Scattered Light Imaging (SLI) is a novel approach for microscopically revealing the fibre architecture of unstained brain sections. The measurements are obtained by illuminating brain sections from different angles and measuring the transmitted (scattered) light under normal incidence. The evaluation of scattering profiles commonly relies on a peak picking technique and feature extraction from the peaks, which allows quantitative determination of parallel and crossing in-plane nerve fibre directions for each image pixel. However, the estimation of the 3D orientation of the fibres cannot be assessed with the traditional methodology. We propose an unsupervised learning approach using spherical convolutions for estimating the 3D orientation of neural fibres, resulting in a more detailed interpretation of the fibre orientation distributions in the brain.@en

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.@en

Purpose: The technique 3D polarized light imaging (3D-PLI) allows to reconstruct nerve fiber orientations of postmortem brains with ultra-high resolution. To better understand the physical principles behind 3D-PLI and improve the accuracy and reliability of the reconstructed fiber orientations, numerical simulations are employed which use synthetic nerve fiber models as input. As the generation of fiber models can be challenging and very time-consuming, we have developed the open source FAConstructor tool which enables a fast and efficient generation of synthetic fiber models for 3D-PLI simulations. Methods : The program was developed as an interactive tool, allowing the user to define fiber pathways with interpolation methods or parametric functions and providing visual feedback. Results : Performance tests showed that most processes scale almost linearly with the amount of fiber points in FAConstructor. Fiber models consisting of < 1.6 million data points retain a frame rate of more than 30 frames per second, which guarantees a stable and fluent workflow. The applicability of FAConstructor was demonstrated on a well-defined fiber model (Fiber Cup phantom) for two different simulation approaches, reproducing effects known from 3D-PLI measurements. Conclusion : We have implemented a user-friendly and efficient tool that enables an interactive and fast generation of synthetic nerve fiber configurations for 3D-PLI simulations. Already existing fiber models can easily be modified, allowing to simulate many different fiber models in a reasonable amount of time.@en

3D Polarized Light Imaging (3D-PLI) is a neuroimaging technique that has opened up new avenues to study the complex architecture of nerve fibers in postmortem brains. The spatial orientations of the fibers are derived from birefringence measurements of unstained histological brain sections that are interpreted by a voxel-based analysis. This, however, implies that a single fiber orientation vector is obtained for each voxel and reflects the net effect of all comprised fibers. The mixture of various fiber orientations within an individual voxel is a priori not accessible by a standard 3D-PLI measurement. In order to better understand the effects of fiber mixture on the measured 3D-PLI signal and to improve the interpretation of real data, we have developed a simulation method referred to as SimPLI. By means of SimPLI, it is possible to reproduce the entire 3D-PLI analysis starting from synthetic fiber models in user-defined arrangements and ending with measurement-like tissue images. For the simulation, each synthetic fiber is considered as an optical retarder, i.e., multiple fibers within one voxel are described by multiple retarder elements. The investigation of different synthetic crossing fiber arrangements generated with SimPLI demonstrated that the derived fiber orientations are strongly influenced by the relative mixture of crossing fibers. In case of perpendicularly crossing fibers, for example, the derived fiber direction corresponds to the predominant fiber direction. The derived fiber inclination turned out to be not only influenced by myelin density but also systematically overestimated due to signal attenuation. Similar observations were made for synthetic models of optic chiasms of a human and a hooded seal which were opposed to experimental 3D-PLI data sets obtained from the chiasms of both species. Our study showed that SimPLI is a powerful method able to test hypotheses on the underlying fiber structure of brain tissue and, therefore, to improve the reliability of the extraction of nerve fiber orientations with 3D-PLI.@en

The correct reconstruction of individual (crossing) nerve fibers is a prerequisite when constructing a detailed network model of the brain. The recently developed technique Scattered Light Imaging (SLI) allows the reconstruction of crossing nerve fiber pathways in whole brain tissue samples with micrometer resolution: the individual fiber orientations are determined by illuminating unstained histological brain sections from different directions, measuring the transmitted scattered light under normal incidence, and studying the light intensity profiles of each pixel in the resulting image series. So far, SLI measurements were performed with a fixed polar angle of illumination and a small number of illumination directions, providing only an estimate of the nerve fiber directions and limited information about the underlying tissue structure. Here, we use a display with individually controllable light-emitting diodes to measure the full distribution of scattered light behind the sample (scattering pattern) for each image pixel at once, enabling scatterometry measurements of whole brain tissue samples. We compare our results to coherent Fourier scatterometry (raster-scanning the sample with a non-focused laser beam) and previous SLI measurements with fixed polar angle of illumination, using sections from a vervet monkey brain and human optic tracts. Finally, we present SLI scatterometry measurements of a human brain section with 3 μm in-plane resolution, demonstrating that the technique is a powerful approach to gain new insights into the nerve fiber architecture of the human brain.@en

