Abstract
For developing a detailed network model of the brain based on image reconstructions, it is necessary to spatially resolve crossing nerve fibers. The accuracy hereby depends on many factors, including the spatial resolution of the imaging technique. 3D Polarized Light Imaging (3D-PLI) allows the three-dimensional reconstruction of nerve fiber tracts in whole brain sections with micrometer in-plane resolution, but leaves uncertainties in pixels containing crossing fibers. Here we introduce Scattered Light Imaging (SLI) to resolve the substructure of nerve fiber crossings. The measurement is performed on the same unstained histological brain sections as in 3D-PLI. By illuminating the brain sections from different angles and measuring the transmitted (scattered) light under normal incidence, light intensity profiles are obtained that are characteristic for the underlying brain tissue structure. We have developed a fully automated evaluation of the intensity profiles, allowing the user to extract various characteristics, like the individual directions of in-plane crossing nerve fibers, for each image pixel at once. We validate the reconstructed nerve fiber directions against results from previous simulation studies, scatterometry measurements, and fiber directions obtained from 3D-PLI. We demonstrate in different brain samples (human optic tracts, vervet monkey brain, rat brain) that the 2D fiber directions can be reliably reconstructed for up to three crossing nerve fiber bundles in each image pixel with an in-plane resolution of up to 6.5 μm. We show that SLI also yields reliable fiber directions in brain regions with low 3D-PLI signals coming from regions with a low density of myelinated nerve fibers or out-of-plane fibers. This makes Scattered Light Imaging a promising new imaging technique, providing crucial information about the organization of crossing nerve fibers in the brain.
Highlights
The human brain consists of about 100 billion neurons [1]
We show that Scattered Light Imaging (SLI) yields reliable fiber directions in brain regions with low 3D Polarized Light Imaging (3D-PLI) signals coming from regions with a low density of myelinated nerve fibers or out-of-plane fibers
The course of crossing nerve fibers can be visually tracked in many regions due to a high contrast of the 3D-PLI images, individual nerve fiber orientations cannot be automatically extracted. 3D-PLI yields a single fiber orientation for each image pixel, which creates uncertainties in fiber orientation if the 60 μm thick brain section at this point is comprised of several crossing nerve fibers with different orientations
Summary
The human brain consists of about 100 billion neurons [1] To this day, neuroscientists are working on disentangling this gigantic, densely grown network of nerve fibers. Diffusion magnetic resonance imaging (dMRI) is the only possibility to map nerve fiber pathways in-vivo. Due to an insufficient knowledge about nerve fiber crossings, fiber tractography algorithms may yield false-positive nerve fiber pathways [6]. The microscopy technique 3D-Polarized Light Imaging (3D-PLI) represents a powerful optical approach to determine the three-dimensional course of nerve fibers in whole, unstained histological brain sections with micrometer in-plane resolution [7, 8]. 3D-PLI yields a single fiber orientation for each image pixel, which creates uncertainties in fiber orientation if the 60 μm thick brain section at this point is comprised of several crossing nerve fibers with different orientations. The resolution is limited by the available scan time and the measurements require special equipment (among others, a synchrotron)
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