Contrast agents and sample preparation strategies for soft tissue imaging in X-ray computed microtomography: Review and experimental validation in mouse tissues

Micro-computed tomography (micro-CT) is an effective technique for non-destructive high-resolution imaging of mineralized tissues; however, imaging of soft tissues is challenging due to their inherently low X-ray attenuation and poor contrast. Therefore, their visualization in absorption micro-CT requires the use of contrast agents (CA) such as iodine-based solutions, osmium tetroxide, phosphotungstic acid, and other polyoxometalates. The aim of this review is to outline the individual steps of sample preparation before micro-CT measurement, such as fixation, dehydration, and contrast enhancement of soft tissues, and summarize the most commonly used CAs in ex vivo visualization. In addition to the literature overview, we conducted comparative staining experiments on mouse organs using several widely applied CAs, with the aim of illustrating practical outcomes and challenges. Based on both the data published and our test results, we recommend the most suitable CAs for different tissue types, with emphasis on minimizing tissue damage and deformation, and devote particular attention to applications in mouse embryos.