo of key glaucomatous structural changes in the retina and the optic nerve head (ONH). Advances in OCT technology have increased the scan speed and enhanced image quality, contributing to early glaucoma diagnosis and monitoring, as well as the visualization of critically important structures deep within the ONH, such as the lamina cribrosa. OCT angiography (OCTA) is a dye-free technique for noninvasively assessing ocular microvasculature, including capillaries within each plexus serving the macula, peripapillary retina and ONH regions, as well as the deeper vessels of the choroid. This layer-specific assessment of the microvasculature has provided evidence that retinal and choroidal vascular impairments can occur during early stages of glaucoma, suggesting that OCTA-derived measurements could be used as biomarkers for enhancing detection of glaucoma and its progression, as well as to reveal novel insights about pathophysiology. Moreover, these innovations have demonstrated that damage to the macula, a critical region for the vision-related quality of life, can be observed in the early stages of glaucomatous eyes, leading to a paradigm shift in glaucoma monitoring. Other advances in software and hardware, such as artificial intelligence-based algorithms, adaptive optics, and visible-light OCT, may further benefit clinical management of glaucoma in the future. This article reviews the utility of OCT and OCTA for glaucoma diagnosis and disease progression detection, emphasizes the importance of detecting macula damage in glaucoma, and highlights the future perspective of OCT and OCTA. We conclude that the OCT and OCTA are essential glaucoma detection and monitoring tools, leading to clinical and economic benefits for patients and society.

Visible wavelength time-stretch optical coherence tomography.

Visible light optical coherence tomography (OCT) is an emerging non-invasive imaging modality that offers new opportunities for anatomical and functional imaging of biological tissues. Time-stretch dispersive Fourier transform, also known as photonic time-stretch, is an all-optical processing method that enables real-time Fourier transformation of ultrafast optical signals and allows for OCT at high A-scan rates. In this work, a working prototype of a photonic time-stretch OCT (TS-OCT) method in the visible wavelength region is proposed and experimentally demonstrated. The proposed visible-light TS-OCT system achieves unprecedented throughput of 100 giga voxels/second and OCT volume rate of 4,000 volumes/second and can be used to expand the range of applications of TS-OCT systems.

Visible-light optical coherence tomography platform for the characterization of the skin barrier.

We demonstrate a free-space, trolley-mountable Fourier domain visible-light optical coherence tomography (OCT) system for studying the stratum corneum in non-palmar human skin. An axial resolution of 1 µm in tissue and at least -75 dB sensitivity have been achieved. High-quality B-scans, containing 1600 A-scans, are acquired at a rate of 39 Hz. Images from the dorsal hand, ventral wrist and ventral forearm areas are obtained, with a clearly resolved stratum corneum layer (typically 5-15 µm thick) presenting as a hypoechogenic dark layer below the bright entrance signal, similar to that found in palmar skin with traditional OCT systems. We find that the appearance of the stratum corneum layer strongly depends on its water content, becoming brighter after occlusive hydration.

Differential roles of FOXC2 in the trabecular meshwork and Schlemm's canal in glaucomatous pathology.

Impaired development and maintenance of Schlemm's canal (SC) are associated with perturbed aqueous humor outflow and intraocular pressure. The angiopoietin (ANGPT)/TIE2 signaling pathway regulates SC development and maintenance, whereas the molecular mechanisms of crosstalk between SC and the neural crest (NC)-derived neighboring tissue, the trabecular meshwork (TM), are poorly understood. Here, we show NC-specific forkhead box (Fox)c2 deletion in mice results in impaired SC morphogenesis, loss of SC identity, and elevated intraocular pressure. Visible-light optical coherence tomography analysis further demonstrated functional impairment of the SC in response to changes in intraocular pressure in NC-Foxc2 -/- mice, suggesting altered TM biomechanics. Single-cell RNA-sequencing analysis identified that this phenotype is predominately characterized by transcriptional changes associated with extracellular matrix organization and stiffness in TM cell clusters, including increased matrix metalloproteinase expression, which can cleave the TIE2 ectodomain to produce soluble TIE2. Moreover, endothelial-specific Foxc2 deletion impaired SC morphogenesis because of reduced TIE2 expression, which was rescued by deleting the TIE2 phosphatase VE-PTP. Thus, Foxc2 is critical in maintaining SC identity and morphogenesis via TM-SC crosstalk.

Alignment of Visible-Light Optical Coherence Tomography Fibergrams with Confocal Images of the Same Mouse Retina.

In recent years, in vivo retinal imaging, which provides non-invasive, real-time, and longitudinal information about biological systems and processes, has been increasingly applied to obtain an objective assessment of neural damage in eye diseases. Ex vivo confocal imaging of the same retina is often necessary to validate the in vivo findings especially in animal research. In this study, we demonstrated a method for aligning an ex vivo confocal image of the mouse retina with its in vivo images. A new clinical-ready imaging technology called visible light optical coherence tomography fibergraphy (vis-OCTF) was applied to acquire in vivo images of the mouse retina. We then performed the confocal imaging of the same retina as the "gold standard" to validate the in vivo vis-OCTF images. This study not only enables further investigation of the molecular and cellular mechanisms but also establishes a foundation for a sensitive and objective evaluation of neural damage in vivo.

