Axially swept dithered light-sheet microscope to reveal cardiac morphology

Understanding cardiac microstructure and vascular networks in their entirety is critical for assessing cardiovascular development, disease progression, and therapeutic interventions. Light-sheet microscopy combined with tissue clearing enables high-resolution volumetric imaging of intact organs but faces limitations in compacted and trabeculated myocardium due to trade-offs among light-sheet thickness, frame rate, and working distance. We developed a quantitative framework that orchestrates illumination-detection dynamics to maintain uniform resolution across multi-scale specimens. Building on this foundation, we implemented high-speed dithered light-sheet (DiLS) illumination, extending the confocal region by at least 40% and enhancing the space-bandwidth product while preserving optical sectioning. Integration of DiLS with an axially swept approach establishes the axially swept dithered light-sheet (AS-DiLS) microscopy, which enhances imaging throughput while preserving axial resolution and enables uniform illumination up to 12.5-millimeter scanning range. AS-DiLS delivers near-isotropic resolution (~2.5 μm) for investigating intricate ventricular trabeculae, pectinate muscles, vasculature, and extracellular matrix, providing scalable platform for comprehensive cardiovascular morphology and topology assessment from embryonic to adult.