Freeform robotic optical coherence tomography beyond the optical field-of-view limit.

Imaging complex, non-planar anatomies with optical coherence tomography (OCT) is limited by the optical field of view (FOV) in a single volumetric acquisition. Combining linear mechanical translation with OCT extends the FOV but suffers from inflexibility in imaging non-planar anatomies. We report the freeform robotic OCT to fill this gap. To address challenges in volumetric reconstruction associated with the robotic movement accuracy being two orders of magnitudes worse than OCT imaging resolution, we developed a volumetric registration algorithm based on simultaneous localization and mapping (SLAM) to overcome this limitation. We imaged the entire aqueous humor outflow pathway, whose imaging has the potential to customize glaucoma surgeries but is typically constrained by the FOV, circumferentially in mice as a test. We acquired volumetric OCT data at different robotic poses and reconstructed the entire anterior segment of the eye. The reconstructed volumes showed heterogeneous Schlemm's canal (SC) morphology in the reconstructed anterior segment and revealed a segmental nature in the circumferential distribution of collector channels (CC) with spatial features as small as a few micrometers.

Dynamic chromosome association with nuclear organelles in living cells.

The development of progressively sophisticated tools complemented by the integration of live cell imaging enhances our understanding of the four-dimensional (4D) nucleome, revealing elaborate molecular interactions and chromatin states. Yet, the dynamics of chromosomes in relation to nuclear organelles or to each other across cell cycle in living cells are underexplored. We have developed photoconvertible GFP H3-Dendra2 stably expressing in PC3M cells. The nuclear lamina and perinucleolar associated heterochromatin or diffuse chromosome regions were photoconverted through a single-point activation using a confocal microscope. The results demonstrated a dynamic nature for both types of chromosomes in the same cell cycle and across mitosis. While some chromosome domains were heritably associated with either nuclear lamina or nucleoli, others changed alliance to different nuclear organelles postmitotically. In addition, co-photoconverted chromosome domains often do not stay together within the same cell cycle and across mitosis, suggesting a transient nature of chromosome neighborhoods. Long-range spreading and movement of chromosomes were also observed. Interestingly, when cells were treated with a low concentration of actinomycin D that inhibits Pol I transcription through intercalating GC-rich DNA, chromosome movement was significantly blocked. Treatment with another Pol I inhibitor, metarrestin, which does not impact DNA, had little effect on the movement, suggesting that the DNA structure itself plays a role in chromosome dynamics. Furthermore, inhibition of Pol II transcription with α-amanitin also reduced the chromosome movement, demonstrating that Pol II, but not Pol I transcription, is important for chromosome dynamics in the nucleus.

Maximizing photon utilization in spectroscopic single-molecule localization microscopy using symmetrically dispersed dual-wedge prisms.

Single-molecule localization microscopy (SMLM) enables super-resolution imaging on conventional fluorescent microscopes. Spectroscopic SMLM (sSMLM) further allows highly multiplexed super-resolution imaging. We report an easy-to-implement symmetrically dispersed dual-wedge prism (SDDWP)-sSMLM design that maximizes photon utilization. We first symmetrically dispersed photons to the -1st and +1st orders in an optical assembly using two identical dual-wedge prisms (DWPs). Then we computationally extracted the fluorophores' spatial position and spectral characteristics using photons in both the -1st and +1st orders. Theoretical analysis and experimental validation showed lateral and spectral precisions of 10.1 nm and 0.3 nm, respectively, representing improvements of 28% and 48% over our previous DWP-based system, where emitted photons are divided separately for spatial and spectral analyses.

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.

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.

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.

Reduced Aqueous Humor Outflow Pathway Arborization in Childhood Glaucoma Eyes.