Longitudinal Analysis of Retinal Ganglion Cell Damage at Individual Axon Bundle Level in Mice Using Visible-Light Optical Coherence Tomography Fibergraphy.

We developed a new analytic tool based on visible-light optical coherence tomography fibergraphy (vis-OCTF) to longitudinally track individual axon bundle transformation as a new in vivo biomarker for retinal ganglion cell (RGC) damage. After acute optic nerve crush injury (ONC) in mice, we analyzed four parameters: lateral bundle width, axial bundle height, cross-sectional area, and the shape of individual bundles. We next correlated the morphological changes in RGC axon bundles with RGC soma loss. We showed that axon bundles became wider and taller at three days post ONC (pONC), which correlated with about 15% RGC soma loss. At six days pONC, axon bundles showed a significant reduction in lateral width and cross-sectional area, followed by a reduction in bundle height at nine days pONC. Bundle shrinking at nine days pONC correlated with about 68% RGC soma loss. Both experimental and simulated results suggested that the cross-sectional area of individual RGC axon bundles is more sensitive than bundle width and height to indicate RGC soma loss. This study is the first to track and quantify individual RGC axon bundles in vivo after ONC injury. Recognizing RGC loss at its earliest stage is crucial for disease diagnosis and treatment. However, current clinical methods to detect the functional and structural changes in the inner retina are not sensitive enough to directly assess RGC health. In this study, we developed vis-OCTF-based parameters to track RGC damage, making possible to establishing a quantifiable biomarker for glaucoma.

Adaptive spectroscopic visible-light optical coherence tomography for clinical retinal oximetry

BackgroundRetinal oxygen saturation (sO2) provides essential information about the eye’s response to pathological changes that can result in vision loss. Visible-light optical coherence tomography (vis-OCT) is a noninvasive tool that has the potential to measure retinal sO2 in a clinical setting. However, its reliability is currently limited by unwanted signals referred to as spectral contaminants (SCs), and a comprehensive strategy to isolate true oxygen-dependent signals from SCs in vis-OCT is lacking.MethodsWe develop an adaptive spectroscopic vis-OCT (ADS-vis-OCT) technique that can adaptively remove SCs and accurately measure sO2 under the unique conditions of each vessel. We also validate the accuracy of ADS-vis-OCT using ex vivo blood phantoms and assess its repeatability in the retina of healthy volunteers.ResultsIn ex vivo blood phantoms, ADS-vis-OCT agrees with a blood gas machine with only a 1% bias in samples with sO2 ranging from 0% to 100%. In the human retina, the root mean squared error between sO2 values in major arteries measured by ADS-vis-OCT and a pulse oximeter is 2.1% across 18 research participants. Additionally, the standard deviations of repeated ADS-vis-OCT measurements of sO2 values in smaller arteries and veins are 2.5% and 2.3%, respectively. Non-adaptive methods do not achieve comparable repeatabilities from healthy volunteers.ConclusionsADS-vis-OCT effectively removes SCs from human images, yielding accurate and repeatable sO2 measurements in retinal arteries and veins with varying diameters. This work could have important implications for the clinical use of vis-OCT to manage eye diseases.

Longitudinal Observation of Retinal Response to Optic Nerve Transection in Rats Using Visible Light Optical Coherence Tomography.

To characterize rat retinal responses after optic nerve transection (ONT) by visible-light optical coherence tomography (vis-OCT). Unilateral ONT was performed in Brown Norway rats (n = 8). In vivo, vis-OCT retinal imaging was performed on the experimental eyes before ONT (baseline), and two days, one week, two weeks, and four weeks (endpoint) after ONT, as well as on fellow eyes at the endpoint. The system was operated at a 70 kHz A-line sampling rate with both raster scans (512 × 2 × 512 A-lines), and circular scans (2048 × 100 A-lines) acquired around the optic disc. Retinal layers were segmented to calculate layer thicknesses and project en face images for visualization and quantifications. Vessel densities and oxygen saturation were used to evaluate the morphologic and functional impact on the retinal vasculature. After ONT, retinal nerve fiber bundles demonstrated significant degeneration, starting at two weeks, with a reduction of thicknesses quantified on the nerve fiber layer, ganglion cell complex, and total retina. Along with that, the activation of macrophage-like cells in the vitreoretinal interface was also observed. Vessel densities for all three retinal plexuses were unaffected over the period of observation. However, oxygen saturation in retinal arteries and veins was significantly reduced at four weeks after ONT. Vis-OCT can provide high-definition, in vivo characterization of retinal responses to ONT in rats. Despite a significant reduction in retinal layer thickness, this was not accompanied by alterations in vascular density. Despite this, oximetry indicates reduced retinal oxygen saturation, suggesting that altered vascular physiology is not reflected in the anatomic appearance of retinal blood vessel density alone.

