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Increased regurgitant blood flow has been linked to endocardial cushion defects and resultant congenital heart diseases (CHDs). Prenatal alcohol exposure (PAE) has been shown to alter early blood flow resulting in abnormal endocardial cushions and CHDs. Compounds, including folic acid (FA), mitigate PAE effects and prevent CHDs, but few studies have assessed their effects on blood flow. We modeled binge drinking in quail embryos at gastrulation. Embryos were exposed to ethanol alone, FA (3.2 μg/egg) alone, and the two simultaneously. We quantified in cardiac looping stages (equivalent to 4 wk of human gestation), the regurgitant blood flow with Doppler optical coherence tomography (OCT) and endocardial cushion volumes using OCT imaging. Incidences of abnormal body curvature and heart rates were also measured. Embryos exposed to ethanol showed significantly increased regurgitant blood flow compared with controls, whereas embryos given FA with ethanol had significantly reduced regurgitant blood flow but without returning to control levels. Ethanol exposure led to significantly smaller, abnormal endocardial cushions, and the addition of FA improved their size, but they remained smaller than controls. Abnormal body curvatures after PAE were reduced in incidence but not fully prevented by FA. FA supplementation partially alleviated PAE-induced abnormal cardiovascular function and morphology. Normal blood flow and endocardial cushions are both required to produce a healthy four-chambered heart. These findings support that FA supplementation should begin early in pregnancy to prevent heart as well as neural tube defects. Investigations into the efficacy of combinations of compounds to prevent PAE-induced defects are warranted. NEW & NOTEWORTHY State-of-the-art biophotonic tools captured blood flow and endocardial cushion volumes in tiny beating quail embryo hearts, an accessible model for studying four-chambered heart development. Both hemodynamic flow and endocardial cushion volumes were altered with ethanol exposure but normalized when folic acid was introduced with ethanol. Folic acid supplementation preserved hemodynamic function that is intimately involved in sculpting the heart from the earliest stages of heart development.

Optical coherence tomography (OCT) has been used to investigate heart development because of its capability to image both structure and function of beating embryonic hearts. Cardiac structure segmentation is a prerequisite for the quantification of embryonic heart motion and function using OCT. Since manual segmentation is time-consuming and labor-intensive, an automatic method is needed to facilitate high-throughput studies. The purpose of this study is to develop an image-processing pipeline to facilitate the segmentation of beating embryonic heart structures from a 4-D OCT dataset. Sequential OCT images were obtained at multiple planes of a beating quail embryonic heart and reassembled to a 4-D dataset using image-based retrospective gating. Multiple image volumes at different time points were selected as key-volumes, and their cardiac structures including myocardium, cardiac jelly, and lumen, were manually labeled. Registration-based data augmentation was used to synthesize additional labeled image volumes by learning transformations between key-volumes and other unlabeled volumes. The synthesized labeled images were then used to train a fully convolutional network (U-Net) for heart structure segmentation. The proposed deep learning-based pipeline achieved high segmentation accuracy with only two labeled image volumes and reduced the time cost of segmenting one 4-D OCT dataset from a week to two hours. Using this method, one could carry out cohort studies that quantify complex cardiac motion and function in developing hearts.

Of all congenital heart defects (CHDs), anomalies in heart valves and septa are among the most common and contribute about fifty percent to the total burden of CHDs. Progenitors to heart valves and septa are endocardial cushions formed in looping hearts through a multi-step process that includes localized expansion of cardiac jelly, endothelial-to-mesenchymal transition, cell migration and proliferation. To characterize the development of endocardial cushions, previous studies manually measured cushion size or cushion cell density from images obtained using histology, immunohistochemistry, or optical coherence tomography (OCT). Manual methods are time-consuming and labor-intensive, impeding their applications in cohort studies that require large sample sizes. This study presents an automated strategy to rapidly characterize the anatomy of endocardial cushions from OCT images. A two-step deep learning technique was used to detect the location of the heart and segment endocardial cushions. The acellular and cellular cushion regions were then segregated by K-means clustering. The proposed method can quantify cushion development by measuring the cushion volume and cellularized fraction, and also map 3D spatial organization of the acellular and cellular cushion regions. The application of this method to study the developing looping hearts allowed us to discover a spatial asymmetry of the acellular cardiac jelly in endocardial cushions during these critical stages, which has not been reported before.

Axially swept light-sheet microscopy (ASLM) is an effective method of generating a uniform light sheet across a large field of view (FOV). However, current ASLM designs are more complicated than conventional light-sheet systems, limiting their adaptation in less experienced labs. By eliminating difficult-to-align components and reducing the total number of components, we show that high-performance ASLM can be accomplished much simpler than existing designs, requiring less expertise and effort to construct, align, and operate. Despite the high simplicity, our design achieved 3.5-µm uniform optical sectioning across a >6-mm FOV, surpassing existing light-sheet designs with similar optical sectioning. With well-corrected chromatic aberration, multi-channel fluorescence imaging can be performed without realignment. This manuscript provides a comprehensive tutorial on building the system and demonstrates the imaging performance with optically cleared whole-mount tissue samples.

