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, 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.Comment: 30 pages, 16 figure

Light Scattering Measurements Enable an Improved Reconstruction of Nerve Fiber Crossings

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

The hippocampus of birds in a view of evolutionary connectomics

The avian brain displays a different brain architecture compared to mammals. This has ledthe first pioneers of comparative neuroanatomy to wrong conclusions about bird brainevolution by assuming that the avian telencephalon is a hypertrophied striatum. Based ongrowing evidence from divers analysis demonstrating that most of the avian forebrain ispallial in nature, this view has substantially changed during the past decades. Further, birdsshow cognitive abilities comparable to or even exceeding those of some mammals, evenwithout a “six-layered” cortex. Beside higher associative regions, most of these cognitivefunctions include the processing of information in the hippocampal formation (HF) thatshares a homologue structure in birds and mammals. Here we show with 3D polarized lightimaging (3D-PLI) that the HF of pigeons like the mammalian HF shows regional specializationsalong the anterioreposterior axis in connectivity. In addition, different levels of adultneurogenesis were observed in the subdivisions of the HF per se and in the most caudalregions pointing towards a functional specialization along the anterioreposterior axis.Taken together our results point to species specific morphologies but still conservedhippocampal characteristics of connectivity, cells and adult neurogenesis if compared to themammalian situation. Here our data provides new aspects for the ongoing discussion onhippocampal evolution and mind

ELASTIC REGISTRATION OF HIGH-RESOLUTION 3D PLI DATA OF THE HUMAN BRAIN

We introduce a new approach for the elastic registration ofhigh-resolution 3D polarized light imaging (3D PLI) data ofhistological sections of the human brain. For accurate regis-tration of different types of 3D PLI modalities, we proposea novel intensity similarity measure that is based on a least-squares formulation of normalized cross-correlation. More-over, we present a fully automatic registration pipeline forrigid and elastic registration of high-resolution 3D PLI imageswith a blockface reference including a preprocessing step. Wehave successfully evaluated our approach using manually ob-tained ground truth for five sections of a human brain and ex-perimentally compared it with previous approaches. We alsopresent experimental results for 60 brain sections

Three-dimensional reconstruction of histological blockface images using ID-encoded markers

Introduction:The alignment of serial histological sections of the human brain into an anatomically realistic space requires prior elimination of various distortions, which are inevitably introduced during tissue processing, e.g. cutting and mounting [2]. Blockface images provide largely undistorted images of brain sections. Thus the aligned volume of blockface images represents an important reference to recover the spatial coherence of the non-linearly deformed histological sections. We introduce a robust and efficient method for an automatic 3D reconstruction of blockface images taken from the brain during sectioning (Fig.1).Materials and Methods:The method is based on the use of ID-encoded markers, which have been established in the fields of augmented reality and computer vision to trace camera positions and orientations in real-time. Each marker represents an individual identifying number and encodes a 12-bit number in a 6x6 array of black and white pixels (Fig.1). This pattern guarantees a robust code identification by a “majority vote”, which is important in an environment, where the markers are likely to be partially covered, e.g. by ice crystals or sectioning residues. The detection of the markers was realized by implementation of the ARToolkitPlus library [3]. The basic idea is to extract the coordinates of the same markers in different blockface images and to align them to each other by means of affine transformations. Using only marker matching in the background causes perspective errors in the brain tissue, since the sectioning plane of the brain and the background containing the marker patterns have decisively different distances to the camera lens. Therefore, the median along the depth of the marker-based registered volume was calculated to eliminate outliers. In a next step, the marker-based registered images were aligned slice by slice onto the median volume using a translation transform estimated by the pixel-based image registration algorithm provided by the ITK library [4]. This procedure was applied to different types of blockface images acquired from both paraffin embedded and frozen brains in different types of microtomes with various camera setups and several marker patterns. The example shown here was taken from a formalin-fixed, left human temporal lobe sectioned in a large-scale cryostat microtome, which was further processed for 3D-PLI [1].Results:While the robustness of the marker detection algorithm showed strong dependencies on the marker size and the distance between the markers in relation to the camera resolution, the robustness of the estimation of affine transformations between two sets of coordinates depended on the amount of markers, which is limited by the camera's field of view. Best results were achieved with a marker size of 4mm, a gap of 2mm and a number of about 750 markers. Using this setup, the algorithm detected 95% of all visible markers on average. This step provided pixel-precise results with respect to the marker alignment. In case of using small-scale microtomes for small tissue samples from rodent or monkey brains, for instance, the perspective errors in the cutting plane were most significant due to the close distance to the camera lens. An elimination of perspective outliers in the cutting plane using an alignment to the median of the marker-based aligned images provides an accuracy of less than 4 pixels. Since the algorithm was parallelized, the processing time of 1000 sections could be reduced to one hour. Fig.2 shows the quality of the finally generated blockface volume of the human temporal lobe in reconstructed sagittal and transversal views

Resolving nerve fiber crossings at micrometer resolution in rat, vervet monkey, and human brain samples

Unstained histological sections obtained from a Wistar rat brain, two vervet monkey brains, and a human optic chiasm were measured with Scattered Light Imaging (SLI) to study nerve fiber crossings at micrometer resolution, e.g. in the corona radiata of the vervet monkey brain. The data provided here contains the original image stacks obtained from the SLI measurements as well as generated parameter maps showing different characteristics of the measured brain sections, e.g. the individual (in-plane) orientations for up to three (crossing) nerve fiber bundles for each measured image pixel (with a resolution down to 6.5 μm). For some of the measured brain sections, we also provide parameter maps generated from 3D-Polarized Light Imaging (3D-PLI) measurements for better comparison

The three-dimensional structure of VIM-31--a metallo-β-lactamase from Enterobacter cloacae in its native and oxidized form.

The metallo-β-lactamase VIM-31 differs from VIM-2 by only two Tyr224His and His252Arg substitutions. Located close to the active site, the Tyr224His substitution is also present in VIM-1, VIM-4, VIM-7 and VIM-12. The VIM-31 variant was reported in 2012 from Enterobacter cloacae and kinetically characterized. It exhibits globally lower catalytic efficiencies than VIM-2. In the present study, we report the three-dimensional structures of VIM-31 in its native (reduced) and oxidized forms. The so-called 'flapping-loop' (loop 1) and loop 3 of VIM-31 were not positioned as in VIM-2 but instead were closer to the active site as in VIM-4, resulting in a narrower active site in VIM-31. Also, the presence of His224 in VIM-31 disrupts hydrogen-bonding networks close to the active site. Moreover, a third zinc-binding site, which also exists in VIM-2 structures, could be identified as a structural explanation for the decreased activity of VIM-MBLs at high zinc concentrations