Spatially resolving the molecular expression of multiple probes in 3D can help us understand complex biological processes. For instance, our group has linked abnormal shear stress patterns caused by regurgitant blood flow with resultant congenital heart defects (CHDs). Abnormal shear stress mapped on images of molecular expression gives additional context to developmentally critical pathways such as epithelial-mesenchymal transition (EMT). However, highly multiplexed 3D imaging is needed to accurately identify regions of interest in the looping heart. In this study, we demonstrate a sequential imaging of a library of HCR FISH probes.
Advances in three-dimensional (3D) microscopy are providing never-before-seen images of coronary microvasculature organization. However, it remains inaccessible to researchers due to difficult sample preparation and image analysis. We present a deep learning network that can segment the coronary microvasculature in 3D microscopy without vessel staining. The network is based on 3D U-net and accepts DAPI (nuclei) and autofluorescence (tissue structure) volumes as inputs. The network detects vessels with high accuracy when compared to the ground truth obtained from isolectin staining. Contrast-free segmentation of vessels simplifies sample preparation, frees fluorescent channels during imaging and opens the door toward user-friendly 3D microscopy.
Infrared (IR) inhibition can induce selective heat block on small-diameter axons. We hypothesize that the selective IR inhibition effect can be reproduced by resistance heating via a heating cuff. We tested the hypothesis in vitro on the pleural-abdominal connective of Aplysia californica. The IR optical fiber, heating cuff, and a thermocouple were co-located to ensure both heating modalities induced a similar temperature increase on the nerve. Electrically stimulated compound action potentials were recorded and segmented to characterize the inhibition effect on different axonal subgroups. The dose-response curve showed that resistance heating can reproduce the selective IR inhibition effect.
Vagus nerve stimulation (VNS) is a method to treat drug-resistant epilepsy and depression, but therapeutic outcomes are often not ideal. Newer electrode designs such as intra-fascicular electrodes offer potential improvements in reducing off-target effects but require a detailed understanding of the fascicular anatomy of the vagus nerve. We have adapted a section-and-image technique, cryo-imaging, with UV excitation to visualize fascicles along the length of the vagus nerve. In addition to offering optical sectioning at the surface via reduced penetration depth, UV illumination also produces sufficient contrast between fascicular structures and connective tissue. Here we demonstrate the utility of this approach in pilot experiments. We imaged fixed, cadaver vagus nerve samples, segmented fascicles, and demonstrated 3D tracking of fascicles. Such data can serve as input for computer models of vagus nerve stimulation.
New techniques are needed to study the coronary microvasculature in the embryo and to quantify the differences between healthy and diseased coronary development. We combine our custom optical clearing method (LIMPID), fluorescent staining with DiI and DAPI, and 3D confocal microscopy to visualize the coronary vasculature at two developmental stages (day 9, 13) in quails. We discovered a highly organized coronary vessel network that is aligned with surrounding myocardium cells even at early developmental stages. By characterizing the normal heart vasculature, this experiment provides a baseline for future studies on how diseases affect coronary and myocardial orientation in the embryo.
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.
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.
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.
Abnormal cell proliferation and migration during heart development can lead to severe congenital heart defects (CHDs). Studying the spatial distribution of cells during embryonic development helps our understanding of how the heart develops and the etiology of certain CHDs. However, imaging large groups of single cells in intact tissue volumes is challenging. No current technique can accomplish this task in both a time-efficient and cost-effective manner. OCT has potential with its large field of view and micron-scale resolution, but even the highest resolution OCT systems have poor contrast for counting cells and have a small field of view compared to conventional OCT. We propose using a conventional OCT system and processing the sample to enhance cellular contrast. Inspired by the recently developed Expansion Microscopy, we permeated whole-mount embryonic tissue with a superabsorbent monomer solution and polymerized into a hydrogel. When hydrated in DI water, the tissue-hydrogel complex was uniformly enlarged (~5X in all dimensions) without distorting the microscopic structure. This had a twofold effect: it increased the resolution by a factor of 5 and decreased scattering, which allowed us to resolve cellular level features deep in the tissue with high contrast using conventional OCT. We noted that cell nuclei caused significantly more backscattering than the other subcellular structures after expansion. Based on this property, we were able to distinguish individual cell nuclei, and thus count cells, in expanded OCT images with simple intensity thresholding. We demonstrate the technique with embryonic quail hearts at various developmental stages.
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