Optical coherence tomography (OCT) employs near-infrared light to image the microstructure of different tissues. Clinically, it has been used to image the walls of coronary arteries. In research settings, one of the applications for OCT is visualizing endocardial and subendocardial structures. The present experiment sought to determine whether OCT can identify native conduction tissues in adult porcine hearts. During the study, the right atrial endocardial surfaces of excised adult porcine hearts were exposed. The triangle of Koch was imaged with the OCT system and the conduction tissue was identified. The area was then prepared for histologic examination with Masson's trichrome stain. The results of histologic preparations and OCT images were then compared. Ultimately, nine porcine hearts were examined using this methodology. OCT imaging successfully identified subendocardial structures presumed to be the compact atrioventricular node. Histologic images of the preparations delineated the different tissue types and conduction tissue was easily identified. The location of distinctive hyporeflective areas in the OCT images correlated with the location of conduction tissue in the histology images. In light of the findings of this study, it is suggested that atrioventricular nodal tissue can be identified by OCT in freshly dissected unfixed porcine hearts. OCT images distinguished the differentiated conduction tissue in close proximity with the endocardium, myofibers, and fibrous tissue, and the success of this was verified with histology. This technology may be useful for the direct visualization of the native conduction system during procedures in the operating room and electrophysiology laboratory. Further studies with perfused tissue samples and live animal experiments are needed to better assess the efficacy of this novel application.

Changes in the protein aggregation within the ocular lens may be responsible for both presbyopia and cataracts. Treatments for these conditions are under development, but are likely to be most effective when administered early in the disease. Therefore, a technique compatible with in vivo use which could detect early changes in aggregations is desired. Here, we assess if phase-decorrelation OCT may be sensitive to cold-induced protein aggregation in ex vivo porcine lenses. A major challenge of this approach is the relatively weak scattering signal obtained from the lens nucleus while the lens is in situ. We observed a substantial increase in decorrelation time as the cold cataract reversed. Backscatter intensity also decreased as the cold cataract reversed, as expected. However, compared to backscatter intensity, decorrelation is better correlated with cataract reversal.

Pulmonary vein isolation with radiofrequency ablation (RFA) has become the most common procedure to treat Atrial Fibrillation (AF). However, current RFA lesion formation is guided only with indirect information (e.g. temperature, impedance, contact force), which does not guarantee transmural lesions. Non-transmural lesions are understood to contribute to AF recurrence. We have previously demonstrated that polarization-sensitive optical coherence tomography (PSOCT) can monitor RFA lesion transmurality in the left atrium (LA) of living swine. However, it requires expert interpretation. Here, we demonstrate quantitative image quality and lesion transmurality evaluation metrics applied to the in vivo LA RFA PSOCT lesion monitoring data.

Transseptal puncture (TSP) is commonly conducted under the guidance of fluoroscopy and/or intracardiac echocardiography (ICE) at the fossa ovalis (FO) to gain percutaneous access to the left atrium for intracardiac procedures. Issues with traditional TSP include: additional vascular access through a sheath, and fluoroscopy exposes patients to ionizing radiation. TSP, if not done appropriately can result in serious complications. We studied the feasibility of optical coherence tomography (OCT) guidance of TSP with ex vivo and in vivo experiments. Results show that OCT can provide detailed structure information to identify FO allowing for safe TSP.

BACKGROUND Fetal alcohol spectrum disorder (FASD) is caused by prenatal alcohol exposure (PAE), the intake of ethanol (C2 H5 OH) during pregnancy. Features of FASD cover a range of structural and functional defects including congenital heart defects (CHDs). Folic acid and choline, contributors of methyl groups to one-carbon metabolism (OCM), prevent CHDs in humans. Using our avian model of FASD, we have previously reported that betaine, another methyl donor downstream of choline, prevents CHDs. The CHD preventions are substantial but incomplete. Ethanol causes oxidative stress as well as depleting methyl groups for OCM to support DNA methylation and other epigenetic alterations. To identify more compounds that can safely and effectively prevent CHDs and other effects of PAE, we tested glutathione (GSH), a compound that regulates OCM and is known as a "master antioxidant." METHODS/RESULTS Quail embryos injected with a single dose of ethanol at gastrulation exhibited congenital defects including CHDs similar to those identified in FASD individuals. GSH injected simultaneously with ethanol not only prevented CHDs, but also improved survival and prevented other PAE-induced defects. Assays of hearts at 8 days (HH stage 34) of quail development, when the heart normally has developed 4-chambers, showed that this single dose of PAE reduced global DNA methylation. GSH supplementation concurrent with PAE normalized global DNA methylation levels. The same assays performed on quail hearts at 3 days (HH stage 19-20) of development, showed no difference in global DNA methylation between controls, ethanol-treated, GSH alone, and GSH plus ethanol-treated cohorts. CONCLUSIONS GSH supplementation shows promise to inhibit effects of PAE by improving survival, reducing the incidence of morphological defects including CHDs, and preventing global hypomethylation of DNA in heart tissues.