Purpose: To compare aqueous humor outflow (AHO) pathway patterns between eyes of childhood glaucoma patients and non-glaucomatous patients receiving cataract surgery. Methods: Aqueous angiography was performed in childhood glaucoma eyes (n = 5) receiving glaucoma surgery and in pediatric (n = 1) and healthy adult (n = 5) eyes receiving cataract surgery. Indocyanine green (0.4%) was introduced into the anterior chamber, and AHO was imaged using an angiographic camera (SPECTRALIS HRA+OCT with Flex Module). Images were acquired and analyzed (ImageJ with Analyze Skeleton 2D/3D plugin) from the nasal sides of the eyes, the usual site of glaucoma angle procedures. Image analysis endpoints included AHO vessel length, maximum vessel length, number of branches, number of branch junctions, and vessel density. Results: Qualitatively, childhood glaucoma eyes demonstrated lesser AHO pathway arborization compared to pediatric and adult eyes without glaucoma. Quantitatively, childhood glaucoma and healthy adult cataract eyes showed similar AHO pathway average branch lengths and maximum branch lengths (P = 0.49-0.99). However, childhood glaucoma eyes demonstrated fewer branches (childhood glaucoma, 198.2 ± 35.3; adult cataract, 506 ± 59.5; P = 0.002), fewer branch junctions (childhood glaucoma, 74.6 ± 13.9; adult cataract, 202 ± 41.2; P = 0.019), and lower vessel densities (childhood glaucoma, 8% ± 1.4%; adult cataract, 17% ± 2.5%; P = 0.01). Conclusions: Childhood glaucoma patients demonstrated fewer distal AHO pathways and lesser AHO pathway arborization. These anatomical alternations may result in a new source of trabecular meshwork-independent AHO resistance in this disease cohort. Translational Relevance: Elevated distal outflow pathway resistance due to decreased AHO pathway arborization may explain some cases of failed trabecular bypass surgery in childhood glaucoma.

Quantifying nanoscopic alterations associated with mitochondrial dysfunction using three-dimensional single-molecule localization microscopy.

Mitochondrial morphology provides unique insights into their integrity and function. Among fluorescence microscopy techniques, 3D super-resolution microscopy uniquely enables the analysis of mitochondrial morphological features individually. However, there is a lack of tools to extract morphological parameters from super-resolution images of mitochondria. We report a quantitative method to extract mitochondrial morphological metrics, including volume, aspect ratio, and local protein density, from 3D single-molecule localization microscopy 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 quantitatively analyze mitochondrial morphological alterations associated with disease progression on an individual basis.

Multiscale imaging of corneal endothelium damage and Rho-kinase inhibitor application in mouse models of acute ocular hypertension.

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 mouse cornea damage from the in-vivo tissue 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 corneal edema, and applying Rho-kinase inhibitor reduced the population of fully-distorted tight junctions under acute ocular hypertension. Together, these data point to the utility of multiscale optical imaging in revealing fundamental biology relevant to disease and therapeutics.

Physically informed Monte Carlo simulation of dual-wedge prism-based spectroscopic single-molecule localization microscopy.

Significance: The 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 the system parameters to maximize the spectral or localization performance for various molecular labels. Aim: We developed a Monte Carlo (MC)-based model that predicts the imaging output of the DWP-based sSMLM system given different system parameters. Approach: We 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 (BSs) with a reflectance (R):transmittance (T) of R50:T50 and R30:T70 and their tradeoffs. Results: Our MC simulation showed average deviations of 2.5 and 2.1 nm for localization and spectral precisions against theoretical equations and 2.3 and 1.0 nm against fluorescent microspheres. An R30:T70 BS improved the spectral precision by 8% but worsened the localization precision by 35% on average compared with an R50:T50 BS. Conclusions: The MC model accurately predicted the 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.

Theoretical and experimental study on the detection limit of the micro-ring resonator based ultrasound point detectors.

Combining the diffusive laser excitation and the photoacoustic signals detection, photoacoustic computed tomography (PACT) is uniquely suited for deep tissue imaging. A diffraction-limited ultrasound point detector is highly desirable for maximizing the spatial resolution and the field-of-view of the reconstructed volumetric images. Among all the available ultrasound detectors, micro-ring resonator (MRR) based ultrasound detectors offer the lowest area-normalized limit of detection (nLOD) in a miniature form-factor, making it an ideal candidate as an ultrasound point detector. However, despite their wide adoption for photoacoustic imaging, the underlying signal transduction process has not been systematically studied yet. Here we report a comprehensive theoretical model capturing the transduction of incident acoustic signals into digital data, and the associated noise propagation process, using experimentally calibrated key process parameters. The theoretical model quantifies the signal-to-noise ratio (SNR) and the nLOD under the influence of the key process variables, including the quality factor (Q-factor) of the MRR and the driving wavelength. While asserting the need for higher Q-factors, the theoretical model further quantifies the optimal driving wavelength for optimizing the nLOD. Given the MRR with a Q-factor of 1 × 105, the theoretical model predicts an optimal SNR of 30.1 dB and a corresponding nLOD of 3.75 × 10-2 mPa mm2/Hz1/2, which are in good agreement with the experimental measurements of 31.0 dB and 3.39 × 10-2 mPa mm2/Hz1/2, respectively. The reported theoretical model can be used in guiding the optimization of MRR-based ultrasonic detectors and PA experimental conditions, in attaining higher imaging resolution and contrast. The optimized operating condition has been further validated by performing PACT imaging of a human hair phantom.

