Tissue clearing and imaging methods for cardiovascular development

Summary Tissue imaging in 3D using visible light is limited and various clearing techniques were developed to increase imaging depth, but none provides universal solution for all tissues at all developmental stages. In this review, we focus on different tissue clearing methods for 3D imaging of heart and vasculature, based on chemical composition (solvent-based, simple immersion, hyperhydration, and hydrogel embedding techniques). We discuss in detail compatibility of various tissue clearing techniques with visualization methods: fluorescence preservation, immunohistochemistry, nuclear staining, and fluorescent dyes vascular perfusion. We also discuss myocardium visualization using autofluorescence, tissue shrinking and expansion. Then we overview imaging methods used to study cardiovascular system and live imaging. We discuss heart and vessels segmentation methods and image analysis. Review covers the whole process of cardiovascular system 3D imaging, starting with tissue clearing and its compatibility with various visualization methods, to the types of imaging methods and resulting image analysis.