We propose the application of Compressed Sensing to Computational Scattered Light Imaging to decrease measurement time and data storage. Computational Scattered Light Imaging (ComSLI) determines three-dimensional fiber orientations and crossings in biomedical tissues like brain tissue. Currently, conventional ComSLI is time-consuming and generates large data. Compressed Sensing reconstructs signals with fewer samples than required by the Shannon-Nyquist theorem with minimal perceptual loss, significantly reducing the number of measurements. We introduce an optimized illumination strategy for ComSLI based on the Discrete Cosine Transform and validate it by reconstructing characteristic scattering patterns in vervet brain tissue, thereby demonstrating the feasibility of Compressed Sensing in ComSLI.@en

The neuroimaging technique three-dimensional polarized light imaging (3D-PLI) provides a high-resolution reconstruction of nerve fibres in human post-mortem brains. The orientations of the fibres are derived from birefringence measurements of histological brain sections assuming that the nerve fibres-consisting of an axon and a surrounding myelin sheath-are uniaxial birefringent and that the measured optic axis is oriented in the direction of the nerve fibres (macroscopic model). Although experimental studies support this assumption, the molecular structure of the myelin sheath suggests that the birefringence of a nerve fibre can be described more precisely by multiple optic axes oriented radially around the fibre axis (microscopic model). In this paper, we compare the use of the macroscopic and the microscopic mode1for simulating 3D-PLI by means of the Jones matrix formalism. The simulations show that the macroscopic mode1ensures a reliable estimation of the fibre orientations as long as the polarimeter does not resolve structures smaller than the diameter of single fibres. In the case of fibre bundles, polarimeters with even higher resolutions can be used without losing reliability. When taking the myelin density into account, the derived fibre orientations are considerably improved.@en

3D-polarized light imaging (3D-PLI) reconstructs nerve fibers in histological brain sections by measuring their birefringence. This study investigates another effect caused by the optical anisotropy of brain tissue – diattenuation. Based on numerical and experimental studies and a complete analytical description of the optical system, the diattenuation was determined to be below 4 % in rat brain tissue. It was demonstrated that the diattenuation effect has negligible impact on the fiber orientations derived by 3D-PLI. The diattenuation signal, however, was found to highlight different anatomical structures that cannot be distinguished with current imaging techniques, which makes Diattenuation Imaging a promising extension to 3D-PLI.@en

The method 3D polarised light imaging (3D-PLI) measures the birefringence of histological brain sections to determine the spatial course of nerve fibres (myelinated axons). While the in-plane fibre directions can be determined with high accuracy, the computation of the out-of-plane fibre inclinations is more challenging because they are derived from the amplitude of the birefringence signals, which depends e.g. on the amount of nerve fibres. One possibility to improve the accuracy is to consider the average transmitted light intensity (transmittance weighting). The current procedure requires effortful manual adjustment of parameters and anatomical knowledge. Here, we introduce an automated, optimised computation of the fibre inclinations, allowing for a much faster, reproducible determination of fibre orientations in 3D-PLI. Depending on the degree of myelination, the algorithm uses different models (transmittance-weighted, unweighted, or a linear combination), allowing to account for regionally specific behaviour. As the algorithm is parallelised and GPU optimised, it can be applied to large data sets. Moreover, it only uses images from standard 3D-PLI measurements without tilting, and can therefore be applied to existing data sets from previous measurements. The functionality is demonstrated on unstained coronal and sagittal histological sections of vervet monkey and rat brains.@en

