Wide-Field Three-Dimensional Depth-Invariant Cellular-Resolution Imaging of the Human Retina.

Three-dimensional (3D) cellular-resolution imaging of the living human retina over a large field of view will bring a great impact in clinical ophthalmology, potentially finding new biomarkers for early diagnosis and improving the pathophysiological understanding of ocular diseases. While hardware-based and computational adaptive optics (AO) optical coherence tomography (OCT) have been developed to achieve cellular-resolution retinal imaging, these approaches support limited 3D imaging fields, and their high cost and intrinsic hardware complexity limit their practical utility. Here, this work demonstrates 3D depth-invariant cellular-resolution imaging of the living human retina over a 3 × 3 mm field of view using the first intrinsically phase-stable multi-MHz retinal swept-source OCT and novel computational defocus and aberration correction methods. Single-acquisition imaging of photoreceptor cells, retinal nerve fiber layer, and retinal capillaries is presented across unprecedented imaging fields. By providing wide-field 3D cellular-resolution imaging in the human retina using a standard point-scan architecture routinely used in the clinic, this platform proposes a strategy for expanded utilization of high-resolution retinal imaging in both research and clinical settings.

Direct Blood Cell Flow Imaging in Microvascular Networks.

Blood flow dynamics in microvascular networks are intimately related to the health of tissues and organs. While numerous imaging modalities and techniques have been developed to assess blood flow dynamics for various applications, their utilization has been hampered by limited imaging speed and indirect quantification of blood flow dynamics. Here, direct blood cell flow imaging (DBFI) is demonstrated that provides visualization of individual motions of blood cells over a field of 0.71 mm × 1.42 mm with a time resolution of 0.69 ms (1450 frames s-1 ) without using any exogenous agents. DBFI enables precise dynamic analysis of blood cell flow velocities and fluxes in various vessels over a large field, from capillaries to arteries and veins, with unprecedented time resolution. Three exemplary applications of DBFI, quantification of blood flow dynamics of 3D vascular networks, analysis of heartbeat induced blood flow dynamics, and analysis of blood flow dynamics of neurovascular coupling, illustrate the potential of this new imaging technology.

Dll4 Suppresses Transcytosis for Arterial Blood-Retinal Barrier Homeostasis.

RATIONALE: Central nervous system has low vascular permeability by organizing tight junction (TJ) and limiting endothelial transcytosis. While TJ has long been considered to be responsible for vascular barrier in central nervous system, suppressed transcytosis in endothelial cells is now emerging as a complementary mechanism. Whether transcytosis regulation is independent of TJ and its dysregulation dominantly causes diseases associated with edema remain elusive. Dll4 signaling is important for various vascular contexts, but its role in the maintenance of vascular barrier in central nervous system remains unknown. OBJECTIVE: To find a TJ-independent regulatory mechanism selective for transcytosis and identify its dysregulation as a cause of pathological leakage. METHODS AND RESULTS: We studied transcytosis in the adult mouse retina with low vascular permeability and employed a hypertension-induced retinal edema model for its pathological implication. Both antibody-based and genetic inactivation of Dll4 or Notch1 induce hyperpermeability by increasing transcytosis without junctional destabilization in arterial endothelial cells, leading to nonhemorrhagic leakage predominantly in the superficial retinal layer. Endothelial Sox17 deletion represses Dll4 in retinal arteries, phenocopying Dll4 blocking-driven vascular leakage. Ang II (angiotensin II)-induced hypertension represses arterial Sox17 and Dll4, followed by transcytosis-driven retinal edema, which is rescued by a gain of Notch activity. Transcriptomic profiling of retinal endothelial cells suggests that Dll4 blocking activates SREBP1 (sterol regulatory element-binding protein 1)-mediated lipogenic transcription and enriches gene sets favorable for caveolae formation. Profiling also predicts the activation of VEGF (vascular endothelial growth factor) signaling by Dll4 blockade. Inhibition of SREBP1 or VEGF-VEGFR2 (VEGF receptor 2) signaling attenuates both Dll4 blockade-driven and hypertension-induced retinal leakage. CONCLUSIONS: In the retina, Sox17-Dll4-SREBP1 signaling axis controls transcytosis independently of TJ in superficial arteries among heterogeneous regulations for the whole vessels. Uncontrolled transcytosis via dysregulated Dll4 underlies pathological leakage in hypertensive retina and could be a therapeutic target for treating hypertension-associated retinal edema.

