Uncovering microstructural architecture from histology.

Microstructural tissue organization underlies the complex connectivity of the brain and controls properties of connective, muscle, and epithelial tissue. However, discerning microstructural architecture with high resolution for large fields of view remains prohibitive. We address this challenge with computational scattered light imaging (ComSLI), which exploits the anisotropic light scattering of aligned structures. Using a rotating lightsource and a high-resolution camera, ComSLI determines fiber architecture with micrometer resolution from histological sections across preparation and staining protocols. We show complex fiber architecture in brain and non-brain sections, including histological paraffin-embedded sections with various stains, and demonstrate its applicability on animal and human tissue, including disease cases with altered microstructure. ComSLI opens new avenues for investigating fiber architecture in new and archived sections across organisms, tissues, and diseases.

Evaluation of surface-based hippocampal registration using ground-truth subfield definitions.

The hippocampus is an archicortical structure, consisting of subfields with unique circuits. Understanding its microstructure, as proxied by these subfields, can improve our mechanistic understanding of learning and memory and has clinical potential for several neurological disorders. One prominent issue is how to parcellate, register, or retrieve homologous points between two hippocampi with grossly different morphologies. Here, we present a surface-based registration method that solves this issue in a contrast-agnostic, topology-preserving manner. Specifically, the entire hippocampus is first analytically unfolded, and then samples are registered in 2D unfolded space based on thickness, curvature, and gyrification. We demonstrate this method in seven 3D histology samples and show superior alignment with respect to subfields using this method over more conventional registration approaches.

A direct fiber approach to model sclera collagen architecture and biomechanics.

Sclera collagen fiber microstructure and mechanical behavior are central to eye physiology and pathology. They are also complex, and are therefore often studied using modeling. Most models of sclera, however, have been built within a conventional continuum framework. In this framework, collagen fibers are incorporated as statistical distributions of fiber characteristics such as the orientation of a family of fibers. The conventional continuum approach, while proven successful for describing the macroscale behavior of the sclera, does not account for the sclera fibers are long, interwoven and interact with one another. Hence, by not considering these potentially crucial characteristics, the conventional approach has only a limited ability to capture and describe sclera structure and mechanics at smaller, fiber-level, scales. Recent advances in the tools for characterizing sclera microarchitecture and mechanics bring to the forefront the need to develop more advanced modeling techniques that can incorporate and take advantage of the newly available highly detailed information. Our goal was to create a new computational modeling approach that can represent the sclera fibrous microstructure more accurately than with the conventional continuum approach, while still capturing its macroscale behavior. In this manuscript we introduce the new modeling approach, that we call direct fiber modeling, in which the collagen architecture is built explicitly by long, continuous, interwoven fibers. The fibers are embedded in a continuum matrix representing the non-fibrous tissue components. We demonstrate the approach by doing direct fiber modeling of a rectangular patch of posterior sclera. The model integrated fiber orientations obtained by polarized light microscopy from coronal and sagittal cryosections of pig and sheep. The fibers were modeled using a Mooney-Rivlin model, and the matrix using a Neo-Hookean model. The fiber parameters were determined by inversely matching experimental equi-biaxial tensile data from the literature. After reconstruction, the direct fiber model orientations agreed well with the microscopy data both in the coronal plane (adjusted R2 = 0.8234) and in the sagittal plane (adjusted R2 = 0.8495) of the sclera. With the estimated fiber properties (C10 = 5746.9 MPa; C01 = -5002.6 MPa, matrix shear modulus 200 kPa), the model's stress-strain curves simultaneously fit the experimental data in radial and circumferential directions (adjusted R2's 0.9971 and 0.9508, respectively). The estimated fiber elastic modulus at 2.16% strain was 5.45 GPa, in reasonable agreement with the literature. During stretch, the model exhibited stresses and strains at sub-fiber level, with interactions among individual fibers which are not accounted for by the conventional continuum methods. Our results demonstrate that direct fiber models can simultaneously describe the macroscale mechanics and microarchitecture of the sclera, and therefore that the approach can provide unique insight into tissue behavior questions inaccessible with continuum approaches.

Imaging crossing fibers in mouse, pig, monkey, and human brain using small-angle X-ray scattering.

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.

Fiber enhancement and 3D orientation analysis in label-free two-photon fluorescence microscopy.

Fluorescence microscopy can be exploited for evaluating the brain's fiber architecture with unsurpassed spatial resolution in combination with different tissue preparation and staining protocols. Differently from state-of-the-art polarimetry-based neuroimaging modalities, the quantification of fiber tract orientations from fluorescence microscopy volume images entails the application of specific image processing techniques, such as Fourier or structure tensor analysis. These, however, may lead to unreliable outcomes as they do not isolate myelinated fibers from the surrounding tissue. In this work, we describe a novel image processing pipeline that enables the computation of accurate 3D fiber orientation maps from both grey and white matter regions, exploiting the selective multiscale enhancement of tubular structures of varying diameters provided by a 3D implementation of the Frangi filter. The developed software tool can efficiently generate orientation distribution function maps at arbitrary spatial scales which may support the histological validation of modern diffusion-weighted magnetic resonance imaging tractography. Despite being tested here on two-photon scanning fluorescence microscopy images, acquired from tissue samples treated with a label-free technique enhancing the autofluorescence of myelinated fibers, the presented pipeline was developed to be employed on all types of 3D fluorescence images and fiber staining.