Spectral domain isolation of ballistic component in visible light OCT based on random matrix description

Visible light optical coherence tomography (vis-OCT) provides a unique tool for imaging both structure and oxygen metabolism in ophthalmology. Working in visible light bandwidth, it suffers from noises due to strong scattering, especially in the blood. This work established the random matrix (RM) description of vis-OCT’s k-space data as ballistic and multiple scattering components. The eigenvalue density of the hybrid RM follows a low-rank biased Marčenko–Pastur law. The ballistic component can thus be separated out using a generalized likelihood ratio test algorithm. The RM-based method was validated by both the Monte Carlo simulation and ex vivo pure blood phantom study. We further demonstrated that the RM-based method could significantly improve the imaging quality in the human fundus, showing more details of the layered structure than current vis-OCT with ∼23.6% increase of signal-to-noise ratio, measuring the blood oxygen value more accurately, and enabling better structure visualization than the traditional method, a 1.6-fold higher contrast-to-noise ratio in raster scan mode. The isolated ballistic component also fits the Beer–Lambert law better, giving more accurate oxygen saturation in arc scan mode. The RM-based method significantly improves the reconstruction quality in 3D and facilitates clinical diagnostics. As a general framework, random matrix description also provides a new separation strategy to estimate the ballistic component in other spectral domain OCT techniques.

Visible Light Optical Coherence Tomography of Peripapillary Retinal Nerve Fiber Layer Reflectivity in Glaucoma.

PurposeTo evaluate the clinical utility of visible light optical coherence tomography (VIS-OCT) and to test whether VIS-OCT reflectivity and spectroscopy of peripapillary retinal nerve fiber layer (pRNFL) are correlated with severity of glaucoma, compared with standard-of-care OCT thickness measurements.MethodsIn total 54 eyes (20 normal, 17 suspect/preperimetric glaucoma [GS/PPG], 17 perimetric glaucoma [PG]) were successfully imaged with complete datasets. All the eyes were scanned by a custom-designed dual-channel device that simultaneously acquired VIS-OCT and near-infrared OCT (NIR-OCT) images. A 5 × 5 mm2 scan was taken of the pRNFL. The pRNFL reflectivity was calculated for both channels and the spectroscopic marker was quantified by pVN, defined as the ratio of VIS-OCT to NIR-OCT pRNFL reflectivity. The results were compared with ophthalmic examinations and Zeiss Cirrus OCT.ResultsVIS-OCT pRNFL reflectivity significantly, sequentially decreased from normal to GS/PPG to PG, as did NIR-OCT pRNFL reflectivity. The pVN had the same decreasing trend among three groups. Normal and GS/PPG eyes were significantly different in VIS-OCT pRNFL reflectivity (P = 0.002) and pVN (P < 0.001), but were not in NIR-OCT pRNFL reflectivity (P = 0.14), circumpapillary RNFL thickness (P = 0.17), or macular ganglion cell layer and inner plexiform layer thickness (P = 0.07) in a mixed linear regression model.ConclusionsVIS-OCT pRNFL reflectivity and pVN better distinguished GS/PPG from normal eyes than Cirrus OCT thickness measurements.Translational RelevanceVIS-OCT pRNFL reflectivity and pVN could be useful metrics in the early detection of glaucoma upon further longitudinal validation.

Visible Light Optical Coherence Tomography Reveals the Relationship of the Myoid and Ellipsoid to Band 2 in Humans.

PurposeWe employ visible light optical coherence tomography (OCT) to investigate the relationship between the myoid, ellipsoid, and band 2 in the living human retina. Rather than refute existing theories, we aim to reveal new bands and better delineate the structures at hand.MethodsAn upgraded spectral/Fourier domain visible light OCT prototype, with 1.0-µm axial resolution, imaged 13 eyes of 13 young adult human subjects (23–40 years old) without a history of ocular pathology. The external limiting membrane (band 1) and band 2 edges were segmented. Reflectivity was examined along the inner segment (IS), defined as extending from band 1 to the band 2 center, and within band 2 itself.ResultsImages highlight a nearly continuously resolved extrafoveal internal limiting membrane, the peripheral single-cell thick ganglion cell layer, and the peripheral photoreceptor axonal fiber layer, a peripheral division of band 2 into bands 2a and 2b, and a reflectivity-based division of the IS into “m” and “e” zones.DiscussionTopography and transverse intensity variations of the outermost band 2b suggest an association with rods. The “m” and “e” zone border is consistent with the myoid–ellipsoid boundary, even recapitulating the well-documented distribution of mitochondria throughout the IS at the foveal center. Theories of outer retinal reflectivity in OCT must adequately explain these observations.Translational RelevanceFindings support that band 2 does partially overlap with the ellipsoid in transversally averaged OCT images due to photoreceptor IS length dispersion but argue that the inner ellipsoid must be inner to band 2, as suggested by prior quantitative measurements.