Robust and easy-to-operate stretched-pulse mode-locked wavelength-swept laser with an all-polarization-maintaining fiber cavity for 10 MHz A-line rate optical coherence tomography.

We demonstrate robust and easy-to-operate stretched-pulse mode-locked laser (SPML) architectures using all-polarization-maintaining fiber laser cavities. Because of the polarization-maintaining construction, the laser performance is unaffected by mechanical perturbation on the cavity fibers. The lasers automatically initiate linear-in-wavenumber sweeps across 100 nm centered at 1290 nm with a 10 MHz repetition rate. OCT imaging with a sensitivity of 98 dB and a single-sided 6 dB coherence length of 2.5 mm is demonstrated. OCT angiography of a mouse brain that visualized three-dimensional cerebral microvasculature over a field of 1.5mm×1.5mm (398 A-lines × 380 B-scans) at a rate of 5.26 volumes per second is also presented. The robust all-PMF SPML lasers are a turnkey, high-performance source for ultrahigh-speed OCT imaging.

Quantification of retinal microvascular parameters by severity of diabetic retinopathy using wide-field swept-source optical coherence tomography angiography.

PURPOSE: To investigate the diagnostic utility of microvascular parameters for grading the severity of diabetic retinopathy (DR) with a range of views using wide-field swept-source optical coherence tomography angiography (SS-OCTA). METHODS: This retrospective study grouped 235 eyes with diabetes into the five grades: diabetes without retinopathy (no-DR), mild non-proliferative DR (NPDR), moderate NPDR, severe NPDR, and proliferative DR (PDR). Foveal avascular zone (FAZ) metrics, vessel density (VD), and the capillary nonperfusion area (NPA) were quantified with a customized, semiautomatic software algorithm. Regions of interest were selected from three rectangular fields of different sizes (i.e., 3 × 3 mm2, 6 × 6 mm2, and 10 × 10 mm2), perpendicular to the fovea-optic disc axis. RESULTS: NPA obtained from the 6 × 6mm2 and 10 × 10mm2 areas was the only discriminating parameter for the three NPDR stages. ROC curve analysis revealed that NPA from the 10 × 10mm2 field exhibited the best performance for grading DR into five stages. The NPA cutoff values were 3.7% (area under the curve (AUC): 0.91), 4.7% (AUC: 0.94), 9.3% (AUC: 0.94), and 21.4% (AUC: 0.90) for grading no-DR, mild from moderate NPDR, moderate from severe NPDR, and severe NPDR from PDR, respectively. CONCLUSIONS: Increasing DR severity as assessed by conventional grading systems is accompanied with increasing retinal ischemia on SS-OCTA. NPA measured from the larger 10 × 10 mm2 scan area showed the highest sensitivity for determining five-grade DR severity. In the future, the addition of quantitative NPA may provide a more clinically feasible DR grading system.

Automatic segmentation of corneal dystrophy on photographic images based on texture analysis.

PURPOSE: To develop an automatic algorithm to analyze dystrophic lesions on photographic images of corneal dystrophy. METHODS: The dataset included 32 images of corneal dystrophy. The dystrophic area was manually segmented twice. Manually labeled dystrophy areas were compared with automatically segmented images. First, we manually removed the light reflex from the image of the cornea. Using an automatic approach, we extracted the brown color of the iris. Then, the program detected the circular region of the pupil and the corneal surface. A whitish dystrophy area was defined based on the image intensity on the iris and the pupil. The sliding square kernel was applied to clearly define the dystrophic region. RESULTS: For the manual analysis and the twice automatic approach, the Dice similarity was 0.804 and 0.801, respectively. The Pearson correlation coefficient was 0.807 and 0.806, respectively. The total number of distinct dystrophic areas showed no significant difference between the manual and automatic approaches according to the Wilcoxon signed-rank test (p < 0.0001, both). CONCLUSIONS: We proposed an automatic algorithm for detecting the dystrophy areas on photographic images with an accuracy of approximately 0.80. This system can be applied to detect and predict the progression of corneal dystrophy.

Wide dynamic range high-speed three-dimensional quantitative OCT angiography with a hybrid-beam scan.

