Three-dimensional virtual histology of human cerebellum by X-ray phase-contrast tomography

Proc Natl Acad Sci U S A. 2018 Jul 3;115(27):6940-6945. doi: 10.1073/pnas.1801678115. Epub 2018 Jun 18.


To quantitatively evaluate brain tissue and its corresponding function, knowledge of the 3D cellular distribution is essential. The gold standard to obtain this information is histology, a destructive and labor-intensive technique where the specimen is sliced and examined under a light microscope, providing 3D information at nonisotropic resolution. To overcome the limitations of conventional histology, we use phase-contrast X-ray tomography with optimized optics, reconstruction, and image analysis, both at a dedicated synchrotron radiation endstation, which we have equipped with X-ray waveguide optics for coherence and wavefront filtering, and at a compact laboratory source. As a proof-of-concept demonstration we probe the 3D cytoarchitecture in millimeter-sized punches of unstained human cerebellum embedded in paraffin and show that isotropic subcellular resolution can be reached at both setups throughout the specimen. To enable a quantitative analysis of the reconstructed data, we demonstrate automatic cell segmentation and localization of over 1 million neurons within the cerebellar cortex. This allows for the analysis of the spatial organization and correlation of cells in all dimensions by borrowing concepts from condensed-matter physics, indicating a strong short-range order and local clustering of the cells in the granular layer. By quantification of 3D neuronal "packing," we can hence shed light on how the human cerebellum accommodates 80% of the total neurons in the brain in only 10% of its volume. In addition, we show that the distribution of neighboring neurons in the granular layer is anisotropic with respect to the Purkinje cell dendrites.

Keywords: 3D virtual histology; X-ray phase-contrast tomography; automatic cell counting; human brain cytoarchitecture.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cerebellum / cytology*
  • Cerebellum / diagnostic imaging*
  • Female
  • Histology*
  • Humans
  • Imaging, Three-Dimensional*
  • Male
  • Tomography, X-Ray Computed*