By employing high‐resolution three‐dimensional (3D) imaging, we aim to detect and quantify monocytes targeting active sites of neovascularization in ischemic myocardium. As a first step, distinguishing exogenously administered cells from highly autofluorescent myocardial tissue and vascular casting material must be addressed. The aim of this study was to optimize visualization of single fluorescent cells with an imaging cryomicrotome.Human vascular endothelial cells (HUVECs) labeled with CellTrackerTM Orange CMTMR (5, 10, 25 and 72μM) were infused into postmortem coronary arteries of rat and rabbit hearts together with replica resin (Mercox) containing fluorescent dye (Potomac Yellow or UV Blue). Following polymerization of replica resin, hearts were frozen at −20ºC and further processed with the imaging cryomicrotome.In 3D reconstructions of hearts cut at 5μm slice thickness, HUVECs labeled with dye concentrations higher than 5μM were visible in the presence of UV Blue cast material, while cells labeled with 72μM dye demonstrated superior brightness.The successful identification of single exogenously administered cells in the presence of vascular cast material will allow quantification of actively growing collaterals which recruit monocytes in ischemic myocardial tissue.Supported by the Center for Translational Molecular Medicine (EMINENCE) and the Netherlands Heart Foundation