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Anterior segment optical coherence tomography (AS-OCT) is a rapidly evolving area of OCT imaging, providing high-resolution and non-invasive volumetric imaging of the anterior segment. This review focuses on recent advances in AS-OCT imaging in small animals, which we categorize into ultrahigh-resolution, spectroscopic, magnetomotive, polarization-sensitive, and angiographic AS-OCTs. We summarize their technical foundations, review their applications to small animal imaging, and briefly discuss their current and future clinical applications.

SignificanceThe dual-wedge prism (DWP)-based spectroscopic single-molecule localization microscopy (sSMLM) system offers improved localization precision and adjustable spectral or localization performance, but its nonlinear spectral dispersion presents a challenge. A systematic method can help understand the challenges and thereafter optimize the DWP system’s performance by customizing system parameters to maximize spectral or localization performance for various molecular labels.AimWe developed an MC-based model which predicts the imaging output of the DWP-based sSMLM system given different system parameters.ApproachWe assessed our MC model’s localization and spectral precisions by comparing our simulation against theoretical equations and fluorescent microspheres. Furthermore, we simulated the DWP-based system using beamsplitters of Reflectance (R):Transmittance (T) of R50:T50 and R30:T70 and their tradeoffs.ResultsOur MC simulation showed average deviations of 2.5 nm and 2.1 nm for localization and spectral precisions against theoretical equations; and 2.3 nm and 1.0 nm against fluorescent microspheres. An R30:T70 beamsplitter improved spectral precision by 8% but worsened localization precision by 35% on average compared to an R50:T50 beamsplitter.ConclusionsThe MC model accurately predicted localization precision, spectral precision, spectral peaks, and spectral widths of fluorescent microspheres, as validated by experimental data. Our work enhances the theoretical understanding of DWP-based sSMLM for multiplexed imaging, enabling performance optimization.

We developed a multiscale optical imaging workflow, integrating and correlating visible-light optical coherence tomography, confocal laser scanning microscopy, and single-molecule localization microscopy to investigate the mouse cornea damages from thein-vivotissue level to the nanoscopic single-molecule level. We used electron microscopy to validate the imaged nanoscopic structures. We imaged wild-type mice and mice with acute ocular hypertension and examined the effects of Rho Kinase inhibitor application. We defined four types of intercellular tight junction structures as healthy, compact, partially-distorted, and fully-distorted types by labeling the Zonula occludens-1 protein in the corneal endothelial cell layer. We correlated the statistics of the four types of tight junction structures with cornea thickness and intraocular pressure. We found that the population of fully-distorted tight junctions correlated well with the level of cornea edema, and applying Rho Kinase inhibitor reduced the population of fully-distorted tight junctions under acute ocular hypertension.

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.

The morphology of mitochondria provides insights into their functions. Three-dimensional (3D) single-molecule localization microscopy (SMLM) uniquely enables the analysis of mitochondrial morphological features individually, thanks to its high resolution. However, nearly all reported studies of mitochondrial morphology have been qualitative and without statistical analysis. We report a quantitative method to extract mitochondrial morphological metrics, including volume, aspect ratio, and local protein density, from 3D SMLM images, with single-mitochondrion sensitivity. We validated our approach using simulated ground-truth SMLM images of mitochondria. We further tested our morphological analysis on mitochondria that have been altered functionally and morphologically in controlled manners. This work sets the stage to analyze mitochondrial morphological alterations associated with disease progression quantitatively.

By manipulating the spectral dispersion of detected photons, spectroscopic single-molecule localization microscopy (sSMLM) permits concurrent high-throughput single-molecular spectroscopic analysis and imaging. Despite its promising potential, using discrete optical components and managing the delicate balance between spectral dispersion and spatial localization compromise its performance, including nonuniform spectral dispersion, high transmission loss of grating, high optical alignment demands, and reduced precision. We designed a dual-wedge prism (DWP)-based monolithic imaging spectrometer to overcome these challenges. We optimized the DWP for spectrally dispersing focused beam without deviation and with minimal wavefront error. We integrated all components into a compact assembly, minimizing total transmission loss and significantly reducing optical alignment requirements. We show the feasibility of DWP using ray-tracing and numerical simulations. We validated our numerical simulations by experimentally imaging individual nanospheres and confirmed that DWP-sSMLM achieved much improved spatial and spectral precisions of grating-based sSMLM. We also demonstrated DWP-sSMLM in 3D multi-color imaging of cells.

