Investigating the diffusion problem of staining agents for contrast-enhanced microCT imaging of whole tissues via engineering solutions

Microfocus X-ray tomography is nowadays one of the most powerful 3D histological techniques for imaging mineralized tissues. In addition, when contrast agents are applied to the tissues before scanning, soft tissues can be visualised. To acquire highly contrasted images, the contrast agent has to be wisely chosen in function of the tissue studied. Moreover, homogeneous staining of the sample is required to ensure the correct and reproducible interpretation of the tissue microstructure. Unfortunately, many factors such as temperature, concentration or pressure can influence the diffusion process, making it easily subject to variability. In addition, the diffusion process depends on the diffusing molecules and the environment, making the diffusion process very case-specific. Consequently, understanding the diffusion process of contrast agents in biological tissues would permit enhancing the staining protocols. This should represent a first step towards a shorter staining time, leading to less deterioration of the tissue due to prolonged incubation in non-ideal conditions. This master thesis aimed to give a first insight on the diffusion process of two contrast agents: Hf-WD 2:2 POM and Mono-WD POM in porcine aortas. The influence of temperature on the diffusion process was studied in two manners: increase of the contrast at a discrete location and progression of the molecules observed as a front. We observed that an increase (decrease) in temperature leads to a faster (slower) penetration of the molecules in the tissue, but the maximal contrast provided to the tissue is lower (higher). These observations could indicate that temperature affects the packing of the elastin sheets of the tissue or that the contrast agent dissociates, forming smaller and less interacting molecules. Furthermore, data from a previous master thesis study, which explored the effect of concentration and volume variation of contrast enhancement was re-examined in terms of penetration front. It appeared that an increase (decrease) in concentration leads to a faster (slower) penetration of the molecules in the tissue. This confirms that the concentration gradient plays an important role in the diffusion process of contrast agents in biological tissues. In conclusion, this master thesis introduces the diffusion process of Mono-WD POM and Hf-WD 2:2 POM through biological tissue. However, we only studied the effect of temperature and concentration on the diffusion behaviour of two CESAs and for only one type of tissue. Further research is required regarding the tissue-molecule interactions, the effect of temperature on the molecule itself and the effect of other factors on the diffusion process. Moreover, a more thorough study of the models used to explain the diffusion of the molecules in the tissue should be carried out.