Nucleoporin downregulation modulates progenitor differentiation independent of nuclear pore numbers.

Nucleoporins (NUPs) comprise nuclear pore complexes, gateways for nucleocytoplasmic transport. As primary human keratinocytes switch from the progenitor state towards differentiation, most NUPs are strongly downregulated, with NUP93 being the most downregulated NUP in this process. To determine if this NUP downregulation is accompanied by a reduction in nuclear pore numbers, we leveraged Stochastic Optical Reconstruction Microscopy. No significant changes in nuclear pore numbers were detected using three independent NUP antibodies; however, NUP reduction in other subcellular compartments such as the cytoplasm was identified. To investigate how NUP reduction influences keratinocyte differentiation, we knocked down NUP93 in keratinocytes in the progenitor-state culture condition. NUP93 knockdown diminished keratinocytes' clonogenicity and epidermal regenerative capacity, without drastically affecting nuclear pore numbers or permeability. Using transcriptome profiling, we identified that NUP93 knockdown induces differentiation genes related to both mechanical and immune barrier functions, including the activation of known NF-κB target genes. Consistently, keratinocytes with NUP93 knockdown exhibited increased nuclear localization of the NF-κB p65/p50 transcription factors, and increased NF-κB reporter activity. Taken together, these findings highlight the gene regulatory roles contributed by differential NUP expression levels in keratinocyte differentiation, independent of nuclear pore numbers.

Adaptive balanced detection spectral domain optical coherence tomography.

Balanced detection optical coherence tomography (BD-OCT) enables near-shot noise-limited imaging by suppressing wavelength-dependent relative intensity noise (RIN) originating from the light source. In spectral-domain BD-OCT (SD-BD-OCT), the level of RIN suppression relies on the co-registration accuracy of the spectra simultaneously captured by two independent spectrometers. However, existing matching methods require careful pre-calibration using a RIN-dominated dataset or subjective post-processing using a signal-dominated dataset. We developed an adaptive subpixel matching approach, referred to as adaptive balance, that can be applied to any SD-BD-OCT dataset regardless of RIN or signal level without the need for pre-calibration. We showed that adaptive balance performed comparable to or better than reported methods by imaging phantoms with varying spectrometer camera gain, exposure time, and supercontinuum laser repetition rate. We further demonstrated the benefits of adaptive balance in human retinal imaging.

Lack of Correlation Between Segmental Trabecular Meshwork Pigmentation and Angiographically Determined Outflow in Ex-Vivo Human Eyes.

PRECIS: Trabecular meshwork pigmentation is not correlated with angiographically determined aqueous humor outflow in an ex-vivo perfusion model using human eyes. PURPOSE: To evaluate whether segmental trabecular meshwork (TM) pigmentation is correlated to segmental aqueous humor outflow (AHO) in human eyes. METHODS: Post-mortem human eyes were acquired, and anterior segments were dissected. TM pigmentation was photographed 360-degrees around the eye. The anterior segments were then mounted onto a perfusion apparatus and perfused with DPBS until a stabile baseline outflow facility was achieved. Aqueous angiography (AHO angiography) was performed using fluorescein (2%), and segmental AHO was documented around the limbus using an angiographic camera (Spectralis HRA+OCT). Circumferential and nasal TM pigmentation were compared to respective angiographic outflow imaging using a Pearson's correlation analysis. RESULTS: Segmental TM pigment distribution and segmental AHO were seen. TM pigment was statistically greatest in the inferior quadrant. AHO angiographic outflow was numerically greatest in the nasal quadrant, but this was not statistically significant. No statistically significant correlation was observed (r=-0.083, P=0.06) between segmental TM pigmentation and segmental AHO angiographic signal. Analyzing just the nasal quadrant, a significant weak negative correlation was found (r=-0.296, P=0.001). DISCUSSION: Segmental TM pigmentation circumferentially around the eye is not a good proxy for segmental AHO circumferentially around the eye and should not be used to guide trabecular minimally invasive glaucoma surgeries.

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.

Investigating Uncertainties in Single-Molecule Localization Microscopy Using Experimentally Informed Monte Carlo Simulation.

