Heart failure remains the leading cause of mortality worldwide, primarily due to the lack of regenerative capacity of mature cardiomyocytes¹. The intricate architecture of the heart plays a crucial role in its function and provides insights into developmental processes. A profound understanding of cardiac structure is essential for elucidating the fundamental processes of cardiac morphogenesis and remodeling in response to myocardial infarction. Recent progress has demonstrated that neonatal mice can restore cardiac function following injury, while adult mice lack such regenerative capacity². This establishes a foundation for investigating cues associated with structural and functional abnormalities in mouse models. Traditional imaging methods, such as confocal microscopy, have technical limitations, including restricted penetration depth, slow point-scanning scheme, and photo damage from prolonged exposure to laser light. These hinder comprehensive three-dimensional (3D) imaging of the intact heart. In this context, light-sheet microscopy (LSM) emerges as a powerful solution, offering the advantages of high-speed imaging, reduced photo damage, and exceptional optical sectioning capabilities³^,⁴^,⁵. The unique features of LSM position it as a promising method to overcome the limitations of conventional techniques, providing unprecedented insights into cardiac development and remodeling processes⁶^,⁷^,⁸.

In this protocol, we introduce an imaging strategy that combines advanced LSM with adapted tissue clearing approaches⁹, allowing for the imaging of entire mouse hearts without the need for specific labeling and mechanical sectioning. We further propose that conventional LSM imaging can be enhanced through multiview deconvolution¹⁰ or axially swept light-sheet microscopy (ASLM) techniques¹¹^,¹²^,¹³^,¹⁴^,¹⁵ to improve axial resolution. Additionally, the integration of image stitching with either of these methods can effectively overcome the trade-off between spatial resolution and field of view (FOV), thereby advancing the imaging of adult mouse hearts. The incorporation of numerous tissue clearing approaches, including hydrophobic, hydrophilic, and hydrogel-based methods, enables deeper light penetration for capturing the morphology of the entire heart¹⁶^,¹⁷^,¹⁸^,¹⁹.

While multiple clearing methods are compatible with current LSM systems, the goal is to minimize photon scattering and enhance light penetration in tissues, like the heart, by replacing lipids with a medium that closely matches its refractive index. iDISCO was chosen as the representative²⁰^,²¹ and adapted for autofluorescence imaging in this protocol due to its rapid processing and high transparency (Figure 1A). Collectively, the integration of the advanced LSM approach with tissue clearing techniques offers a promising framework to unravel intricate cardiac anatomy in rodent hearts, holding significant potential for advancing our understanding of cardiac morphogenesis and pathogenesis.

Light-Sheet Imaging of Rodent Hearts