We sought to develop a reversible staining protocol using micro-computed tomography (micro-CT) paired with a radiopaque contrast agent that allows for three-dimensional in situ visualization and characterization of atherosclerotic plaques. Atherosclerotic porcine coronary arteries were dissected from surrounding myocardium and incubated in iohexol at various concentrations and incubation times and then imaged using direct radiography. Line profiles were generated across the artery x-ray to determine effectiveness of the radiopaque contrast agent to penetrate the tissue. Our studies revealed that, to sufficiently delineate tissue constructs, the minimum effective iohexol concentration and incubation time were 240mgI/mL for 1hour. Among all groups, 24hours of de-staining brought radiopacity back to control levels. After iohexol incubation, micro-CT was performed. Our findings demonstrate that extended staining times and a minimum iohexol concentration of 240mgI/mL are required for effective tissue perfusion, which eliminates the diffusion distribution profile inherent to the ability of the contrast agent to traverse tissue layers.Iohexol enhances ex vivo micro-CT imaging of atherosclerotic coronary arteriesIohexol allows for improved tissue segmentation during micro-CT image analysisEffectiveness of iohexol penetration of the tissue was dependent on concentration and duration of incubation.