Nearly all benchtop studies of corneal biomechanics have relied on protocols which stiffen the cornea, such as riboflavin-UV crosslinking, as a way of providing contrast and validation of biomechanical measurements. However, there are strong clinical motivations to detect softening of the cornea. In this work, we present the evidence that phase-decorrelation OCT (PhD-OCT) is able to detect a small degree of corneal softening due to enzymatic digestion. This benchtop study supports the idea that PhD-OCT may detect keratoconus and early ectasia clinically.

Corneal ectasias, such as keratoconus, are modeled ex vivo by the application of enzymes found to be upregulated in ectasia. Phase-decorrelation OCT is shown to non-invasively detect a small degree of enzymatically-induced weakening.

Smartphone microscopes can be effective tools for a broad range of imaging applications. In this manuscript, we demonstrate the first practical implementation of Microscopy with Ultraviolet Surface Excitation (MUSE) in a compact smartphone microscope called Pocket MUSE, resulting in a remarkably effective design. Fabricated with parts from consumer electronics that are readily available at low cost, the small optical module attaches directly over the rear lens in a smartphone and enables high quality multichannel fluorescence microscopy with submicron resolution over a 10X equivalent field of view. In addition to the novel optical configuration, Pocket MUSE is compatible with a series of simple, portable and user-friendly sample preparation strategies that can be directly implemented for various microscopy applications for point-of-care diagnostics, at-home health monitoring, plant biology, STEM education, environmental studies, etc.

Atrial fibrillation (AF) is the most common sustained arrhythmia in the western world. Pulmonary vein isolation (PVI) using radiofrequency ablation (RFA) is frequently conducted to treat AF. However, current PVI procedures for lesion formation are guided only with indirect information, which may lead to non-transmural lesions, and contribute to the high recurrence of AF. Therefore, direct lesion quality feedback may potentially improve PVI efficacy. To study the real-time direct guidance capability of polarization sensitive optical coherence tomography (PSOCT), a custom-designed integrated PSOCT-RFA catheter was prototyped and tested in RFA procedures in the left atria of living swine.

Background Despite decades of public education about dire consequences of prenatal alcohol exposure, drinking alcohol during pregnancy remains prevalent. As high as 40% of live-born infants exposed to alcohol during gestation and diagnosed with Fetal Alcohol Syndrome have congenital heart defects that can be life-threatening. In animal models, the methyl donor betaine, found in foods such as wheat bran, quinoa, beets and spinach, ameliorated neurobehavioral deficits associated with prenatal alcohol exposure (PAE) but effects on heart development are unknown. Methods Previously we modeled a binge drinking episode during the first trimester in avian embryos. Here we investigated whether betaine could prevent adverse effects of alcohol on heart development. Embryos exposed to ethanol with and without an optimal dose of betaine (5 μM) were analyzed at late developmental stages. Cardiac morphology parameters were rapidly analyzed and quantified using optical coherence tomography. DNA methylation at early stages was detected by immunofluorescent staining for 5-methylcytosine in sections of embryos treated with ethanol or co-treated with betaine. Results Compared to ethanol-exposed embryos, betaine-supplemented embryos had higher late-stage survival rates and fewer gross head and body defects than seen after alcohol exposure alone. Betaine also reduced the incidence of late-stage cardiac defects such as absent vessels, abnormal atrio-ventricular (AV) valves, and hypertrophic ventricles. Furthermore, betaine co-treatment brought measurements of great vessel diameters, interventricular septum (IVS) thickness, and AV leaflet volumes in betaine-supplemented embryos close to control values. Early-stage 5-methycytosine staining revealed that DNA methylation levels were reduced by ethanol exposure and normalized by co-administration with betaine. Conclusions This is the first study demonstrating efficacy of the methyl donor betaine in alleviating cardiac defects associated with PAE. These findings highlight the therapeutic potential of low-concentration betaine doses in mitigating PAE induced birth defects and has implications for prenatal nutrition policies, especially for women who may not be responsive to folate supplementation. This article is protected by copyright. All rights reserved.