Retinal ganglion cells (RGCs) are heterogeneous projection neurons that convey distinct visual features from the retina to brain. Here, we present a high-throughput in vivo RGC activity assay in response to light stimulation using noninvasive Ca 2+ imaging of thousands of RGCs simultaneously in living mice. Population and single-cell analyses of longitudinal RGC Ca 2+ imaging reveal distinct functional responses of RGCs and unprecedented individual RGC activity conversions during traumatic and glaucomatous degeneration. This study establishes a foundation for future in vivo RGC function classifications and longitudinal activity evaluations using more advanced imaging techniques and visual stimuli under normal, disease, and neural repair conditions. These analyses can be performed at both the population and single-cell levels using temporal and spatial information, which will be invaluable for understanding RGC pathophysiology and identifying functional biomarkers for diverse optic neuropathies.

Super-resolution microscopy can capture spatiotemporal organizations of protein interactions with resolution down to 10 nm; however, the analyses of more than two proteins involving low-abundance protein are challenging because spectral crosstalk and heterogeneities of individual fluorescent labels result in molecular misidentification. Here we developed a deep learning-based imaging analysis method for spectroscopic single-molecule localization microscopy to minimize molecular misidentification in three-color super-resolution imaging. We characterized the 3-fold reduction of molecular misidentification in the new imaging method using pure samples of different photoswitchable fluorophores and visualized three distinct subcellular proteins in U2-OS cell lines. We further validated the protein counts and interactions of TOMM20, DRP1, and SUMO1 in a well-studied biological process - Staurosporine-induced apoptosis by comparing the imaging results with Western-blot analysis of different subcellular portions.

Single-molecule localization microscopy (SMLM) enables the detailed visualization of nuclear pore complexes (NPC) in vitro with sub-20 nm resolution. However, it is challenging to translate the localized coordinates in SMLM images to NPC functions because different algorithms to cluster localizations as individual NPCs can be biased without ground truth for validation. We developed a Monte-Carlo simulation to generate synthetic SMLM images of NPC and used the simulated NPC images as the ground truth to evaluate the performance of six clustering algorithms. We identified HDBSCAN as the optimal clustering algorithm for NPC counting and sizing. Furthermore, we compared the clustering results between the experimental and synthetic data for NUP133, a subunit in the NPC, and found them to be in good agreement.

We developed a new analytic tool based on visible-light optical coherence tomography fibergraphy (vis-OCTF) to longitudinally track individual axon bundle transformation following 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 showed that axon bundles became wider and thicker at 3-days post ONC. The bundle swelling at 3-days post-ONC has correlated with about 15% retinal ganglion cell (RGC) soma loss. At 6-days post-ONC, axon bundles showed a significant reduction in lateral width and cross-sectional area, followed by a reduction in bundle height at 9-days post-ONC. Bundle shrinking at 9-days post-ONC has 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 the bundle width and height to indicate RGC soma loss. This study is the first to track and quantify individual RGC axon bundlesin vivofollowing ONC injury and establish the correlation between the morphological changes of RGC axon bundles and RGC soma loss.

