Vascular retinopathy is a leading cause of preventable blindness in diabetes. While most studies focus on individual neural or vascular structures, there remains a knowledge gap in understanding the retinal neurovascular interactions due to injury and repair. We seek to demonstrate 3-D neurovascular interactions with a multi-view light-sheet imaging system. Intact 3-D retinas from 2-month-old mice were fixed and permeabilized followed by the optical clearance. Retinal vessels and neuron cells were stained with fluorescent labels. After refractive index matching, multi-view light-sheet imaging was performed at different angles followed by 3-D registration and reconstruction. This enabled 3-D co-registration of the vasculature in relation to the neuron cells for virtualization. Therefore, the 3-D neuro-microvascular network could be revealed with both deep tissue penetration and high spatial resolution for investigating hyperoxia and diabetes-associated vascular retinopathy.
Compressed sensing has emerged as a promising technique for high-speed 3-D imaging. Recent work proposed light field tomography (LIFT) to acquire en-face one-dimension (1-D) projections instead of 2-D images and reformulate as a computed tomography problem. The light field with reduced dimension brings high temporal resolution and synthetic refocusing ability in post-processing. We hereby propose a scanning light sheet system with LIFT detection, specifically tailored for achieving kilohertz 3-D fluorescent microscopy. The selective illumination introduces signal sparsity, and thus better reconstruction quality for the compressive detection system. And the high-speed light field imager replaces the active focusing unit in scanning light sheet system and increases the volume rate of 3D detection.
Combining functional optical contrast with high spatiotemporal resolution, photoacoustic computed tomography (PACT) benefits mainstream cardiac imaging modalities for preclinical research. However, PACT has not revealed detailed vasculature or hemodynamics of the whole heart without surgical tissue penetration. Here, we present non-invasive imaging of rat hearts using the recently developed three-dimensional PACT (3D-PACT) platform. 3D-PACT utilizes optimized illumination and detection schemes to reduce the effects of optical attenuation and acoustic distortion through the chest wall, thus visualizing cardiac anatomy and intracardiac hemodynamics within a 10-second scan. We then applied 3D-PACT to investigate different structural and functional variations in healthy, hypertensive, and obese rat hearts. 3D-PACT provides high imaging speed and nonionizing penetration to capture the whole heart for diagnosing animal models, holding promises for clinical translation to human neonatal cardiac imaging without sedation or ionizing radiation.
A key challenge when imaging whole biomedical specimens is how to quickly obtain massive cellular information over a large field of view (FOV). We report a subvoxel light-sheet microscopy (SLSM) method enabling high-throughput volumetric imaging of mesoscale specimens at cellular resolution. A nonaxial, continuous scanning strategy is developed to rapidly acquire a stack of large-FOV images with three-dimensional (3-D) nanoscale shifts encoded. Then, by adopting a subvoxel-resolving procedure, the SLSM method models these low-resolution, cross-correlated images in the spatial domain and can iteratively recover a 3-D image with improved resolution throughout the sample. This technique can surpass the optical limit of a conventional light-sheet microscope by more than three times, with high acquisition speeds of gigavoxels per minute. By fast reconstruction of 3-D cultured cells, intact organs, and live embryos, SLSM method presents a convenient way to circumvent the trade-off between mapping large-scale tissue (>100 mm3) and observing single cell (∼1-μm resolution). It also eliminates the need of complicated mechanical stitching or modulated illumination, using a simple light-sheet setup and fast graphics processing unit-based computation to achieve high-throughput, high-resolution 3-D microscopy, which could be tailored for a wide range of biomedical applications in pathology, histology, neuroscience, etc.
There currently is a limited ability to interactively study developmental cardiac mechanics and physiology. We therefore combined light-sheet fluorescence microscopy (LSFM) with virtual reality (VR) to provide a hybrid platform for 3- dimensional (3-D) architecture and time-dependent cardiac contractile function characterization. By taking advantage of the rapid acquisition, high axial resolution, low phototoxicity, and high fidelity in 3-D and 4-D (3-D spatial + 1-D time or spectra), this VR-LSFM hybrid methodology enables interactive visualization and quantification otherwise not available by conventional methods such as routine optical microscopes. We hereby demonstrate multi-scale applicability of VR-LSFM to 1) interrogate skin fibroblasts interacting with a hyaluronic acid-based hydrogel, 2) navigate through the endocardial trabecular network during zebrafish development, and 3) localize gene therapy-mediated potassium channel expression in adult murine hearts. We further combined our batch intensity normalized segmentation (BINS) algorithm with deformable image registration (DIR) to interface a VR environment for the analysis of cardiac contraction. Thus, the VR-LSFM hybrid platform demonstrates an efficient and robust framework for creating a user-directed microenvironment in which we uncovered developmental cardiac mechanics and physiology with high spatiotemporal resolution.
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