BACKGROUND The relationship between changes in endocardial cushion and resultant congenital heart diseases (CHD) has yet to be established. It has been shown that increased regurgitant flow early in embryonic heart development leads to endocardial cushion defects, but it remains unclear how abnormal endocardial cushions during the looping stages might affect the fully septated heart. The goal of this study was to reproducibly alter blood flow in vivo and then quantify the resultant effects on morphology of endocardial cushions in the looping heart and on CHDs in the septated heart. METHODS Optical pacing was applied to create regurgitant flow in embryonic hearts, and optical coherence tomography (OCT) was utilized to quantify regurgitation and morphology. Embryonic quail hearts were optically paced at 3 Hz (180 bpm, well above intrinsic rate 60-110 bpm) at stage 13 of development (3-4 weeks human) for 5 min. Pacing fatigued the heart and led to at least 1 h of increased regurgitant flow. Resultant morphological changes were quantified with OCT imaging at stage 19 (cardiac looping-4-5 weeks human) or stage 35 (4 chambered heart-8 weeks human). RESULTS All paced embryos imaged at stage 19 displayed structural changes in cardiac cushions. The amount of regurgitant flow immediately after pacing was inversely correlated with cardiac cushion size 24-h post pacing (P value

The study of corneal biomechanics is motivated by the tight relationship between biomechanical properties and visual function within the ocular system. For instance, variation in collagen fibril alignment and non-enzymatic crosslinks rank high among structural factors which give rise to the cornea's particular shape and ability to properly focus light. Gradation in these and other factors engender biomechanical changes which can be quantified by a wide variety of techniques. This review summarizes what is known about both the changes in corneal structure and associated changes in corneal biomechanical properties in aging, keratoconic, and photochemically crosslinked corneas. In addition, methods for measuring corneal biomechanics are discussed and the topics are related to both clinical studies and biomechanical modeling simulations.

Introduction: Optical coherence tomography (OCT) is a high-resolution imaging modality which can be used to acquire detailed elastograms of biological tissue. In this investigation, we demonstrate the use of OCT to generate µm-scale strain maps of articular cartilage (AC) under compressive and shear deformations. AC is a dense connective tissue which provides a low-friction surface in synovial joints. The specific alignment of collagen fibrils and proteoglycans (which contribute primarily to shear and compressive stiffness, respectively) give rise to depth-dependent mechanical properties. Methods: Six 6mm diameter samples of articular cartilage were harvested from a calf femur. A custom-designed biaxial loading apparatus applied compressive and shear displacements. Three-dimensional images of the tissue were obtained using a spectral-domain OCT system as the sample was loaded at constant rate of displacement. Both speckle-tracking and phase-shift methods were used to generate strain maps from these images. Results: Under both shear and compressive loading, clear differences in local strain distribution were observed between the superficial, transitional, and radial zones of the cartilage. In shear, the superficial/transitional zones are stiffest while in compression these regions are more compliant than the radial zone. These distributions correspond with existing literature and the known orientation of collagen in the AC. Conclusion: It is feasible to rapidly acquire strain maps in AC using OCT. This technique may be extended to high-throughput screening to nondestructively determine the functionality and failure modes of engineered AC as compared to native tissue.

Three dimensional (3D) microvascular imaging of cubic millimeter to centimeter size volumes often requires much time and expensive instruments. By combining optical clearing with a novel scatter-based optical coherence tomography (OCT) contrast agent, we have greatly extended OCT imaging depth in excised tissues while maintaining a simple and low cost approach that does not require in-depth OCT knowledge. The new method enables fast 3D microvascular mapping in large tissue volumes, providing a promising tool for investigating organ level microvascular abnormalities in large cohorts.

Purpose Millions of people suffer from diseases that involve corneal nerve dysfunction, caused by various conditions, including dry eye syndrome, neurotrophic keratopathy, diabetes, herpes simplex, glaucoma, and Alzheimer's disease. The morphology of corneal nerves has been studied extensively. However, corneal nerve function has only been studied in a limited fashion owing to a lack of tools. Here, we present a new system for studying corneal nerve function. Methods Optical imaging was performed on the cornea of excised murine globes taken from a model animal expressing a genetically encoded calcium indicator, GCaMP6f, to record calcium transients. A custom perfusion and imaging chamber for ex vivo murine globes was designed to maintain and stabilize the cornea, while allowing the introduction of chemical stimulation during imaging. Results Imaging of calcium signals in the ex vivo murine cornea was demonstrated. Strong calcium signals with minimal photobleaching were observed in experiments lasting up to 10 minutes. Concentrated potassium and lidocaine solutions both modulated corneal nerve activity. Similar responses were observed in the same neurons across multiple chemical stimulations, suggesting the feasibility of using chemical stimulations to test the response of the corneal nerves. Conclusions Our studies suggest that this tool will be of great use for studying functional changes to corneal nerves in response to disease and ocular procedures. This process will enable preclinical testing of new ocular procedures to minimize damage to corneal innervation and therapies for diminished neural function.

Blood-induced shear stress influences gene expression. Abnormal shear stress patterns on the endocardium of the early-stage heart tube can lead to congenital heart defects. To have a better understanding of these mechanisms, it is essential to include shear stress measurements in longitudinal cohort studies of cardiac development. Previously reported approaches are computationally expensive and nonpractical when assessing many animals. Here, we introduce a new approach to estimate shear stress that does not rely on recording 4D image sets and extensive post processing. Our method uses two adjacent optical coherence tomography frames (B-scans) where lumen geometry and flow direction are determined from the structural data and the velocity is measured from the Doppler OCT signal. We validated our shear stress estimate by flow phantom experiments and applied it to live quail embryo hearts where observed shear stress patterns were similar to previous studies.