Spectroscopic single-molecule localization microscopy (sSMLM, or spectroscopic nanoscopy) has been established as a key tool in functional super-resolution imaging by providing spatial and spectral information of single molecules at nanoscale resolution. A recently developed dual-wedge prism (DWP) imaging spectrometer, a monolithic optical component, has broadened the accessibility of sSMLM with an improved imaging resolution of more than 40%. It also improved the system reliability by reducing the number of discrete optical components. However, achieving its optimal performance requires the comprehensive understanding of the underlying constraints of the key system parameters, such as the refractive index of the DWP, spectral dispersion (SD), axial separation for three-dimensional (3D) biplane reconstruction, and the overall dimensional constraints. In this work, we present a generalized design principle for the DWP imaging spectrometer. Specifically, we develop the theoretical framework capturing the influence of the primary design parameters, including the achievable SD and localization performance, for different design cases. It further establishes the workflow to design and optimize the DWP imaging spectrometer for better multi-color functional imaging. This will give practical guidance for users to easily design the DWP imaging spectrometer, allowing for straightforward 3D sSMLM implementation.

The optical properties of blood encode oxygen-dependent information. Noninvasive optical detection of these properties is increasingly desirable to extract biomarkers for tissue health. Recently, visible-light optical coherence tomography (vis-OCT) demonstrated retinal oxygen saturation (sO2) measurements by inversely measuring the oxygen-dependent absorption and scattering coefficients of whole blood. However, vis-OCT may be sensitive to optical scattering properties of whole blood, different from those reported in the literature. Incorrect assumptions of such properties can add additional uncertainties or biases to vis-OCT’s sO2 model. This work investigates whole blood’s scattering coefficient measured by vis-OCT. Using Monte Carlo simulation of a retinal vessel, we determined that vis-OCT almost exclusively detects multiple-scattered photons in whole blood. Meanwhile, photons mostly forward scatter in whole blood within the visible spectral range, allowing photons to maintain ballistic paths and penetrate deeply, leading to a reduction in the measured scattering coefficient. We defined a scattering scaling factor (SSF) to account for such a reduction and found that SSF varied with measurement conditions, such as numerical aperture, depth resolution, and depth selection. We further experimentally validated SSF in ex vivo blood phantoms with pre-set sO2 levels and in the human retina, both of which agreed well with our simulation.

Optical properties of blood encode oxygen-dependent information. Noninvasive optical detection of these properties is increasingly desirable to extract biomarkers for tissue health. Recently, visible-light optical coherence tomography (vis-OCT) demonstrated retinal oxygen saturation (sO2) measurements using the depth-resolved spectrum of blood. Such measurements rely on differences between the absorption and scattering coefficients of oxygenated and deoxygenated blood. However, there is still broad disagreement, both theoretically and experimentally, on how vis-OCT measures blood’s scattering coefficient. Incorrect assumptions of blood’s optical properties can add additional uncertainties or biases into vis-OCT’s sO2 model. Using Monte Carlo simulation of a retinal vessel, we determined that vis-OCT almost exclusively detects multiple-scattered photons in blood. Meanwhile, photons mostly forward scatter in blood within the visible spectral range, allowing photons to maintain ballistic paths and penetrate deeply, leading to a reduction in the measured scattering coefficient. We defined a scattering scaling factor (SSF) to account for such a reduction and found that SSF varied with measurement conditions, such as numerical aperture, depth resolution, and depth selection. We further experimentally validated SSF in ex vivo blood phantoms pre-set sO2 levels and in the human retina, both of which agreed well with our simulation.

Aniridia is a panocular condition characterized by impaired eye development and vision, which is mainly due to the haploinsufficiency of the paired-box-6 (PAX6) gene. Like what is seen in aniridia patients, Pax6-deficient mice Pax6(Sey-Neu/+) exhibit a varied degree of ocular damage and impaired vision. Our previous studies showed that theses phenotypes were partially rescued by PD0325901, a mitogen-activated protein kinase kinase (MEK or MAP2K) inhibitor. In this study, we assessed the long-term efficacy of PD0325901 treatment in retinal health and visual behavior. At about one year after the postnatal treatment with PD0325901, Pax6(Sey-Neu/+) mice showed robust improvements in retina size and visual acuity, and the elevated intraocular pressure (IOP) was also alleviated, compared to age-matched mice treated with vehicles only. Moreover, the Pax6(Sey-Neu/+) eyes showed disorganized retinal ganglion cell (RGC) axon bundles and retinal layers, which we termed as hotspots. We found that the PD treatment reduced the number and size of hotspots in the Pax6(Sey-Neu/+) retinas. Taken together, our results suggest that PD0325901 may serve as an efficacious intervention in protecting retina and visual function in aniridia-afflicted subjects.

