Virtual histology of an entire mouse brain from formalin fixation to paraffin embedding. Part 2: Volumetric strain fields and local contrast changes

Background: Fixation and embedding of post mortem brain tissue is a pre-requisite for both gold-standard conventional histology and X-ray virtual histology. This process alters the morphology and density of the brain microanatomy. New method: To quantify these changes, we employed synchrotron radiation-based hard X-ray tomography with 3 ism voxel length to visualize the same mouse brain after fixation in 4% formalin, immersion in ethanol solutions (50%, 70%, 80%, 90%, and 100%), xylene, and finally after embedding in a paraffin block. The volumetric data were non-rigidly registered to the initial formalin-fixed state to align the microanatomy within the entire mouse brain. Results: Volumetric strain fields were used to characterize local shrinkage, which was found to depend on the anatomical region and distance to external surface. X-ray contrast was altered and enhanced by preparation induced inter-tissue density changes. The preparation step can be selected to highlight specific anatomical features. For example, fiber tract contrast is amplified in 100% ethanol. Comparison with existing methods: Our method provides volumetric strain fields, unlike approaches based on feature-to-feature or volume measurements. Volumetric strain fields are produced by non-rigid registration, which is less labor-intensive and observer-dependent than volume change measurements based on manual segmentations. X-ray microtomography provides spatial resolution at least an order of magnitude higher than magnetic resonance microscopy, allowing for analysis of morphology and density changes within the brain's microanatomy. Conclusion: Our approach belongs to three-dimensional virtual histology with isotropic micrometer spatial resolution and therefore complements atlases based on a combination of magnetic resonance microscopy and optical micrographs of serial histological sections.

Automated summary

This study employed synchrotron radiation-based hard X-ray tomography to visualize changes in morphology and density of mouse brain tissue. It was found that X-ray contrast was altered and enhanced by preparation induced inter-tissue density changes, highlighting specific anatomical features. Additionally, the method provides volumetric strain fields, which are less labor-intensive and observer-dependent compared to manual segmentation based volume measurements.