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

Griffin Rodgers; Christine Tanner; Georg Schulz; Alexandra Migga; Willy Kuo; Christos Bikis; Mario Scheel; Vartan Kurtcuoglu; Timm Weitkamp; Bert Müller

doi:10.1016/j.jneumeth.2021.109385

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

J Neurosci Methods. 2022 Jan 1:365:109385. doi: 10.1016/j.jneumeth.2021.109385. Epub 2021 Oct 9.

Authors

Affiliations

¹ Biomaterials Science Center, Department of Biomedical Engineering, University of Basel, 4123 Allschwil, Switzerland; Biomaterials Science Center, Department of Clinical Research, University Hospital Basel, 4031 Basel, Switzerland.
² Biomaterials Science Center, Department of Biomedical Engineering, University of Basel, 4123 Allschwil, Switzerland; Biomaterials Science Center, Department of Clinical Research, University Hospital Basel, 4031 Basel, Switzerland. Electronic address: christine.tanner@unibas.ch.
³ The Interface Group, Institute of Physiology, University of Zurich, 8057 Zurich, Switzerland; National Centre of Competence in Research, Kidney.CH, 8057 Zurich, Switzerland.
⁴ Biomaterials Science Center, Department of Biomedical Engineering, University of Basel, 4123 Allschwil, Switzerland; Biomaterials Science Center, Department of Clinical Research, University Hospital Basel, 4031 Basel, Switzerland; Integrierte Psychiatrie Winterthur - Zürcher Unterland, 8408 Winterthur, Switzerland.
⁵ Synchrotron Soleil, 91192 Gif-sur-Yvette, France.

PMID: 34637810
DOI: 10.1016/j.jneumeth.2021.109385

Abstract

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 μm 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.

Keywords: Embedding media for contrast enhancement in brain tissue; Neuroimaging; Non-rigid registration; Non-uniform shrinkage; Synchrotron radiation-based microtomography.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Animals
Brain* / diagnostic imaging
Formaldehyde*
Mice
Paraffin Embedding
Synchrotrons
X-Ray Microtomography / methods

Substances

Formaldehyde