Experimental biomechanics - Clinical translation of X-ray-based 3D histology: optimizing the washing step to allow classical 2D histology after contrast-enhanced microCT imaging

Details

Supervisors

Kerckhofs, Greet

Faculty

Ecole polytechnique de Louvain

Degree label

Master [120] : ingénieur civil biomédical, à finalité spécialisée

Abstract

enBackground: Contrast-enhanced X-ray micro-computed tomography (CECT) has emerged as a promising approach for X-ray-based 3D histology, enabling non-destructive visualization of soft biological tissues at micrometric resolution. However, the contrast-enhancing staining agents (CESAs) required for imaging may remain within the tissue after scanning and interfere with subsequent conventional histological processing, limiting the compatibility between 3D CECT imaging and classical 2D histology. Optimizing post-imaging washing procedures is therefore essential to facilitate the integration of both techniques. Objective: This study investigated the removal of the hafnium-substituted Wells-Dawson polyoxometalate (Hf-WD POM) after CECT imaging. The objectives were to characterize washing kinetics over time, evaluate the influence of different washing solutions, and determine whether periodic solution renewal improves contrast-agent removal. Methods: Mouse ear tissues were stained with Hf-WD POM and subsequently washed using six different solutions: phosphate-buffered saline (PBS), Tris buffer, acetate buffer, EDTA, NaCl 1 M, and NaCl 2 M. Each solution was tested under two conditions: with and without periodic solution renewal. Samples were monitored by repeated CECT imaging throughout the washing process. Gray values were quantified in the epidermis, dermis, and cartilage and used as an indirect measure of residual contrast-agent content. Washing rates and final normalized grayscale values were compared statistically between solutions and washing strategies. Results: Washing efficiency strongly depended on the composition of the washing solution. Tris consistently exhibited the highest initial washing rates and produced final grayscale values closest to those of unstained control tissues. EDTA also demonstrated favorable performance, particularly when combined with solution renewal. In contrast, acetate showed poor washing efficiency, characterized by limited grayscale reduction and the highest residual contrast at the end of the experiment. Increasing NaCl concentration from 1 M to 2 M did not result in a consistent improvement in washing performance. The effect of solution renewal was highly solution-dependent: renewal improved intermediate washing kinetics for Tris and EDTA but provided little or no benefit for PBS and NaCl solutions, while negatively affecting acetate. Overall, Tris and renewed EDTA yielded the most favorable final outcomes across tissue compartments. Conclusion: The composition of the washing solution plays a major role in the removal of Hf-WD POM following CECT imaging. Among the investigated conditions, Tris and EDTA emerged as the most promising alternatives to the current PBS-based approach. These findings provide a foundation for the development of optimized post-CECT washing protocols aimed at improving the compatibility between X-ray-based 3D histology and conventional 2D histological analysis. Further work including direct evaluation of subsequent histological staining quality is needed to confirm the practical benefits of these washing strategies.