  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.
