Abstract
Light-sheet fluorescence microscopy (LSFM), with its innovative design of selective plane illumination and orthogonal detection optics, significantly reduces phototoxicity and photobleaching inherent in conventional microscopy, providing a revolutionary tool for long-term dynamic imaging of living specimens. This review focuses on throughput enhancement strategies of LSFM, systematically summarizing advancements in optical architecture optimization and multimodal integration. Key technological innovations include: improved sample compatibility, large-field imaging via optimized light-sheet generation, microfluidics-coupled high-throughput automation, and hyperspectral imaging for multiplexed analysis. Through adaptive light-sheet modulation, remote focusing synchronization, and AI-driven algorithmic optimization, LSFM achieves multiscale 3D imaging spanning subcellular structures to centimeter-scale tissues at speeds exceeding hundreds of volumetric frames per second. In biomedical applications, LSFM has successfully resolved complex processes such as cellular lineage dynamics during embryogenesis, whole-brain neuronal activity mapping, and structure-function correlations in cardiovascular systems, while enabling high-throughput drug screening and pathological model analysis. These breakthroughs establish LSFM as a cornerstone technology for intravital imaging, offering an integrated solution that combines high spatiotemporal resolution, minimal photodamage, and big-data throughput. By bridging molecular, cellular, and organ-level observations, LSFM drives paradigm shifts in developmental biology, neuroscience, and translational medicine, empowering unprecedented exploration of living systems across scales.