Three-dimensional mouse brain cytoarchitecture revealed by laboratory-based x-ray phase-contrast tomography

Mareike Töpperwien,Jürgen Goldschmidt,Martin Krenkel,Tim Salditt,Daniel Vincenz,Anja M Oelschlegel,Franziska Stöber

doi:10.1038/srep42847

Abstract

Studies of brain cytoarchitecture in mammals are routinely performed by serial sectioning of the specimen and staining of the sections. The procedure is labor-intensive and the 3D architecture can only be determined after aligning individual 2D sections, leading to a reconstructed volume with non-isotropic resolution. Propagation-based x-ray phase-contrast tomography offers a unique potential for high-resolution 3D imaging of intact biological specimen due to the high penetration depth and potential resolution. We here show that even compact laboratory CT at an optimized liquid-metal jet microfocus source combined with suitable phase-retrieval algorithms and a novel tissue preparation can provide cellular and subcellular resolution in millimeter sized samples of mouse brain. We removed water and lipids from entire mouse brains and measured the remaining dry tissue matrix in air, lowering absorption but increasing phase contrast. We present single-cell resolution images of mouse brain cytoarchitecture and show that axons can be revealed in myelinated fiber bundles. In contrast to optical 3D techniques our approach does neither require staining of cells nor tissue clearing, procedures that are increasingly difficult to apply with increasing sample and brain sizes. The approach thus opens a novel route for high-resolution high-throughput studies of brain architecture in mammals.

Highlights

Resolution, conveniently in the range of a few micron to the sub-micron range, while simultaneously requiring a lower dose[18,19]
A region of interest tomogram with 1000 projections over 180° was recorded in the area of the hippocampus as well as the cortex with a pixel size of 0.47 μm and an exposure time of 50 s per projection
The results demonstrate that the combination of a new tissue preparation and optimized propagation-based phase-contrast tomography enables imaging of the central nervous system at large scales with contrast and resolution high enough to identify single neurons, both at synchrotrons as well as laboratory sources

Summary

Introduction

Resolution, conveniently in the range of a few micron to the sub-micron range, while simultaneously requiring a lower dose[18,19]. We present propagation-based phase-contrast tomography at an optimized combination of a liquid-metal jet laboratory source[20], instrumentation, sample preparation and reconstruction algorithm, to image large volumes of brain tissue from mice. We can show that high contrast within the reconstructed volume can be achieved without labeling and that within a region of interest (ROI) individual neurons can be visualized. This gives access to the intact 3D cytoarchitecture of the brain. In contrast to staining with high-Z elements, this technique, which we term EOS (evaporation-of-organic-solvent)-preparation, substantially lowers absorption due to removal of water and lipids while generating a novel contrast between the tissue protein-matrix and surrounding air. Φθ(r⊥) Due to the denote the intensity and phase distribution laplacian, phase contrast typically appears idnirtehcitslyrebgeihminedastheedogbe-jeecnthaanndcekm=en2λtπ

Methods

Results

Conclusion