Scale matters: The nested human connectome.

A comprehensive description of how neurons and entire brain regions are interconnected is fundamental for a mechanistic understanding of brain function and dysfunction. Neuroimaging has shaped the way to approaching the human brain's connectivity on the basis of diffusion magnetic resonance imaging and tractography. At the same time, polarization, fluorescence, and electron microscopy became available, which pushed spatial resolution and sensitivity to the axonal or even to the synaptic level. New methods are mandatory to inform and constrain whole-brain tractography by regional, high-resolution connectivity data and local fiber geometry. Machine learning and simulation can provide predictions where experimental data are missing. Future interoperable atlases require new concepts, including high-resolution templates and directionality, to represent variants of tractography solutions and estimates of their accuracy.

Post mortem mapping of connectional anatomy for the validation of diffusion MRI.

Diffusion MRI (dMRI) is a unique tool for the study of brain circuitry, as it allows us to image both the macroscopic trajectories and the microstructural properties of axon bundles in vivo. The Human Connectome Project ushered in an era of impressive advances in dMRI acquisition and analysis. As a result of these efforts, the quality of dMRI data that could be acquired in vivo improved substantially, and large collections of such data became widely available. Despite this progress, the main limitation of dMRI remains: it does not image axons directly, but only provides indirect measurements based on the diffusion of water molecules. Thus, it must be validated by methods that allow direct visualization of axons but that can only be performed in post mortem brain tissue. In this review, we discuss methods for validating the various features of connectional anatomy that are extracted from dMRI, both at the macro-scale (trajectories of axon bundles), and at micro-scale (axonal orientations and other microstructural properties). We present a range of validation tools, including anatomic tracer studies, Klingler's dissection, myelin stains, label-free optical imaging techniques, and others. We provide an overview of the basic principles of each technique, its limitations, and what it has taught us so far about the accuracy of different dMRI acquisition and analysis approaches.

Automated computation of nerve fibre inclinations from 3D polarised light imaging measurements of brain tissue.

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.

Additional fiber orientations in the sagittal stratum-noise or anatomical fine structure?

The sagittal stratum is a prominent and macroscopically clearly visible white-matter structure within occipital and parietal lobes with a highly organized structure of parallel fibers running in rostro-caudal direction. Apart from the major tract running through, i.e., the optic radiation, the source and arrangement of other fibers within the sagittal stratum is only partially understood. Recent diffusion imaging studies in-vivo suggest additional minor fiber directions, perpendicular to the major rostro-caudal ones, but the spatial resolution does not allow to resolve them, and to unambiguously distinguish it from noise. Taking this previous evidence as motivation, the present study used 3D polarized light imaging (3D-PLI) for micrometer resolution analysis of nerve fibers in postmortem specimens of a vervet monkey brain. The analysis of coronal occipital and parietal sections revealed that the sagittal stratum consisted of an external and an internal layer, which are joined and crossed by fibers from the surrounding white matter and the tapetum. Fibers from different parietal and occipital regions entered the sagittal stratum in the dorsal, ventral or middle sector, as solid large bundles or as several small fiber aggregations. These patterns were remarkably similar to published results of tracer experiments in macaques. Taking this correspondence as external validation of 3D-PLI enabled translation to the human brain, where a similarly complex fiber architecture within the sagittal stratum could be exemplified in a human hemisphere in our study. We thus argue in favor of a dedicated fiber microstructure within the sagittal stratum as a correlate of the additional fiber directions typically seen in in-vivo diffusion imaging studies.

Scatterometry Measurements With Scattered Light Imaging Enable New Insights Into the Nerve Fiber Architecture of the Brain.

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.

Distribution and orientation of nerve fibers and myelin assembly in a brain section retrieved by small-angle neutron scattering.

The structural connectivity of the brain has been addressed by various imaging techniques such as diffusion weighted magnetic resonance imaging (DWMRI) or specific microscopic approaches based on histological staining or label-free using polarized light (e.g., three-dimensional Polarized Light Imaging (3D-PLI), Optical Coherence Tomography (OCT)). These methods are sensitive to different properties of the fiber enwrapping myelin sheaths i.e. the distribution of myelin basic protein (histology), the apparent diffusion coefficient of water molecules restricted in their movements by the myelin sheath (DWMRI), and the birefringence of the oriented myelin lipid bilayers (3D-PLI, OCT). We show that the orientation and distribution of nerve fibers as well as myelin in thin brain sections can be determined using scanning small angle neutron scattering (sSANS). Neutrons are scattered from the fiber assembly causing anisotropic diffuse small-angle scattering and Bragg peaks related to the highly ordered periodic myelin multilayer structure. The scattering anisotropy, intensity, and angular position of the Bragg peaks can be mapped across the entire brain section. This enables mapping of the fiber and myelin distribution and their orientation in a thin brain section, which was validated by 3D-PLI. The experiments became possible by optimizing the neutron beam collimation to highest flux and enhancing the myelin contrast by deuteration. This method is very sensitive to small microstructures of biological tissue and can directly extract information on the average fiber orientation and even myelin membrane thickness. The present results pave the way toward bio-imaging for detecting structural aberrations causing neurological diseases in future.