Significance: Pulmonary vein isolation with catheter-based radiofrequency ablation (RFA) is carried out frequently to treat atrial fibrillation. However, RFA lesion creation is only guided by indirect information (e.g., temperature, impedance, and contact force), which may result in poor lesion quality (e.g., nontransmural) and can lead to reoccurrence or complications. Aim: The feasibility of guiding intracardiac RFA with an integrated polarization-sensitive optical coherence tomography (PSOCT)-RFA catheter in the right atria (RA) of living swine is demonstrated. Approach: In total, 12 sparse lesions were created in the RA of three living swine using an integrated PSOCT-RFA catheter with standard ablation protocol. PSOCT images were displayed in real time to guide catheter-tissue apposition. After experiments, post-processed PSOCT images were analyzed to assess lesion quality and were compared with triphenyltetrazolium chloride (TTC) lesion quality analysis. Results: Five successful lesions identified with PSOCT images were all confirmed by TTC analysis. In two ablations, PSOCT imaging detected gas bubble formation, indicating overtreatment. Unsuccessful lesions observed with PSOCT imaging were confirmed by TTC analysis. Conclusions: The results demonstrate that the PSOCT-RFA catheter provides real-time feedback to guide catheter-tissue apposition, monitor lesion quality, and possibly help avoid complications due to overtreatment, which may enable more effective and safer RFA treatment.

Cardiac arrhythmias are a major source of mortality in the United States. Ablation is the only curative therapy for cardiac arrhythmias, and catheter-based radiofrequency ablation (RFA) through percutaneous access has emerged as the standard care for many arrhythmias. However, current procedures only monitor temperature, impedance and pressure measurements during ablation, which results in incomplete lesion formation (e.g., high recurrence rate for atrial fibrillation patients) and complications. In our previous work, we have shown integration of a commercially available RFA catheter with polarization sensitive optical coherence tomography (PSOCT). This was accomplished by housing the PSOCT probe inside of the RFA catheter with a window at the catheter tip that allows for forward viewing PSOCT imaging. Data from ex vivo experiments have shown that the integrated RFA catheter can be used to confirm catheter tissue apposition and monitor tissue change during ablation by measuring retardance. It was also demonstrated that the PSOCT window does not interfere with normal lesion formation. However, only the catheter tip was integrated, enabling only in vitro experiments. Therefore, we have developed a fully integrated PSOCT RFA catheter based on a commercially available catheter to enable experiments in living swine via percutaneous access. By properly choosing a middle layer sheath to house the PSOCT probe, we reduced the non-uniform rotation distortion (NURD), reduced PSOCT probe tip longitudinal movement due to winding back, improved image quality and stability. Further validation of functionality by simulating RFA procedures in living swine through percutaneous access is ongoing.

Optical clearing is an effective tool for investigating spatial distribution of molecules in embryonic tissue. Unfortunately, it has not been broadly adapted in the field of development biology. One reason is that most current optical clearing methods involve complicated procedures that are more difficult compared to common lab procedures. To address this problem, we developed an easy and convenient optical clearing method, termed lipid-preserving index matching for prolonged imaging depth (LIMPID), that involves only one major step in the entire procedure. Since all LIMPID ingredient are water-soluble, it can directly diffuse into the fixed tissue and match the refractive index. Because no dehydration, organic solvent exchange or lipid extraction is required, LIMPID also well preserves fluorescent signal and tissue morphology while maintaining high clearing capability. In addition, LIMPID clearing solution has low viscosity that allows fast diffusion of the chemicals into the tissue. We have tested LIMPID using embryonic quail tissue at various developmental stages. By simply immersing the fixed and stained tissue in excess LIMPID solution, it is capable of clearing whole-mount stage 20 quail embryos in 10 minutes, stage 36 quail hearts overnight, and stage 36 quail brains in 24 hours. Verified by confocal microscopy, fluorescent signals from eYFP, DAPI, LysoTracker and several Alexa Fluor tagged primary antibodies were all well preserved. Imaging depth of LIMPID is only limited by the working distance of the optical system, up to multiple millimeters. Many small embryo tissues can be imaged all the way through using common confocal setups.

