3D virtual histology using contrast-enhanced microCT : screening its benefits and limitations for different tissues

Details

Supervisors

Kerckhofs, Greet ; Hoffmann, Delia

Faculty

Ecole polytechnique de Louvain

Degree label

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

Abstract

The correct functioning of organs is influenced by the composition and the 3D organization of their constituents. Histology, a destructive method requiring colorimetric stains, is the standard technique used to assess this composition in two dimensions (2D). It gives images of high resolution, but it loses information concerning the spatial distribution of the constituents. Due to this limitation, contrast-enhanced computed tomography (CECT) is currently being developed. This non-destructive technique gives a three-dimensional (3D) dataset that can be processed to obtain/segment the individual 3D volumes of constituents composing the organ. Since the method is based on X-rays, it requires the use of contrast-enhancing staining agents (CESAs) to increase the coefficient of attenuation of soft tissues and allow their visualization. The CESAs have often unknown binding mechanisms and their comparison is currently missing. These information are crucial to determine which CESA should be used to study a specific tissue. Therefore, this project aimed to characterize the benefits and limitations of CESAs on the contrast of different tissue types. Murine auricles, having a highly heterogeneous composition, were stained with different CESAs, i.e. Lugol’s iodine, CA4+, or Hafnium-substituted Wells-Dawson polyoxometalate (Hf-WD POM), and scanned with a microCT scanner to compare and assess the contrast impact of the CESAs on the auricle constituents. Results showed differences in the contrast enhancement between the CESAs. However, no matter the CESA, almost all the constituents have been contrasted and segmented. In histology, the colorimetric stains are often washed to improve the contrast. The same principle could be applied to CESAs to see if the difference of contrast is enhanced after washing. Additionally, it was not known if CESAs could be entirely washed out from tissues. Nude murine auricles were stained with Lugol’s iodine, CA4+, and Hf-WD POM and then washed with PBS. CECT scans of the samples were made at the different steps. It seems that no CESA was fully washed out from the tissues and that washing Lugol’s iodine for one day improved the image contrast. Finally, the combination of positively- and negatively-charged CESAs was never assessed in literature while it is often done in histology. Nude murine auricles were stained either with Hf-WD POM or CA4+, then washed with PBS to be restained with the other CESA. It appeared that the order of staining was important and that, depending on the tissue constituent, the contrast could either be summed up or CESAs seemed to interact together. We determined in this project which CESA can be used to highlight and segment which constituents of the outer ear. However, the washing and double staining protocols need to be optimized and more tissue types should be studied. It would also be useful to determine the binding mechanisms of the CESAs to the different constituents.