Autofluorescence enhancement for label-free imaging of myelinated fibers in mammalian brains.

Analyzing the structure of neuronal fibers with single axon resolution in large volumes is a challenge in connectomics. Different technologies try to address this goal; however, they are limited either by the ineffective labeling of the fibers or in the achievable resolution. The possibility of discriminating between different adjacent myelinated axons gives the opportunity of providing more information about the fiber composition and architecture within a specific area. Here, we propose MAGIC (Myelin Autofluorescence imaging by Glycerol Induced Contrast enhancement), a tissue preparation method to perform label-free fluorescence imaging of myelinated fibers that is user friendly and easy to handle. We exploit the high axial and radial resolution of two-photon fluorescence microscopy (TPFM) optical sectioning to decipher the mixture of various fiber orientations within the sample of interest. We demonstrate its broad applicability by performing mesoscopic reconstruction at a sub-micron resolution of mouse, rat, monkey, and human brain samples and by quantifying the different fiber organization in control and Reeler mouse's hippocampal sections. Our study provides a novel method for 3D label-free imaging of nerve fibers in fixed samples at high resolution, below micrometer level, that overcomes the limitation related to the myelinated axons exogenous labeling, improving the possibility of analyzing brain connectivity.

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

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.

A cortex-like canonical circuit in the avian forebrain.

Although the avian pallium seems to lack an organization akin to that of the cerebral cortex, birds exhibit extraordinary cognitive skills that are comparable to those of mammals. We analyzed the fiber architecture of the avian pallium with three-dimensional polarized light imaging and subsequently reconstructed local and associative pallial circuits with tracing techniques. We discovered an iteratively repeated, column-like neuronal circuitry across the layer-like nuclear boundaries of the hyperpallium and the sensory dorsal ventricular ridge. These circuits are connected to neighboring columns and, via tangential layer-like connections, to higher associative and motor areas. Our findings indicate that this avian canonical circuitry is similar to its mammalian counterpart and might constitute the structural basis of neuronal computation.

Anatomy of nerve fiber bundles at micrometer-resolution in the vervet monkey visual system.

Although the primate visual system has been extensively studied, detailed spatial organization of white matter fiber tracts carrying visual information between areas has not been fully established. This is mainly due to the large gap between tracer studies and diffusion-weighted MRI studies, which focus on specific axonal connections and macroscale organization of fiber tracts, respectively. Here we used 3D polarization light imaging (3D-PLI), which enables direct visualization of fiber tracts at micrometer resolution, to identify and visualize fiber tracts of the visual system, such as stratum sagittale, inferior longitudinal fascicle, vertical occipital fascicle, tapetum and dorsal occipital bundle in vervet monkey brains. Moreover, 3D-PLI data provide detailed information on cortical projections of these tracts, distinction between neighboring tracts, and novel short-range pathways. This work provides essential information for interpretation of functional and diffusion-weighted MRI data, as well as revision of wiring diagrams based upon observations in the vervet visual system.

Analytical and fast Fiber Orientation Distribution reconstruction in 3D-Polarized Light Imaging.

Three dimensional Polarized Light Imaging (3D-PLI) is an optical technique which allows mapping the spatial fiber architecture of fibrous postmortem tissues, at sub-millimeter resolutions. Here, we propose an analytical and fast approach to compute the fiber orientation distribution (FOD) from high-resolution vector data provided by 3D-PLI. The FOD is modeled as a sum of K orientations/Diracs on the unit sphere, described on a spherical harmonics basis and analytically computed using the spherical Fourier transform. Experiments are performed on rich synthetic data which simulate the geometry of the neuronal fibers and on human brain data. Results indicate the analytical FOD is computationally efficient and very fast, and has high angular precision and angular resolution. Furthermore, investigations on the right occipital lobe illustrate that our strategy of FOD computation enables the bridging of spatial scales from microscopic 3D-PLI information to macro- or mesoscopic dimensions of diffusion Magnetic Resonance Imaging (MRI), while being a means to evaluate prospective resolution limits for diffusion MRI to reconstruct region-specific white matter tracts. These results demonstrate the interest and great potential of our analytical approach.

FAConstructor: an interactive tool for geometric modeling of nerve fiber architectures in the brain.

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.

Publisher Correction: Diattenuation Imaging reveals different brain tissue properties.

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