Imaging crossing fibers in mouse, pig, monkey, and human brain using small-angle X-ray scattering

Abstract

Myelinated axons (nerve fibers) efficiently transmit signals throughout the brain via action potentials. Multiple methods that are sensitive to axon orientations, from microscopy to magnetic resonance imaging, aim to reconstruct the brain's structural connectome. As billions of nerve fibers traverse the brain with various possible geometries at each point, resolving fiber crossings is necessary to generate accurate structural connectivity maps. However, doing so with specificity is a challenging task because signals stemming from oriented fibers can be influenced by brain (micro)structures unrelated to myelinated axons. X-ray scattering can specifically probe myelinated axons due to the periodicity of the myelin sheath, which yields distinct peaks in the scattering pattern. Here, we show that scanning small-angle X-ray scattering (SAXS) can be used to detect myelinated, axon-specific fiber crossings. We first demonstrate the capability using strips of human corpus callosum to create artificial double and triple crossing fiber geometries, and then apply the method in mouse, pig, vervet monkey, and human brains. Given its specificity, capability of 3-dimensional sampling and high resolution, scanning SAXS can serve as a ground truth for validating MRI as well as microscopy-based methods.

Micron-resolution fiber mapping in histology independent of sample preparation

“…Given its independence from myelin birefringence, ComSLI can yield neuropathologic insights when combined with myelin-sensitive microscopic methods such as 3D-PLI [18][19][20] . polarizationsensitive optical coherence tomography 16,17 , and X-ray scattering 9,10,36,37 . Especially compared to 3D-PLI, which is the method-of-choice for micron-resolution fiber mapping on sections, ComSLI can be applied to virtually any section independent of sample preparation or birefringence, including FFPE sections, and can resolve crossing fibers per pixel, with an easyto-implement and cost-effective setup.…”

“…Electron microscopy resolves nerve cells at nanometer resolution 5,6 , but tracking the fiber pathways is limited to small tissue volumes 7,8 . Small-angle X-ray scattering (SAXS) can directly probe multi-directional fiber pathways 9,10 , but cannot provide whole-brain micrometer resolution and requires expensive equipment such as synchrotrons. Various light microscopy techniques visualize nerve fibers at high resolution and over larger fields of view, using staining 11,12 , fluorescence 13 , or other contrast mechanisms.…”

“…Electron microscopy resolves nerve cells at sub-micron resolution (8,10), but tracking the fiber pathways is time consuming and limited to small tissue volumes (57)(58). Small-angle X-ray scattering (SAXS) can directly probe multi-directional fiber pathways (20,59), but cannot provide micrometer resolution for larger samples and requires expensive equipment. To visualize nerve fibers at high resolution and over larger fields of view, there exist various light microscopy techniques.…”

Using light and X-ray scattering to untangle complex neuronal orientations and validate diffusion MRI

“…The method can be tomographic (SAXS tensor tomography – Liebi et al, 2015 ; Gao et al, 2019; Georgiadis et al, 2021 ), and 3D-scanning SAXS (3D-sSAXS) can provide 3D distributions of axon orientations in tissue sections ( Georgiadis et al, 2020 ). Recent studies further revealed that SAXS can exploit the modulations in the azimuthal position of the myelin-specific Bragg peaks to resolve crossing nerve fiber populations across species ( Georgiadis et al, 2022 ).…”