X-ray micro-computed tomography (µCT) is highly suited to non-destructive 3D imaging of biological samples. While bones, teeth and other hard biomaterials sit at the ‘highly feasible’ end of the µCT imaging spectrum, the low (and typically homogenous) X-ray attenuation of soft tissues pushes them into a more challenging realm in terms of achieving contrast. While tissue staining is commonplace in the fields of optical and electron imaging for the biological sciences, heavy metal staining (for example with iodine, phosphotungstic acid or osmium tetroxide) can also be adopted for introducing contrast in X-ray imaging.
Iodine staining can be a fast, relatively non-toxic and reliable way of introducing contrast to soft tissues. However, factors such as the penetration rate, staining time, tissue shrinkage and the affinity of iodine for certain tissue types, are poorly characterised. Diffusible iodine contrast enhanced computed tomography (diceCT) is a relatively new term that has been coined to describe a more rigorous approach to the application of iodine-based staining for biological samples. Here we present examples of iodine staining in a range of animal and plant specimens, with a focus on rates of stain penetration, contrast enhancement and tissue shrinkage when a diceCT protocol is employed.