We demonstrate a novel hybrid-beam scanning-based quantitative optical coherence tomography angiography (OCTA) that provides high-speed wide dynamic range blood flow speed imaging. The hybrid-beam scanning scheme enables multiple OCTA image acquisitions with a wide range of multiple time intervals simultaneously providing wide dynamic range blood flow speed imaging independent of the blood vessel orientation, which was quantified over a speed range of 0.6∼104 mm/s through the blood flow phantom experiments. A fully automated high-speed hybrid-beam scanning-based quantitative OCTA system demonstrates visualization of blood flow speeds in various vessels from the main arteries to capillaries in a 4 mm×4 mm area (1024 A-lines × 512 B-scans) in vivo in 20 s, showing its potential as a useful imaging tool for various biomedical applications.

Intracoronary dual-modal optical coherence tomography-near-infrared fluorescence structural-molecular imaging with a clinical dose of indocyanine green for the assessment of high-risk plaques and stent-associated inflammation in a beating coronary artery.

AIMS: Inflammation plays essential role in development of plaque disruption and coronary stent-associated complications. This study aimed to examine whether intracoronary dual-modal optical coherence tomography (OCT)-near-infrared fluorescence (NIRF) structural-molecular imaging with indocyanine green (ICG) can estimate inflammation in swine coronary artery. METHODS AND RESULTS: After administration of clinically approved NIRF-enhancing ICG (2.0 mg/kg) or saline, rapid coronary imaging (20 mm/s pullback speed) using a fully integrated OCT-NIRF catheter was safely performed in 12 atheromatous Yucatan minipigs and in 7 drug-eluting stent (DES)-implanted Yorkshire pigs. Stronger NIRF activity was identified in OCT-proven high-risk plaque compared to normal or saline-injected controls (P = 0.0016), which was validated on ex vivo fluorescence reflectance imaging. In vivo plaque target-to-background ratio (pTBR) was much higher in inflamed lipid-rich plaque compared to fibrous plaque (P < 0.0001). In vivo and ex vivo peak pTBRs correlated significantly (P < 0.0022). In vitro cellular ICG uptake and histological validations corroborated the OCT-NIRF findings in vivo. Indocyanine green colocalization with macrophages and lipids of human plaques was confirmed with autopsy atheroma specimens. Two weeks after DES deployment, OCT-NIRF imaging detected strong NIRF signals along stent struts, which was significantly higher than baseline (P = 0.0156). Histologically, NIRF signals in peri-strut tissue co-localized well with macrophages. CONCLUSION: The OCT-NIRF imaging with a clinical dose of ICG was feasible to accurately assess plaque inflammation and DES-related inflammation in a beating coronary artery. This highly translatable dual-modal molecular-structural imaging strategy could be relevant for clinical intracoronary estimation of high-risk plaques and DES biology.

Multi-functional angiographic OFDI using frequency-multiplexed dual-beam illumination.

Detection of blood flow inside the tissue sample can be achieved by measuring the local change of complex signal over time in angiographic optical coherence tomography (OCT). In conventional angiographic OCT, the transverse displacement of the imaging beam during the time interval between a pair of OCT signal measurements must be significantly reduced to minimize the noise due to the beam scanning-induced phase decorrelation at the expense of the imaging speed. Recent introduction of dual-beam scan method either using polarization encoding or two identical imaging systems in spectral-domain (SD) OCT scheme shows potential for high-sensitivity vasculature imaging without suffering from spurious phase noise caused by the beam scanning-induced spatial decorrelation. In this paper, we present multi-functional angiographic optical frequency domain imaging (OFDI) using frequency-multiplexed dual-beam illumination. This frequency multiplexing scheme, utilizing unique features of OFDI, provides spatially separated dual imaging beams occupying distinct electrical frequency bands that can be demultiplexed in the frequency domain processing. We demonstrate the 3D multi-functional imaging of the normal mouse skin in the dorsal skin fold chamber visualizing distinct layer structures from the intensity imaging, information about mechanical integrity from the polarization-sensitive imaging, and depth-resolved microvasculature from the angiographic imaging that are simultaneously acquired and automatically co-registered.

All-fiber wavelength swept ring laser based on Fabry-Perot filter for optical frequency domain imaging.

Innovations in laser engineering have yielded several novel configurations for high repetition rate, broad sweep range, and long coherence length wavelength swept lasers. Although these lasers have enabled high performance frequency-domain optical coherence tomography, they are typically complicated and costly and many require access to proprietary materials or devices. Here, we demonstrate a simplified ring resonator configuration that is straightforward to construct from readily available materials at a low total cost. It was enabled by an insight regarding the significance of isolation against bidirectional operation and by configuring the sweep range of the intracavity filter to exceed its free spectral range. The design can easily be optimized to meet a range of operating specifications while yielding robust and stable performance. As an example, we demonstrate 240 kHz operation with 125 nm sweep range and >70 mW of average output power and demonstrate high quality frequency domain OCT imaging. The complete component list and directions for assembly of the laser are posted on-line at www.octresearch.org.

