3D Histological Analysis of Soft Tissues by Contrast-Enhanced X-Ray Microfocus Computed Tomography: Screening and Staining Optimization of Contrast-Enhancing Staining Agents

Abstract The gold standard for studying biological soft tissues at the microscale (i.e., histology) is tissue sectioning with subsequent colorimetric or fluorescent staining and visual inspection under the microscope. When tissue integrity must be maintained for 3D histological assessment, contrast-enhanced microfocus X-ray computed tomography (CECT) is a promising solution, but there is still a lack of staining protocol optimization of contrast-enhancing staining agents (CESAs). Therefore, in this study, mouse auricles were incubated with Hafnium-substituted Wells–Dawson polyoxometalate, cationic iodinated contrast agent, or Lugol's iodine and were imaged with high-resolution CECT. Alignment with corresponding H&E-stained sections enabled the identification and segmentation of different tissue types. Contrast differences between tissue types were increased by washing the samples after staining or by combining CESAs. Finally, we proved that the latter could be used to quantitatively assess the 3D thickness distribution of the epidermis in the ears of a mouse model of psoriasis-like dermatitis. In conclusion, CECT and bright-field microscopy are complementary and not mutually exclusive techniques for the histological assessment of biological tissues. While bright-field microscopy gives detailed information about the cellular composition of tissues, CECT provides a better insight into the spatial interrelationship of tissues and is a powerful tool for performing 3D structural quantification.