The zebrafish (Danio rerio) is a widely used model organism for studying cardiac development, physiology, and repair due to its optical transparency, genetic tractability, and regenerative capacity¹^,²^,³^,⁴. Upon myocardial infarction, while structural and functional changes impact the cardiac ejection and hemodynamics, technical limitations continue to hinder the ability to investigate the dynamic process during cardiac regeneration with the high spatiotemporal resolution. For example, conventional imaging methods, such as confocal microscopy, have limitations in terms of imaging depth, temporal resolution, or phototoxicity for capturing the dynamic changes and assessing cardiac contractile function during multiple cardiac cycles⁵.

Light-sheet microscopy represents a state-of-the-art imaging method that successfully addresses these issues by quickly sweeping the laser across the heart's ventricle and atrium, achieving detailed images with enhanced spatiotemporal resolution and negligible photo-bleaching and photo-toxic effects⁶^,⁷^,⁸^,⁹^,¹⁰^,¹¹.

This protocol introduces a comprehensive imaging strategy that includes LSM system construction, 4D image reconstruction, 3D cell tracking, and interactive analysis to capture and analyze the dynamics of cardiomyocytes across the entire heart during multiple cardiac cycles¹². The customized imaging system and computational methodology allow one to track the myocardial microstructure and contractile function at the single-cell level in transgenic Tg(myl7:nucGFP) zebrafish larvae. Furthermore, small molecule compounds were delivered into the embryos using microinjection to assess drug-induced cardiac injury and subsequent regeneration. This holistic strategy provides an entry point to in vivo investigate structural, functional, and mechanical properties of myocardium at the single-cell level during cardiac development and regeneration.

4D Imaging Reconstruction of Zebrafish Larva, 3D Cell Segmentation, and Cardiac Contractility Analysis in Virtual Reality