The infiltration of polymorphonuclear leukocytes (PMNs) in ischemia-reperfusion injury (I/RI) has been implicated as a critical component of inflammatory damage following ischemic stroke. However, successful blockade of PMN transendothelial migration (TEM) in preclinical studies has not translated to meaningful clinical outcomes. To investigate this further, leukocyte infiltration patterns were quantified, and these patterns were modulated by blocking platelet endothelial cell adhesion molecule-1 (PECAM), a key regulator of TEM. LysM-eGFP mice and microscopy were used to visualize all myeloid leukocyte recruitment following ischemia/reperfusion. Visual examination showed heterogeneous leukocyte distribution across the infarct at both 24 and 72 hours after I/RI. A semiautomated process was designed to precisely map PMN position across brain sections. Treatment with PECAM function-blocking antibodies did not significantly affect total leukocyte recruitment but did alter their distribution, with more observed at the cortex at both early and later time points (24 hours: 89% PECAM blocked vs. 72% control; 72 hours: 69% PECAM blocked vs. 51% control). This correlated with a decrease in infarct volume. These findings suggest that TEM, in the setting of I/RI in the cerebrovascular, occurs primarily at the cortical surface. The reduction of stroke size with PECAM blockade suggests that infiltrating PMNs may exacerbate I/RI and indicate the potential therapeutic benefit of regulating the timing and pattern of leukocyte infiltration after stroke.

Impaired development and maintenance of the Schlemm’s Canal (SC) is associated with perturbed aqueous humor outflow regulation and glaucoma progression. Key molecular mechanisms, such as ANGPT/TIE2, PROX1, and VEGF-C/VEGFR-3 regulate SC development and maintenance, but mechanisms of paracrine signaling from neighboring tissues, including the trabecular meshwork (TM) are poorly understood. Here, we show Foxc2 is critical within the neural crest (NC)-derived TM and SC endothelium for development of the aqueous humor outflow pathway. In mice, NC- specific deletion of Foxc2 results in abnormal anterior eye segment development, including impaired SC morphogenesis and functional maintenance, loss of SC identity, and impaired maintenance of intraocular pressure (IOP). Visible light optical coherence tomography angiography analysis also demonstrated functional impairment of the SC in response to changes in IOP in NC-Foxc2-/- mice, suggesting increased TM stiffness. Utilization of single-cell RNA-sequencing (scRNA-seq) analysis then identified that this phenotype is predominately characterized by transcriptional changes associated with extracellular matrix organization and stiffness in TM-associated cell clusters, including increased matrix metalloproteinase (MMP) expression, which can generate soluble TIE2 that acts as an ANGPT trap. As FOXC2 is also critically involved in development of the lymphatic vasculature in other tissues, we also show that endothelial-specific deletion of Foxc2 resulted in impaired SC morphogenesis due to loss of TIE2 expression, which was rescued by deletion of the TIE2 phosphatase VE-PTP. Thus, NC-Foxc2 is critical for development of the TM, and both NC- and endothelial-Foxc2 are key for maintenance of SC identity and its morphogenesis.

