During the development and progression of coronary artery disease not only the large epicardial coronary arteries but also the more distal resistance vessels are affected. Studying the role of the entire coronary vasculature is a major challenge due to heart motion and the large vascular diameter range. Knowledge about the microvasculature, that stays invisible using conventional coronary imaging techniques, is essential in the search for early disease markers and targeted treatment. In this thesis the imaging cryomicrotome was used to assess the structure of vasculature in high detail. This imaging technology allows for slice-by-slice high resolution imaging of whole organs using fluorescent markers.This thesis expanded the knowledge on the coronary small vessel vascular structure and function. The performance and versatility of the imaging cryomicrotome technique was further exploited when studying the vascular structure in acardiac twinning and additionally in assessing the vascular structure in acardiac twinning and additionally in assessing the vascular structure and interstitial fluid clearance in the brain.Combining the imaging cryomicrotome with a broad spectrum of other imaging techniques in future studies, can yield information that leads to a broader understanding of the changes that occur within the arterial network in health, disease and during therapeutic intervention, and the impact on tissue perfusion.