Scattered Light Imaging: Resolving the substructure of nerve fiber crossings in whole brain sections with micrometer resolution

TL;DR

This paper introduces Scattered Light Imaging (SLI), a technique that enhances the resolution of nerve fiber crossing substructures in brain sections, complementing existing 3D-PLI methods for detailed neural network modeling.

Contribution

SLI provides a novel, automated method to resolve multiple crossing nerve fibers at micrometer resolution, validated across various brain samples and compatible with 3D-PLI.

Findings

SLI reliably reconstructs up to three crossing fibers per pixel.

The technique works in regions with low 3D-PLI signals.

SLI complements 3D-PLI for full 3D nerve fiber mapping.

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, SLI provides information about the underlying nerve fiber structure. A fully automated evaluation of the resulting light intensity profiles has been developed, 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 $\mu$m. 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. In combination with 3D-PLI, the technique can be used for a full reconstruction of the three-dimensional nerve fiber architecture in the brain.