Brain virtual histology with X-ray phase-contrast tomography Part I: whole-brain myelin mapping in white-matter injury models

AbstractWhite-matter injury leads to severe functional loss in many neurological diseases. Myelin staining on histological samples is the most common technique to investigate white-matter fibers. However, tissue processing and sectioning may affect the reliability of 3D volumetric assessments. The purpose of this study was to propose an approach that enables myelin fibers to be mapped in the whole rodent brain with microscopic resolution and without the need for strenuous staining. With this aim, we coupled in-line (propagation-based) X-ray phase-contrast tomography (XPCT) to ethanol-induced brain sample dehydration. We here provide the proof-of-concept that this approach enhances myelinated axons in rodent and human brain tissue. In addition, we demonstrated that white-matter injuries could be detected and quantified with this approach, using three animal models: ischemic stroke, premature birth and multiple sclerosis. Furthermore, in analogy to diffusion tensor imaging (DTI), we retrieved fiber directions and DTI-like diffusion metrics from our XPCT data to quantitatively characterize white-matter microstructure. Finally, we showed that this non-destructive approach was compatible with subsequent complementary brain sample analysis by conventional histology. In-line XPCT might thus become a novel gold-standard for investigating white-matter injury in the intact brain. This is Part I of a series of two articles reporting the value of in-line XPCT for virtual histology of the brain; Part II shows how in-line XPCT enables the whole-brain 3D morphometric analysis of amyloid-β (Aβ) plaques.HighlightsX-ray phase-contrast tomography (XPCT) enables myelin mapping of the whole brainXPCT detects and quantifies white-matter injuries in a range of diseasesFiber directions and anisotropy metrics can be retrieved from XPCT dataXPCT is compatible with subsequent conventional histology of brain samplesXPCT is a powerful virtual histology tool that requires minimal sample preparationGraphical Abstract

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

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1801678115 ◽

2018 ◽

Vol 115 (27) ◽

pp. 6940-6945 ◽

Cited By ~ 41

Author(s):

Mareike Töpperwien ◽

Franziska van der Meer ◽

Christine Stadelmann ◽

Tim Salditt

Keyword(s):

Phase Contrast ◽

Spatial Organization ◽

Granular Layer ◽

Three Dimensional ◽

Cell Segmentation ◽

X Ray ◽

Human Cerebellum ◽

Conventional Histology ◽

3D Information ◽

Phase Contrast Tomography

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.

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Brain virtual histology with X-ray phase-contrast tomography Part II: 3D morphologies of amyloid-β plaques in Alzheimer's disease models

10.1101/2021.03.25.436908 ◽

2021 ◽

Author(s):

Matthieu Chourrout ◽

Margaux Roux ◽

Carlie Boisvert ◽

Coralie Gislard ◽

David Legland ◽

...

Keyword(s):

Alzheimer’S Disease ◽

Alzheimer's Disease ◽

Phase Contrast ◽

Amyloid Β ◽

Mouse Strains ◽

3D Analysis ◽

Virtual Histology ◽

X Ray ◽

Phase Contrast Tomography ◽

The Brain

While numerous transgenic mouse strains have been produced to model the formation of amyloid-β (Aβ) plaques in the brain, efficient methods for whole-brain 3D analysis of Aβ deposits are lacking. Moreover, standard immunohistochemistry performed on brain slices precludes any shape analysis of Aβ plaques. The present study shows how in-line (propagation-based) X-ray phase-contrast tomography (XPCT) combined with ethanol-induced brain sample dehydration enables hippocampus-wide detection and morphometric analysis of Aβ plaques. Performed in three distinct Alzheimer mouse strains, the proposed workflow identified differences in signal intensity and 3D shape parameters: 3xTg displayed a different type of Aβ plaques, with a larger volume and area, greater elongation, flatness and mean breadth, and more intense average signal than J20 and APP/PS1. As a label-free non-destructive technique, XPCT can be combined with standard immunohistochemistry. XPCT virtual histology could thus become instrumental in quantifying the 3D spreading and the morphological impact of seeding when studying prion-like properties of Aβ aggregates in animal models of Alzheimer's disease. This is Part II of a series of two articles reporting the value of in-line XPCT for virtual histology of the brain; Part I shows how in-line XPCT enables 3D myelin mapping in the whole rodent brain and in human autopsy brain tissue.

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