Despite advances in imaging, image-based vascular systems biology has remained challenging because blood vessel data is often available only from a single modality or at a given spatial scale, and cross-modality data are difficult to integrate. Therefore, there is an exigent need for a multimodality pipeline that enables ex vivo vascular imaging with MRI, CT and optical microscopy of the same sample, while permitting imaging with complementary contrast mechanisms from the whole-organ to endothelial cell spatial scales. To achieve this, we developed ‘VascuViz’ – an easy-to-use method for simultaneous 3D imaging and visualization of the vascular microenvironment using MRI, CT and optical microscopy in the same intact, unsectioned tissue. The VascuViz workflow permits multimodal imaging with a single labeling step using commercial reagents and is compatible with diverse tissue types and protocols. VascuViz’s interdisciplinary utility in conjunction with novel data visualization approaches opens up new vistas in image-based vascular systems biology.