While major coronary artery development and pathologies affecting them have been extensively studied, understanding the development and organization of the coronary microvasculature beyond the earliest developmental stages requires new tools. Without techniques to image the coronary microvasculature over the whole heart, it is likely we are underestimating the microvasculature's impact on normal development and diseases. We present a new imaging and analysis toolset to visualize the coronary microvasculature in intact embryonic hearts and quantify vessel organization. The fluorescent dyes DiI and DAPI were used to stain the coronary vasculature and cardiomyocyte nuclei in quail embryo hearts during rapid growth and morphogenesis of the left ventricular wall. Vessel and cardiomyocytes orientation were automatically extracted and quantified, and vessel density was calculated. The coronary microvasculature was found to follow the known helical organization of cardiomyocytes in the ventricular wall. Vessel density in the left ventricle did not change during and after compaction. This quantitative and automated approach will enable future cohort studies to understand the microvasculature's role in diseases such as hypertrophic cardiomyopathy where misalignment of cardiomyocytes has been observed in utero.

Department of Biomedical Engineering School of Medicine Case Western Reserve University Wood Building WG28 2109 Adelbert Road Cleveland OH 44106 USA

Department of Pediatrics School of Medicine Case Western Reserve University Cleveland USA

Institute of Anatomy 1st Faculty of Medicine Charles University Prague Czech Republic

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Tissue clearing and imaging methods for cardiovascular development