Background
The brain’s nerve fiber network is disturbed in neurodegeneration, but resolving fiber trajectories over large fields-of-view to study connectivity changes remains prohibitive. Current methods study small volumes (electron microscopy), have limited resolution (diffusion MRI), or need birefringence-preserving sample preparation and cannot resolve crossings (polarization microscopy). Here we show that computational scattered light imaging (ComSLI) resolves neuronal trajectories, including degenerating hippocampal tracts, with micron resolution in any histology section independent of sample preparation.

Methods
We studied standard-sized 5-10μm formalin-fixed paraffin-embedded (FFPE) sections prepared using various protocols (cf. text/figures) and two whole-brain sections (Figure 1 – FFPE, from the Jülich BigBrain, 20μm, silver-stained, and Figure 2E - celloidin-embedded and myelin-stained in 1904, from the Institute for Brain Research, Düsseldorf, Germany).

Computational scattered light imaging (ComSLI) was performed in Stanford and Jülich. The setup (Figure 1A,B) includes a micron-resolution low angle-of-acceptance camera-adapter-lens system and a rotating LED lightsource. Images were acquired at 5-15o rotation steps (24-72 images/sample), with 3-9μm pixel size. Motorized stages enable tile-scanning. SLIX software quantified orientations, MATLAB was used for orientation analysis, and MRtrix3 for creating orientation distribution functions and subsequent tractography.

Result
ComSLI produced a micron-resolution whole-brain fiber orientation map (Figure 1C). Figures 1D-E show zoomed-in fiber orientations in corpus callosum/fornix and corona radiata. Microscopic resolutions enabled generating fiber orientation distributions at multiple scales (Figure 1F), leading to microstructure-informed whole-brain tracts (Figure 1G-H).

ComSLI works for various sample preparation protocols (Figure 2). Consecutive human hippocampal sections with different stains (iron, microglia, tau, and amyloid) show identical orientations (Figure 2B,C), quantified after co-registration in Figure 2D. Orientations were also derived from a 120-year-old human section (Figure 2E-F), and were consistent at various sample preparation steps (Figure 2G).

ComSLI can study neurodegenerating tracts, such as the hippocampal perforant pathway (Figure 3). A healthy hippocampus includes strong perforant pathway connections through the subiculum and CA1 subfields (Figure 3A-F), which almost entirely disappear in a sclerotic hippocampus (Figure 3G-L), and are severely compromised in Alzheimer’s disease (Figure 3M-R).

Conclusion
ComSLI is a cost-effective method to study intricate fiber networks at micron-resolution in any histological tissue section, and can reveal subtle changes in neurodegeneration.

Mapping the brain’s fiber network is crucial for understanding its function and malfunction, but resolving nerve trajectories over large fields of view is challenging. Here, we show that computational scattered light imaging (ComSLI) can map fiber networks in histology independent of sample preparation, also in formalin-fixed paraffin-embedded (FFPE) tissues including whole human brain sections. We showcase this method in new and archived, animal and human brain sections, for different sample preparations (in paraffin, deparaffinized, various stains, unstained fresh-frozen). We convert microscopic orientations to microstructure-informed fiber orientation distributions (μFODs). Adapting tractography tools from diffusion magnetic resonance imaging (dMRI), we trace axonal trajectories revealing white and gray matter connectivity. These allow us to identify altered microstructure or deficient tracts in demyelinating or neurodegenerating pathology, and to show key advantages over dMRI, polarization microscopy, and structure tensor analysis. Finally, we map fibers in non-brain tissues, including muscle, bone, and blood vessels, unveiling the tissue’s function. Our cost-effective, versatile approach enables micron-resolution studies of intricate fiber networks across tissues, species, diseases, and sample preparations, offering new dimensions to neuroscientific and biomedical research.

Background
The brain's nerve fiber network is disturbed in neurodegeneration, but resolving fiber trajectories over large fields-of-view to study connectivity changes remains prohibitive. Current methods study small volumes (electron microscopy), have limited resolution (diffusion MRI), or need birefringence-preserving sample preparation and cannot resolve crossings (polarization microscopy). Here we show that computational scattered light imaging (ComSLI) resolves neuronal trajectories, including degenerating hippocampal tracts, with micron resolution in any histology section independent of sample preparation.

Methods
We studied standard-sized 5-10μm formalin-fixed paraffin-embedded (FFPE) sections prepared using various protocols (cf. text/figures) and two whole-brain sections (Figure 1 – FFPE, from the Jülich BigBrain, 20μm, silver-stained, and Figure 2E - celloidin-embedded and myelin-stained in 1904, from the Institute for Brain Research, Düsseldorf, Germany).

Computational scattered light imaging (ComSLI) was performed in Stanford and Jülich. The setup (Figure 1A,B) includes a micron-resolution low angle-of-acceptance camera-adapter-lens system and a rotating LED lightsource. Images were acquired at 5-15o rotation steps (24-72 images/sample), with 3-9μm pixel size. Motorized stages enable tile-scanning. SLIX software quantified orientations, MATLAB was used for orientation analysis, and MRtrix3 for creating orientation distribution functions and subsequent tractography.

Result
ComSLI produced a micron-resolution whole-brain fiber orientation map (Figure 1C). Figures 1D-E show zoomed-in fiber orientations in corpus callosum/fornix and corona radiata. Microscopic resolutions enabled generating fiber orientation distributions at multiple scales (Figure 1F), leading to microstructure-informed whole-brain tracts (Figure 1G-H).

ComSLI works for various sample preparation protocols (Figure 2). Consecutive human hippocampal sections with different stains (iron, microglia, tau, and amyloid) show identical orientations (Figure 2B,C), quantified after co-registration in Figure 2D. Orientations were also derived from a 120-year-old human section (Figure 2E-F), and were consistent at various sample preparation steps (Figure 2G).

ComSLI can study neurodegenerating tracts, such as the hippocampal perforant pathway (Figure 3). A healthy hippocampus includes strong perforant pathway connections through the subiculum and CA1 subfields (Figure 3A-F), which almost entirely disappear in a sclerotic hippocampus (Figure 3G-L), and are severely compromised in Alzheimer's disease (Figure 3M-R).

Conclusion
ComSLI is a cost-effective method to study intricate fiber networks at micron-resolution in any histological tissue section, and can reveal subtle changes in neurodegeneration.

We present a method for direct imaging of nerve fiber orientations in cell-body stained histological brain sections, which was not yet possible for paraffin-treated tissue.

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 originating 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 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 we then apply the method in mouse, pig, vervet monkey, and human brains. We compare results to polarized light imaging (3D-PLI), tracer experiments, and to outputs from diffusion MRI that sometimes fails to detect crossings. Given its specificity, capability of 3-dimensional sampling and high resolution, SAXS could serve as a ground truth for validating fiber orientations derived using diffusion MRI as well as microscopy-based methods. Statement of significance: To study how the nerve fibers in our brain are interconnected, scientists need to visualize their trajectories, which often cross one another. Here, we show the unique capacity of small-angle X-ray scattering (SAXS) to study these fiber crossings without use of labeling, taking advantage of SAXS's specificity to myelin - the insulating sheath that is wrapped around nerve fibers. We use SAXS to detect double and triple crossing fibers and unveil intricate crossings in mouse, pig, vervet monkey, and human brains. This non-destructive method can uncover complex fiber trajectories and validate other less specific imaging methods (e.g., MRI or microscopy), towards accurate mapping of neuronal connectivity in the animal and human brain.

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, respectivelyHere, 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.