Optical clearing is a useful tool for investigating large biological tissues in 3D, but it has not been widely adapted in regular biomedical research community, partially due to the high complexity and low speed of the current optical clearing methods. Therefore, we developed an optical clearing technique, termed lipid-preserving index matching for prolonged imaging depth (LIMPID), that simplifies the clearing procedure while maintaining the advantages of the state of the art clearing methods. (1) LIMPID is designed as an aqueous solution that directly diffuses into the tissue and makes the refractive indices uniform. It is capable of clearing the tissue in a single step, simply by immersing fixed and pre-labeled samples in the clearing solution. In contrast, most current clearing techniques involve multiple steps and some of steps are complicated and time consuming. (2) LIMPID clears the tissue quickly. The solution has low viscosity and rapidly diffuses into the tissue at room temperature. For samples with submillimeter thickness, it clears the tissue within an hour. Clearing times for larger samples are also impressive. (3) LIMPID preserves fluorescence and tissue morphology while maintaining high transparency. No dehydration, organic solvent exchange or lipid extraction is required. We have used LIMPID to study several animal and disease models. For instance, it revealed abnormal peripheral nerve innervation in the embryonic quail heart in a fetal alcohol syndrome model. We believe this simple, quick method with no discernable disadvantages could become the optical clearing protocol of choice for many microscopy applications.

Altered hemodynamics in developing embryonic hearts lead to congenital heart diseases, motivating close monitoring of blood flow over several stages of development. Doppler OCT can assess blood flow in tubular hearts where blood velocity increases drastically during the period of cardiac cushion (valve precursors) formation. The blood-induced shear stress undergoes dramatic changes as well, which affects gene expression by the endothelial cells. Previously, we built a high-speed OCT system using an FDML laser (Optores GmbH, Germany) at a sweep rate of 1.68 MHz (axial resolution - 12 μm, sensitivity - 105 dB, phase stability - 96 mrad). The ultra-fast A-line rate of this laser may be used to collect real-time volumetric images of the heart, or can be traded off to obtain dense B-scans for more accurate Doppler measurements with a larger dynamic range using Doppler complex regression. Since we cannot achieve volumetric imaging with dense B-scans, an image-based retrospective gating technique was developed to register the asynchronously acquired dense B-scans to the 4D volumes. The direction of flow was determined by finding the centroid of the Doppler signal from the rearranged B-scans along the heart tube to compute absolute velocity. Subsequently, the cross-section from which the shear stress is calculated was realigned orthogonal to the direction of blood flow to approximate the velocity gradient normal to the wall. In conclusion, our high-speed OCT system will enable semi-automated measurement of the absolute blood velocity, as well as mapping the shear stress exerted on the inner walls of the embryonic hearts.

The study of the mechanical properties of embryonic tissues has become an area of increasing need as more relationships between structure and function are discovered. However, there are no appropriate tools currently available to study mechanical properties of soft, millimeter-scale structures. Here, we present work on a micro-scale compression optical coherence elastography (C-OCE) system which can make quantitative measurements of the mechanical properties of small biological tissues. Inspired by "sensor" C-OCE, in which a hydrogel is laid over the sample of interest to calibrate the deformation, here we fully embed a small sample in hydrogel, then apply compressive or tensile force. Meanwhile, a phase-stable optical coherence tomography (OCT) system images the sample. Nanometer-scale displacements are extracted from the phase signal and used to generate a cross-sectional strain map. The strain map is then interpreted to provide information about the absolute mechanical properties of the sample at micron-level resolution. To date, we have demonstrated sensitivity to 20 kPa differences in Young's modulus in soft gelatin phantoms. Additionally, we have used this method to measure the mechanical properties of de-lamination of endocardial cushions as they develop into cardiac valves in quail embryos. Further work will include 3D property characterizations, use of finite element modeling to calculate absolute mechanical properties of complex structures, and deeper investigation into the role of mechanics during valvulogenesis and other developmental processes.

Congenital coronary anomalies can result in severe consequences such as arrhythmias and sudden death. However, the etiology of abnormal embryonic coronary microvasculature development is understudied. Using a novel contrast-agent-based optical coherence tomography (OCT) technique, scatter labeled imaging of microvasculature in excised tissue (SLIME), we compared diseased and normal embryonic quail coronary microvasculature in 3D. Congenital heart defects associated with fetal alcohol syndrome (FAS) were induced in a quail model by injecting 40 uL of 50% ethanol solution into eggs during gastrulation. These and saline-injected quail eggs were incubated until stage 36. SLIME contrast agent was perfused through the aortas of embryos and fixed in the vessels with a crosslinking agent. Dissected hearts were treated with a CUBIC-1 clearing agent and the scattering contrast labeled vasculature were imaged using customized spectral domain OCT systems. SLIME data revealed that coronary microvasculature of the control group was organized as parallel bundles over the left ventricle near the apex, whereas in a majority of the ethanol-treated embryos coronary microvasculature had chaotic patterns in similar regions. These differences in alignment of microvasculature have not been previously described in this disease model. Quantitative and statistical assessment will aid in evaluating the significance of this coronary defect. Future investigations will determine whether coronary mispatterning may reflect misalignment of cardiomyocytes that could lead to other negative consequences.