Introduction: The spatiotemporal pattern of microvascular reperfusion in vivo after large vessel recanalization is poorly understood. Combining chronic cranial window with embedded microprism (CCW-MP) and visible-light optical coherence tomography angiography (Vis-OCTA) enables the study of mouse cortical cerebrovasculature up to 60 days post-stroke. Vis-OCTA/CCW-MP can identify the presence or absence of microvascular flow, without labelling, up to 1 mm in cortical depth of the mouse brain at 1.3 micron resolution. We sought to build on these findings to understand the pattern of microvascular no-reflow in a mouse model of stroke that simulates large vessel occlusion and reperfusion. Hypothesis: Different regions of the cortex have striking variation in flow characteristics after macrovascular reperfusion. Methods: All mice studied were 3-4 mo old, C57/Bl6 background, and subject to IMPROVE guideline. Control mice (n=3) were studied to confirm absence of cortical or vascular changes induced by CCW-MP surgery. Test mice (n =6) underwent CCW-MP as previously published. After 2 weeks to allow for healing post-surgery, mice were subject to transient middle cerebral artery occlusion (tMCAO) to model large vessel occlusion and reperfusion. Vis-OCTA measured cortical flow at baseline, 24 hours post-stroke, and 72 hours post-stroke. Cortical mouse vascular regions were divided into layers 1-3: layer 4 : layer 5-6 from top to bottom. Results: Control mice showed no neuronal death, astrogliosis, microgliosis, or neutrophil recruitment induced by CCW-MP implantation. There was no significant difference between vascular density for prism-adjacent cortex and prism-remote cortex. We also determined that the CCW-MP images cortical vascular territory supplied by the middle cerebral artery. For the test mice, all six mice survived combined surgery. Vis-OCTA showed four of six mice had reduction of microvascular flow > 20% from baseline by 72h post-stroke at layer 5-6. Conclusions: Microvascular reperfusion post large vessel recanalization can be incomplete up to 72 hours post-stroke, especially at deeper cortical levels. Further studies should define associated changes in inflammatory cells as well as oxygenation of vessels.

We present a deep-learning-based computational approach for multicolor spectroscopic single-molecule localization microscopy. Artificial neural networks trained on simulated data to localize the stochastically blinking single-molecules and enhance the corresponding emission spectra show improved simultaneous multicolor imaging.

Due to their similarities in anatomy, physiology, and pharmacology to humans, mice are a valuable model system to study the generation and mechanisms modulating conventional outflow resistance and thus intraocular pressure. In addition, mouse models are critical for understanding the complex nature of conventional outflow homeostasis and dysfunction that results in ocular hypertension. In this review, we describe a set of minimum acceptable standards for developing, characterizing, and utilizing mouse models of open-angle ocular hypertension. We expect that this set of standard practices will increase scientific rigor when using mouse models and will better enable researchers to replicate and build upon previous findings.

Purpose Growing evidence suggests that dendrite retraction or degeneration in a subpopulation of the retinal ganglion cells (RGCs) may precede detectable soma abnormalities and RGC death in glaucoma. Visualization of the lamellar structure of the inner plexiform layer (IPL) could advance clinical management and fundamental understanding of glaucoma. We investigated whether visible-light optical coherence tomography (vis-OCT) could detect the difference in the IPL sublayer thicknesses between small cohorts of healthy and glaucomatous subjects. Method We imaged nine healthy and five glaucomatous subjects with vis-OCT. Four of the healthy subjects were scanned three times each in two separate visits, and five healthy and five glaucoma subjects were scanned three times during a single visit. IPL sublayers were manually segmented using averaged A-line profiles. Results The mean ages of glaucoma and healthy subjects are 59.6 ± 13.4 and 45.4 ± 14.4 years (P = 0.02.) The visual field mean deviations (MDs) are −26.4 to −7.7 dB in glaucoma patients and −1.6 to 1.1 dB in healthy subjects (P = 0.002). Median coefficients of variation (CVs) of intrasession repeatability for the entire IPL and three sublayers are 3.1%, 5.6%, 6.9%, and 5.6% in healthy subjects and 1.8%, 6.0%, 7.7%, and 6.2% in glaucoma patients, respectively. The mean IPL thicknesses are 36.2 ± 1.5 µm in glaucomatous and 40.1 ± 1.7 µm in healthy eyes (P = 0.003). Conclusions IPL sublayer analysis revealed that the middle sublayer could be responsible for the majority of IPL thinning in glaucoma. Vis-OCT quantified IPL sublayers with good repeatability in both glaucoma and healthy subjects.