A low-cost, high-throughput pipeline for 3D imaging of embryonic mouse hearts using lightsheet microscopy.

Lightsheet microscopy is a powerful tool for three-dimensional imaging of both live and fixed specimens, spanning small to large scales. However, the time-intensive sample preparation and mounting process often limit its use in high-throughput studies. Given our focus on cardiac development and identifying cardiovascular abnormalities after teratogenic exposure, we have developed an efficient, low-cost, and user-friendly system for specimen fixation, clearing, and mounting. This system enables rapid 3D imaging of a mouse embryonic heart within 1 min using the Zeiss Lightsheet Z.1 microscopy and supports imaging of at least 30 hearts per hour with high resolution. With this system, we obtained high-quality images of embryonic hearts at various stages, visualizing internal structures like the aortic valve and coronary arteries with this system. We further demonstrated its capability for quantitative analysis, including endocardial cushion cell density at E10.5 and volumetric measurements of valve morphology. As an extended application, the system was also applied to postnatal P10 hearts and extra-cardiac organs like kidney and ovary, showing clear structural detail. Additionally, integration of the water-soluble clearing agent, EZ Clear, alongside Cre-loxP-mediated genetic lineage tracing, enabled 3D visualization of cellular contributions to heart development with high resolution. The sample preparation system described here promises broader applications in embryology, anatomy, and pathology research, especially in studies requiring both high throughput and high resolution of 3D imaging.