Background The most commonly used method to analyze congenital heart defects involves serial sectioning and histology. However, this is often a time-consuming process where the quantification of cardiac defects can be difficult due to problems with accurate section registration. Here we demonstrate the advantages of using optical coherence tomography, a comparatively new and rising technology, to phenotype avian embryo hearts in a model of Fetal Alcohol Syndrome where a binge-like quantity of alcohol/ethanol was introduced at gastrulation.

A robust method to measure viscosity of microquantities of biological samples, such as blood and mucus, could lead to a better understanding and diagnosis of diseases. Microsamples have presented persistent challenges to conventional rheology, which requires bulk quantities of a sample. Alternatively, fluid viscosity can be probed by monitoring microscale motion of particles. Here, we present a decorrelation-based method using M-mode phase-sensitive optical coherence tomography (OCT) to measure particle Brownian motion. This is similar to previous methods using laser speckle decorrelation but with sensitivity to nanometer-scale displacement. This allows for the measurement of decorrelation in less than 1 millisecond and significantly decreases sensitivity to bulk motion, thereby potentially enabling in vivo and in situ applications. From first principles, an analytical method is established using M-mode images obtained from a 47 kHz spectral-domain OCT system. A g(1) first-order autocorrelation is calculated from windows containing several pixels over a time frame of 200-1000 microseconds. Total imaging time is 500 milliseconds for averaging purposes. The autocorrelation coefficient over this short time frame decreases linearly and at a rate proportional to the diffusion constant of the particles, allowing viscosity to be calculated. In verification experiments using phantoms of microbeads in 200 µL glycerol-water mixtures, this method showed insensitivity to 2 mm/s lateral bulk motion and accurate viscosity measurements over a depth of 400 µm. In addition, the method measured a significant decrease of the apparent diffusion constant of soft tissue after formalin fixation, suggesting potential applications in mapping tissue stiffness.

Over 500,000 women per year in the United States drink during pregnancy, and 1 in 5 of this population also binge drink. Up to 40% of live-born children with prenatal alcohol exposure (PAE) present with congenital heart defects (CHDs) including life-threatening outflow and valvuloseptal anomalies. Previously we established a PAE model in the avian embryo and used optical coherence tomography (OCT) imaging to assay looping-stage (early) cardiac function/structure and septation-stage (late) cardiac defects. Early-stage ethanol-exposed embryos had smaller cardiac cushions (valve precursors) and increased retrograde flow, while late-stage embryos presented with gross head/body defects, and exhibited smaller atrio-ventricular (AV) valves, interventricular septae, and aortic vessels. However, supplementation with the methyl donor betaine reduced gross defects, prevented cardiac defects such as ventricular septal defects and abnormal AV valves, and normalized cardiac parameters. Immunofluorescent staining for 5-methylcytosine in transverse embryo sections also revealed that DNA methylation levels were reduced by ethanol but normalized by co-administration of betaine. Furthermore, supplementation with folate, another methyl donor, in the PAE model appeared to normalize retrograde flow levels which are typically elevated by ethanol exposure. Studies are underway to correlate retrograde flow numbers for folate with associated cushion volumes. Finally, preliminary findings have revealed that glutathione, a key endogenous antioxidant which also regulates methyl group donation, is particularly effective in improving alcohol-impacted survival and gross defect rates. Current investigations will determine whether glutathione has any positive effect on PAE-related CHDs. Our studies could have significant implications for public health, especially related to prenatal nutrition recommendations.

Congenital heart defects (CHDs) are the most common birth defect, affecting between 4 and 75 per 1,000 live births depending on the inclusion criteria. Many of these defects can be traced to defects of cardiac cushions, critical structures during development that serve as precursors to many structures in the mature heart, including the atrial and ventricular septa, and all four sets of cardiac valves. Epithelial-mesenchymal transition (EMT) is the process through which cardiac cushions become populated with cells. Altered cushion size or altered cushion cell density has been linked to many forms of CHDs, however, quantitation of cell density in the complex 3D cushion structure poses a significant challenge to conventional histology. Optical coherence tomography (OCT) is a technique capable of 3D imaging of the developing heart, but typically lacks the resolution to differentiate individual cells. Our goal is to develop an algorithm to quantitatively characterize the density of cells in the developing cushion using 3D OCT imaging. First, in a heart volume, the atrioventricular (AV) cushions were manually segmented. Next, all voxel values in the region of interest were pooled together to generate a histogram. Finally, two populations of voxels were classified using either K-means classification, or a Gaussian mixture model (GMM). The voxel population with higher values represents cells in the cushion. To test the algorithm, we imaged and evaluated avian embryonic hearts at looping stages. As expected, our result suggested that the cell density increases with developmental stages. We validated the technique against scoring by expert readers.

