Three-dimensional x-ray microscopy of a mouse brain: quantifying morphological changes from formalin fixation via ascending alcohol series to the dried state

Biological matter may change shape via water absorption or loss. For example, brain tissue shows non-uniform shrinkage during formalin fixation and paraffin embedding, which is the most common tissue preparation for conventional histological analysis. Local deformations can be analyzed with non-rigid registration of non-destructive three-dimensional imaging datasets. We utilized synchrotron radiation microtomography at the ANATOMIX beamline of Synchrotron SOLEIL to image a mouse brain with 3 micron voxel length after formalin fixation, immersion in ascending alcohol series and xylene, and after paraffin embedding. We created a pipeline for non- rigid registration to align the volumes and extract volumetric strain fields. In this way, we could visualize the swelling/shrinkage of anatomical features. This method avoids time-consuming segmentation of brain regions, however it is sensitive to the registration parameters. In this proceedings paper, we discuss the selection of registration parameters in order to generate plausible volumetric strain fields. This protocol can be deployed to any type of shape change of biological matter and allows for the quantification of the related processes.