Single-molecule localization microscopy (SMLM) enables the visualization of cellular nanostructures in vitro with sub-20 nm resolution. While substructures can generally be imaged with SMLM, the structural understanding of the images remains elusive. To better understand the link between SMLM images and the underlying structure, we developed a Monte Carlo (MC) simulation based on experimental imaging parameters and geometric information to generate synthetic SMLM images. We chose the nuclear pore complex (NPC), a nanosized channel on the nuclear membrane which gates nucleo-cytoplasmic transport of biomolecules, as a test geometry for testing our MC model. Using the MC model to simulate SMLM images, we first optimized our clustering algorithm to separate >106 molecular localizations of fluorescently labeled NPC proteins into hundreds of individual NPCs in each cell. We then illustrated using our MC model to generate cellular substructures with different angles of labeling to inform our structural understanding through the SMLM images obtained.

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.

Highly sensitive ultrasound detection using nanofabricated polymer micro-ring resonators.

Photoacoustic (PA) imaging enables noninvasive volumetric imaging of biological tissues by capturing the endogenous optical absorption contrast. Conventional ultrasound detectors using piezoelectric materials have been widely used for transducing ultrasound signals into the electrical signals for PA imaging reconstruction. However, their inherent limitations in detection bandwidth and sensitivity per unit area have unfortunately constrained the performance of PA imaging. Optical based ultrasound detection methods emerge to offer very promising solutions. In particular, polymer micro-ring resonators (MRRs) in the form of integrated photonic circuits (IPC) enable significant reduction for the sensing area to 80 µm in diameter, while maintaining highly sensitive ultrasound detection with noise equivalent pressure (NEP) of 0.49 Pa and a broad detection frequency range up to 250 MHz. The continued engineering innovation has further transformed MRRs to be transparent to the light and thus, opens up a wide range of applications, including multi-modality optical microscope with isometric resolution, PA endoscope, photoacoustic computed tomography (PACT), and more. This review article summarizes and discusses the evolution of polymer MRR design and the associated nanofabrication process for improving the performance of ultrasound detection. The resulting novel imaging applications will also be reviewed and discussed.

Greater Outflow Facility Increase After Targeted Trabecular Bypass in Angiographically Determined Low-low Regions.

PURPOSE: To investigate the impact of trabecular bypass surgery targeted to angiographically determined high- vs. low-aqueous humor outflow areas on outflow facility (C) and intraocular pressure (IOP). DESIGN: Ex vivo comparative study. SUBJECTS: Postmortem ex vivo porcine and human eyes. METHODS: Porcine (n = 14) and human (n = 13) whole globes were acquired. In both species, anterior segments were dissected, mounted onto a perfusion chamber, and perfused using Dulbecco's phosphate buffered solution containing glucose in a constant flow paradigm to achieve a stable baseline. Fluorescein was perfused into the anterior chamber and used to identify baseline segmental high- and low-flow regions of the conventional outflow pathways. The anterior segments were divided into 2 groups, and a 5 mm needle goniotomy was performed in either a high- or low-flow area. Subsequently, C and IOP were quantitatively reassessed and compared between surgery in baseline "high-flow" and "low-flow" region eyes followed by indocyanine green angiography. MAIN OUTCOME MEASURES: Outflow facility. RESULTS: In all eyes, high- and low-flow segments could be identified. Performing a 5-mm goniotomy increased outflow facility to a variable extent depending on baseline flow status. In the porcine high-flow group, C increased from 0.31 ± 0.09 to 0.39 ± 0.09 µL/mmHg/min (P = 0.12). In the porcine low-flow group, C increased from 0.29 ± 0.03 to 0.56 ± 0.10 µL/mmHg/min (P < 0.001). In the human high-flow group, C increased from 0.38 ± 0.20 to 0.41 ± 0.20 µL/mmHg/min (P = 0.02). In the human low-flow group, C increased from 0.25 ± 0.11 to 0.32 ± 0.11 µL/mmHg/min (<0.001). There was statistically significant greater increase in C for eyes where surgery was targeted to baseline low-flow regions in both porcine (0.07 ± 0.09 vs. 0.27 ± 0.13, P = 0.007 µL/mmHg/min, high vs low flow) and human eyes (0.03 ± 0.03 vs. 0.07 ± 0.02, P = 0.03 µL/mmHg/min, high vs. low flow). CONCLUSIONS: Targeting surgery to low-flow areas of the trabecular meshwork yields higher overall facility increase and IOP reduction compared to surgery in high-flow areas. FINANCIAL DISCLOSURE(S): Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article.

Multiscale imaging of corneal endothelium damage and effects of Rho Kinase inhibitor application in mouse models of acute ocular hypertension.

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 the in-vivo tissue 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.