Abnormal coronary development causes various health problems. However, coronary development remains one of the highly neglected areas in developmental cardiology due to limited technology. Currently, there is not a robust method available to map the microvasculature throughout the entire embryonic heart in 3D. This is a challenging task because it requires both micron level resolution over a large field of view and sufficient imaging depth. Speckle-variance optical coherence tomography (OCT) has reasonable resolution for coronary vessel mapping, but limited penetration depth and sensitivity to bulk motion made it impossible to apply this method to late-stage beating hearts. Some success has been achieved with coronary dye perfusion, but smaller vessels are not efficiently stained and penetration depth is still an issue. To address this problem, we present an OCT imaging procedure using optical clearing and a contrast agent (titanium dioxide) that enables 3D mapping of the coronary microvasculature in developing embryonic hearts. In brief, the hearts of stage 36 quail embryos were perfused with a low viscosity mixture of polyvinyl acetate (PVA) and titanium dioxide through the aorta using micropipette injection. After perfusion, the viscosity of the solution was increased by crosslinking the PVA polymer chains with borate ions. The tissue was then optically cleared. The titanium dioxide particles remaining in the coronaries provided a strong OCT signal, while the rest of the cardiac structures became relatively transparent. Using this technique, we are able to investigate coronary morphologies in different disease models.

The limited dynamic range of optical coherence tomography (OCT) Doppler velocity measurements makes it difficult to conduct experiments on samples requiring a large dynamic range without phase wrapping at high velocities or loss of sensitivity at slow velocities. Hemodynamics and wall motion undergo significant increases in velocity as the embryonic heart develops. Experimental studies indicate that altered hemodynamics in early-stage embryonic hearts can lead to congenital heart diseases (CHDs), motivating close monitoring of blood flow over several stages of development. We have built a high-speed OCT system using an FDML laser (Optores GmbH, Germany) at a sweep rate of 1.68 MHz (axial resolution - 12 μm, sensitivity - 105 dB, phase stability - 17 mrad). The speed of this OCT system allows us to acquire high-density B-scans to obtain an extended velocity dynamic range without sacrificing the frame rate (100 Hz). The extended dynamic range within a frame is achieved by varying the A-scan interval at which the phase difference is found, enabling detection of velocities ranging from tens of microns per second to hundreds of millimeters per second. The extra lines in a frame can also be utilized to improve the structural and Doppler images via complex averaging. In structural images where the presence of blood causes additional scattering, complex averaging helps retrieve features located deeper in the tissue. Moreover, high-density frames can be registered to 4D volumes to determine the orthogonal direction of flow for calculating shear stress as well as estimating the cardiac output. In conclusion, high density B-scans acquired by our high-speed OCT system enable image enhancement and direct measurement of biological parameters in cohort studies.

The extensive development of frequency-domain optical coherence tomography (OCT) for more than a decade has enabled A-scan rates in the MHz range. Furthermore, frequency-domain OCT gives access to the amplitude and phase of the OCT signal. These characteristics have opened the possibilities of doing different kinds of averaging in order to improve OCT imaging. It is well known that multiple scattering in OCT reduces image contrast and resolution especially at greater depths within the tissue. Here, we demonstrate that complex averaging can decrease the effect of multiple scattering and improve OCT imaging contrast, in addition to increasing the dynamic range due to reducing the noise floor as previously demonstrated. We take advantage of the fact that complex averaging, in contrast to conventional magnitude averaging, is sensitive to phase changes, as one averages the complex-valued Fourier-transformed spectral fringe signals before calculating the magnitude. Any motion that leads to higher phase variance will lead to lower magnitude when performing complex averaging. Also, motion preferentially increases the phase variance of multiply scattered photons when compared to singly scattered photons with each scattering event spreading the phase. This indicates that we may reduce multiple scattering by implementing complex averaging to preferentially reduce the magnitude of the multiply scattered light signal in OCT images. We have performed several experiments on liquid phantoms that give experimental evidence for this hypothesis.

Viscosity is often a critical characteristic of biological fluids such as blood and mucus. However, traditional rheology is often inadequate when only small quantities of sample are available. A robust method to measure viscosity of microquantities of biological samples could lead to a better understanding and diagnosis of diseases. Here, we present a method to measure viscosity by observing particle Brownian motion within a sample. M-mode optical coherence tomography (OCT) imaging, obtained with a phase-sensitive 47 kHz spectral domain system, yields a viscosity measurement from multiple 200-1000 microsecond frames. This very short period of continuous acquisition, as compared to laser speckle decorrelation, decreases sensitivity to bulk motion, thereby potentially enabling in vivo and in situ applications. The theory linking g(1) first-order image autocorrelation to viscosity is derived from first principles of Brownian motion and the Stokes-Einstein relation. To improve precision, multiple windows acquired over 500 milliseconds are analyzed and the resulting linear fit parameters are averaged. Verification experiments were performed with 200 µL samples of glycerol and water with polystyrene microbeads. Lateral bulk motion up to 2 mm/s was tolerated and accurate viscosity measurements were obtained to a depth of 400 µm or more. Additionally, the method measured a significant decrease of the apparent diffusion constant of soft tissue after formalin fixation, suggesting potential for mapping tissue stiffness over a volume.