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.@en

Unraveling the structure and function of the brain requires a detailed knowledge about the neuronal connections, i.e., the spatial architecture of the nerve fibers. One of the most powerful histological methods to reconstruct the three-dimensional nerve fiber pathways is 3D-polarized light imaging (3D-PLI). The technique measures the birefringence of histological brain sections and derives the spatial fiber orientations of whole human brain sections with micrometer resolution. However, the technique yields only a single fiber orientation for each measured tissue voxel even if it is composed of fibers with different orientations, so that in-plane crossing fibers are misinterpreted as out-of-plane fibers. When generating a detailed model of the three-dimensional nerve fiber architecture in the brain, a correct detection and interpretation of nerve fiber crossings is crucial. Here, we show how light scattering in transmission microscopy measurements can be leveraged to identify nerve fiber crossings in 3D-PLI data and demonstrate that measurements of the scattering pattern can resolve the substructure of brain tissue like the crossing angles of the nerve fibers. For this purpose, we develop a simulation framework that permits the study of transmission microscopy measurements - in particular, light scattering - on large-scale complex fiber structures like brain tissue, using finite-difference time-domain (FDTD) simulations and high-performance computing. The simulations are used not only to model and explain experimental observations, but also to develop new analysis methods and measurement techniques. We demonstrate in various experimental studies on brain sections from different species (rodent, monkey, and human) and in FDTD simulations that the polarization-independent transmitted light intensity (transmittance) decreases significantly (by more than 50%) with an increasing out-of-plane angle of the nerve fibers and that it is mostly independent of the in-plane crossing angle. Hence, the transmittance can be used to distinguish regions with low fiber density and in-plane crossing fibers from regions with out-of-plane fibers, solving a major problem in 3D-PLI and allowing for a much better reconstruction of the complex nerve fiber architecture in the brain. Finally, we show that light scattering (oblique illumination) in the visible spectrum reveals the underlying structure of brain tissue like the crossing angle of the nerve fibers with micrometer resolution, enabling an even more detailed reconstruction of nerve fiber crossings in the brain and opening up new fields of research.@en

When transmitting polarised light through histological brain sections, different types of diattenuation (polarisation-dependent attenuation of light) can be observed: In some brain regions, the light is minimally attenuated when it is polarised parallel to the nerve fibres (referred to as D+), in others, it is maximally attenuated (referred to as D−). The underlying mechanisms of these effects and their relationship to tissue properties were so far unknown. Here, we demonstrate in experimental studies that diattenuation of both types D+ and D− can be observed in brain tissue samples from different species (rodent, monkey, and human) and that the strength and type of diattenuation depend on the nerve fibre orientations. By combining finite-difference time-domain simulations and analytical modelling, we explain the observed diattenuation effects and show that they are caused both by anisotropic absorption (dichroism) and by anisotropic light scattering. Our studies demonstrate that the diattenuation signal depends not only on the nerve fibre orientations but also on other brain tissue properties like tissue homogeneity, fibre size, and myelin sheath thickness. This allows to use the diattenuation signal to distinguish between brain regions with different tissue properties and establishes Diattenuation Imaging as a valuable imaging technique.@en

We show that light scattering measurements of brain tissue reveal valuable information about the underlying tissue structure such as the crossing angle of the nerve fibers.@en

We present SLI Scatterometry: Scattered Light Imaging performed with individually controllable LEDs, allowing the measurement of full scattering patterns for each image pixel in a brain tissue sample, revealing complex nerve fiber architectures.@en

We improve the determination of nerve fiber orientations in brain tissue sections that have been measured with Computational Scattered Light Imaging by close examination of low intensity signals with iterative thresholding.@en

We improve the determination of nerve fiber orientations in brain tissue sections that have been measured with Computational Scattered Light Imaging by close examination of low intensity signals with iterative thresholding.@en

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.@en

3D Polarized Light Imaging is a neuroimaging technique that provides a high-resolution reconstruction of nerve fiber pathways in human postmortem brains. The spatial fiber orientations are derived from birefringence measurements of histological brain sections which are interpreted by a macroscopic model of uniaxial birefringence. In order to validate the macroscopic model and to investigate possible limitations, numerical simulations have been used. Simulations of a fiber bundle with different inclination angles and optical resolutions have shown that the macroscopic model ensures a reliable estimation of the fiber orientations as long as the polarimeter does not resolve structures smaller than the fiber radius.@en

In this work, we employ an integrated label-free dual approach that combines 3DPolarized light imaging with two-photon fluorescence microscopy to study the mixture of various fiber orientations within the sample of interest.@en

We explore the polarization-(in)dependent transmitted light intensity of histological brain sections. Using experimental and simulation studies, we demonstrate that it contains valuable information about nerve fiber architecture and tissue structure.@en