Screening and development of contrast‐enhancing staining agents for CECT imaging
(2023)
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- The visualization of the composition and the spatial organization of tissues is of paramount importance to better understand their functioning and the impact of a disease. Microfocus X‐ray computed tomography (microCT) is a valuable tool for 3D imaging to complement classical 2D histological imaging techniques. In the last two decades, the incorporation of contrast‐enhancing staining agents (CESAs) has augmented the applicability of microCT for soft tissue imaging, which is referred to as contrast‐enhanced computed tomography (CECT). This PhD aspires to advance the field of CECT imaging of biological tissues. This is achieved by exploring the capabilities of existing compounds as contrast-enhancing staining agents and by synthesizing new CESAs. The goal is to develop CESAs that can effectively differentiate between different tissue constituents without causing distinct tissue deformation. First, polyoxometalates were explored for imaging murine bone marrow and kidney. Monolacunary Wells-Dawson polyoxometalate combined with LiCl showed good tissue penetration and resulted in high contrast enhancement of soft tissue without inducing substantial tissue shrinkage. Second, novel cationic iodine-based CESAs with varying levels of iodine content and charge density were developed to improve glycosaminoglycan (GAG) detection, while existing synthetic procedures were optimized in terms of efficiency and sustainability. Time-lapsed staining experiments of osteochondral plugs revealed that contrast enhancement was positively correlated with the staining time, and the CESA concentration, iodine content and positive charge density. Moreover, the spatial distribution of the CESA at diffusion equilibrium was consistent with the depth-wise GAG concentration in healthy cartilage. Third, novel CESAs were designed for protein-specific imaging. Moving away from antibodies as detecting probes, short peptide sequences were explored in the first part. By means of phage display, peptide sequences showing affinity towards target proteins were identified. Nevertheless, it was noted that the isolated peptide demonstrated lower target specificity compared to the corresponding phage. Increasing the avidity of this peptide-based probe could lead to better binding, and hence serve as a good alternative for antibody-based labeling. In the second part, X-ray attenuating revealing agents were developed for 3D immunostaining by CECT. Although iodinated revealing agents were successfully synthesized, their poor water solubility hampered their use to detect antibody-labeled proteins. Improving the water solubility of revealing agents would be a next step towards increasing their efficacy. In this PhD, the research revolved around exploring the potential of both existing and novel compounds as CESAs for general visualization of soft tissues as well as for targeted imaging. This work paves the way for the application of CECT as a tool for X-ray based histology at high resolution, complementary to classical 2D histology.
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Vangrunderbeeck, S. (2023). Screening and development of contrast‐enhancing staining agents for CECT imaging. https://hdl.handle.net/2078.5/233587