Polarization-sensitive OFDI using polarization-multiplexed wavelength-swept laser.

We demonstrate a novel polarization-sensitive optical frequency domain imaging system employing passive polarization multiplexing. Simple modification of a fiber delay line in the wavelength-swept light source enables illumination with two perpendicular polarizations that are required for determination of the Stokes vector components of the light reflected from each depth of the tissue. This simple all-passive approach provides a robust and low-cost solution for PS imaging replacing relatively complex conventional schemes such as polarization modulation or frequency-encoded polarization multiplexing.

Dual-wavelength band spectroscopic optical frequency domain imaging using plasmon-resonant scattering in metallic nanoparticles.

We demonstrate a dual-wavelength band optical frequency domain imaging (OFDI) system that provides high-resolution spectroscopic imaging with metallic nanoparticles as exogenous contrast agents. The local increase of the OFDI signal by elastic scattering from two different custom-fabricated nonspherical nanoparticles resonant at each wavelength band of the system was successfully detected, and we were able to distinguish and visualize the location of each of the nanoparticles in a scattering sample and in biological tissue.

Imaging peritoneal blood vessels through optical coherence tomography angiography for laparoscopic surgery.

Laparoscopic surgery presents challenges in identifying blood vessels due to lack of tactile feedback. The image-guided laparoscopic surgical tool (IGLaST) integrated with optical coherence tomography (OCT) has potential for in vivo blood vessel imaging; however, distinguishing vessels from surrounding tissue remains a challenge. In this study, we propose utilizing an inter-A-line intensity differentiation-based OCT angiography (OCTA) to improve visualization of blood vessels. By evaluating a tissue phantom with varying flow speeds, we optimized the system's blood flow imaging capabilities in terms of minimum detectable flow and contrast-to-noise ratio. In vivo experiments on rat and porcine models, successfully visualized previously unidentified blood vessels and concealed blood flows beneath the 1 mm depth peritoneum. Qualitative comparison of various OCTA algorithms indicated that the intensity differentiation-based algorithm performed best for our application. We believe that implementing IGLaST with OCTA can enhance surgical outcomes and reduce procedure time in laparoscopic surgeries.

Numerical compensation of system polarization mode dispersion in polarization-sensitive optical coherence tomography.

Polarization mode dispersion (PMD), which can be induced by circulators or even moderate lengths of optical fiber, is known to be a dominant source of instrumentation noise in fiber-based PS-OCT systems. In this paper we propose a novel PMD compensation method that measures system PMD using three fixed calibration signals, numerically corrects for these instrument effects and reconstructs an improved sample image. Using a frequency multiplexed PS-OFDI setup, we validate the proposed method by comparing birefringence noise in images of intralipid, muscle, and tendon with and without PMD compensation.

Complex wavefront shaping for optimal depth-selective focusing in optical coherence tomography.

We report on an approach to exploit multiple light scattering by shaping the incident wavefront in optical coherence tomography (OCT). Most of the reflected signal from biological tissue consists of multiply scattered light, which is regarded as noise in OCT. A digital mirror device (DMD) is utilized to shape the incident wavefront such that the maximal energy is focused at a specific depth in a highly scattering sample using a coherence-gated reflectance signal as feedback. The proof-of-concept experiment demonstrates that this approach enhances depth-selective focusing in the presence of optical inhomogeneity, and thus extends the penetration depth in spectral domain-OCT (SD-OCT).

Spectral binning for mitigation of polarization mode dispersion artifacts in catheter-based optical frequency domain imaging.

Polarization mode dispersion (PMD) has been recognized as a significant barrier to sensitive and reproducible birefringence measurements with fiber-based, polarization-sensitive optical coherence tomography systems. Here, we present a signal processing strategy that reconstructs the local retardation robustly in the presence of system PMD. The algorithm uses a spectral binning approach to limit the detrimental impact of system PMD and benefits from the final averaging of the PMD-corrected retardation vectors of the spectral bins. The algorithm was validated with numerical simulations and experimental measurements of a rubber phantom. When applied to the imaging of human cadaveric coronary arteries, the algorithm was found to yield a substantial improvement in the reconstructed birefringence maps.

Artifacts in polarization-sensitive optical coherence tomography caused by polarization mode dispersion.

