High-throughput, high-fidelity reconstruction of whole mouse-heart cytoarchitecture at cellular scale

Understanding the three-dimensional organisation of the myocardium requires imaging approaches that can resolve cellular structures across the entire heart. Existing methods are limited by trade-offs between spatial resolution, imaging depth and throughput. Here we present a high-throughput imaging pipeline for high-fidelity reconstruction of whole mouse hearts. The approach combines a CUBIC tissue clearing protocol optimized for cardiac tissue, a dual-camera selective plane-illumination microscope (dual-mesoSPIM) and an automated fusion algorithm that selects the highest-contrast information from the opposing detection views. Whole hearts (n = 9) were imaged label free (using myocardial autofluorescence) at a voxel size of 3.25 × 3.25 × 3 µm³ in approximately 25 min per sample. Dual-view fusion significantly reduces depth-dependent information loss compared to single-camera acquisitions, enabling robust three-dimensional reconstruction and morphometry of cardiomyocyte orientations and laminar tissue organisation across the ventricular walls. Quantitative morphometry of fibre and sheet orientations confirmed the expected transmural organisation of the myocardium, including the progressive rotation of the helix angle and the regional variation in sheet architecture. The protocol is compatible with endogenous fluorescent reporters and whole-mount staining, allowing, for example, the reconstruction of the cardiac sympathetic nervous system throughout the intact mouse organ. Our new pipeline provides a rapid and scalable strategy for mesoscale mapping of cardiac cytoarchitecture in physiological and pathological models. KEY POINTS: Understanding cardiac function requires mapping the three-dimensional arrangement of cardiomyocytes throughout the whole organ, yet reconstructing this cytoarchitecture at cellular resolution across an intact heart remains technically demanding. We combined CUBIC tissue clearing with a dual-camera light-sheet microscope (dual-mesoSPIM) to reconstruct the entire adult mouse heart in three dimensions at cellular scale, rapidly and with high fidelity. Acquiring and fusing images from two opposing cameras substantially reduced the loss of information at depth, allowing the local orientation of cardiomyocytes and myocardial sheetlets to be quantified throughout the ventricular walls, including the expected transmural rotation of the fibre helix angle. The approach preserves endogenous fluorescent reporters and is compatible with whole-heart immunostaining, which we used to reconstruct the cardiac sympathetic innervation across the entire organ, opening the way to mapping cellular organization and its remodelling in health and disease.