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

Abstract

Disentangling human brain connectivity requires an accurate description of nerve fiber trajectories, unveiled via detailed mapping of axonal orientations. However, this is challenging because axons can cross one another on a micrometer scale. Diffusion magnetic resonance imaging (dMRI) can be used to infer axonal connectivity because it is sensitive to axonal alignment, but it has limited spatial resolution and specificity. Scattered light imaging (SLI) and small-angle X-ray scattering (SAXS) reveal axonal orientations with microscopic resolution and high specificity, respectively. Here, we apply both scattering techniques on the same samples and cross-validate them, laying the groundwork for ground-truth axonal orientation imaging and validating dMRI. We evaluate brain regions that include unidirectional and crossing fibers in human and vervet monkey brain sections. SLI and SAXS quantitatively agree regarding in-plane fiber orientations including crossings, while dMRI agrees in the majority of voxels with small discrepancies. We further use SAXS and dMRI to confirm theoretical predictions regarding SLI determination of through-plane fiber orientations. Scattered light and X-ray imaging can provide quantitative micrometer 3D fiber orientations with high resolution and specificity, facilitating detailed investigations of complex fiber architecture in the animal and human brain.

Micron-resolution fiber mapping in histology independent of sample preparation

“…In this study, we only show in-plane fiber orientations, but the method also provides information of fiber inclination ( 14–16 ), and our future work will focus on quantifying out-of-plane angles. While measurements were performed in transmission mode through thin sections, we aim to extend measurements to back-scattering mode to analyze fiber orientations on surfaces without sectioning.…”

“…Compared to Nissl-ST, ComSLI provides significantly higher resolution in a label-free manner, including multiple crossing fiber orientations per image pixel, and the possibility for out-of-plane orientation information (14)(15)(16). Diffusion MRI can image living human subjects tomographically but lacks cellular and microscopic details.…”

“…Thus, there is a need for an easily accessible, widely applicable method to elucidate the intricate nerve fiber architecture of brain tissue, independent of sample preparation, also in regions with highly interwoven and crossing fibers. Previous studies have shown that anisotropic scattering of light can be exploited to reveal such interwoven fiber networks in unstained cryo-sections with micrometer resolution (14)(15)(16)26). As the distribution of scattered light seems mostly affected by the overall arrangement of fibers and not by their birefringence, it might also be a promising approach for reconstructing fiber architectures in FFPE sections.…”

“…Nanometer-resolution methods like electron microscopy achieve exquisite results in small volumes of various tissues (8)(9)(10). 3D-polarized light imaging (3D-PLI) and scattered light imaging (SLI) have delivered micron-resolution maps in unstained brain sections (11)(12)(13)(14)(15)(16). While 3D-PLI shows specific sensitivity to myelination and allows tracing fiber tracts from deep white matter into the depth of the cerebral cortex, SLI provides superior insights into complex fiber tract crossings.…”