Polarization mode dispersion (PMD) severely degrades images of biological tissue measured with polarization-sensitive optical coherence tomography. It adds a bias to the local retardation value that can be spatially confined, resulting in regions of seemingly high sample birefringence that are purely artificial. Here, we demonstrate and analyze this effect, both experimentally and with numerical simulations, and show that artifacts can be avoided by limiting the system PMD to less than the system axial resolution. Even then, spatial averaging over a dimension larger than that characteristic of speckle is required to remove a PMD-induced bias of the local retardation values.

Deep learning-based image enhancement in optical coherence tomography by exploiting interference fringe.

Optical coherence tomography (OCT), an interferometric imaging technique, provides non-invasive, high-speed, high-sensitive volumetric biological imaging in vivo. However, systemic features inherent in the basic operating principle of OCT limit its imaging performance such as spatial resolution and signal-to-noise ratio. Here, we propose a deep learning-based OCT image enhancement framework that exploits raw interference fringes to achieve further enhancement from currently obtainable optimized images. The proposed framework for enhancing spatial resolution and reducing speckle noise in OCT images consists of two separate models: an A-scan-based network (NetA) and a B-scan-based network (NetB). NetA utilizes spectrograms obtained via short-time Fourier transform of raw interference fringes to enhance axial resolution of A-scans. NetB was introduced to enhance lateral resolution and reduce speckle noise in B-scan images. The individually trained networks were applied sequentially. We demonstrate the versatility and capability of the proposed framework by visually and quantitatively validating its robust performance. Comparative studies suggest that deep learning utilizing interference fringes can outperform the existing methods. Furthermore, we demonstrate the advantages of the proposed method by comparing our outcomes with multi-B-scan averaged images and contrast-adjusted images. We expect that the proposed framework will be a versatile technology that can improve functionality of OCT.

In vivo imaging of renal microvasculature in a murine ischemia-reperfusion injury model using optical coherence tomography angiography.

Optical coherence tomography angiography (OCTA) provides three-dimensional structural and semiquantitative imaging of microvasculature in vivo. We developed an OCTA imaging protocol for a murine kidney ischemia-reperfusion injury (IRI) model to investigate the correlation between renal microvascular changes and ischemic damage. Mice were divided into mild and moderate IRI groups according to the duration of ischemia (10 and 35 mins, respectively). Each animal was imaged at baseline; during ischemia; and at 1, 15, 30, 45, and 60 mins after ischemia. Amplitude decorrelation OCTA images were constructed with 1.5-, 3.0-, and 5.8-ms interscan times, to calculate the semiquantitative flow index in the superficial (50-70 µm) and the deep (220-340 µm) capillaries of the renal cortex. The mild IRI group showed no significant flow index change in both the superfial and the deep layers. The moderate IRI group showed a significantly decreased flow index from 15 and 45 mins in the superficial and deep layers, respectively. Seven weeks after IRI induction, the moderate IRI group showed lower kidney function and higher collagen deposition than the mild IRI group. OCTA imaging of the murine IRI model revealed changes in superficial blood flow after ischemic injury. A more pronounced decrease in superficial blood flow than in deep blood flow was associated with sustained dysfunction after IRI. Further investigation on post-IRI renal microvascular response using OCTA may improve our understanding of the relationship between the degree of ischemic insult and kidney function.

Increased capillary stalling is associated with endothelial glycocalyx loss in subcortical vascular dementia.

Proper regulation and patency of cerebral microcirculation are crucial for maintaining a healthy brain. Capillary stalling, i.e., the brief interruption of microcirculation has been observed in the normal brain and several diseases related to microcirculation. We hypothesized that endothelial glycocalyx, which is located on the luminal side of the vascular endothelium and involved in cell-to-cell interaction regulation in peripheral organs, is also related to cerebral capillary stalling. We measured capillary stalling and the cerebral endothelial glycocalyx (cEG) in male mice using in vivo optical coherence tomography angiography (OCT-A) and two-photon microscopy. Our findings revealed that some capillary segments were prone to capillary stalling and had less cEG. In addition, we demonstrated that the enzymatic degradation of the cEG increased the capillary stalling, mainly by leukocyte plugging. Further, we noted decreased cEG along with increased capillary stalling in a mouse model of subcortical vascular dementia (SVaD) with impaired cortical microcirculation. Moreover, gene expression related to cEG production or degradation changed in the SVaD model. These results indicate that cEG mediates capillary stalling and impacts cerebral blood flow and is involved in the pathogenesis of SVaD.