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We investigate structural properties of neurons in the granular layer of human cerebellum with respect to their involvement in multiple sclerosis (MS). To this end we analyze data recorded by X-ray phase contrast tomography from tissue samples collected post mortem from a MS and a healthy control group. Using automated segmentation and histogram analysis based on optimal transport theory (OT) we find that the distributions representing nuclear structure in the granular layer move to a more compact nuclear state, i.e. smaller, denser and more heterogeneous nuclei in MS. We have previously made a similar observation for neurons of the dentate gyrus in Alzheimer’s disease, suggesting that more compact structure of neuronal nuclei which we attributed to increased levels of heterochromatin, may possibly represent a more general phenomenon of cellular senescence associated with neurodegeneration.

World journal of gastroenterology 28(29), 3994 - 4006 (2022). doi:10.3748/wjg.v28.i29.3994, BACKGROUNDThe enteric nervous system (ENS) is situated along the entire gastrointestinal tract and is divided into myenteric and submucosal plexuses in the small and large intestines. The ENS consists of neurons, glial cells, and nerves assembled into ganglia, surrounded by telocytes, interstitial cells of Cajal, and connective tissue. Owing to the complex spatial organization of several interconnections with nerve fascicles, the ENS is difficult to examine in conventional histological sections of 3-5 μm.AIMTo examine human ileum full-thickness biopsies using X-ray phase-contrast nanotomography without prior staining to visualize the ENS.METHODSSix patients were diagnosed with gastrointestinal dysmotility and neuropathy based on routine clinical and histopathological examinations. As controls, full-thickness biopsies were collected from healthy resection ileal regions after hemicolectomy for right colon malignancy. From the paraffin blocks, 4-µm thick sections were prepared and stained with hematoxylin and eosin for localization of the myenteric ganglia under a light microscope. A 1-mm punch biopsy (up to 1 cm in length) centered on the myenteric plexus was taken and placed into a Kapton® tube for mounting in the subsequent investigation. X-ray phase-contrast tomography was performed using two custom-designed laboratory setups with micrometer resolution for overview scanning. Subsequently, selected regions of interest were scanned at a synchrotron-based end-station, and high-resolution slices were reported. In total, more than 6000 virtual slices were analyzed from nine samples.RESULTSIn the overview scans, the general architecture and quality of the samples were studied, and the myenteric plexus was localized. High-resolution scans revealed details, including the ganglia, interganglional nerve fascicles, and surrounding tissue. The ganglia were irregular in shape and contained neurons and glial cells. Spindle-shaped cells with very thin cellular projections could be observed on the surface of the ganglia, which appeared to build a network. In the patients, there were no alterations in the general architecture of the myenteric ganglia. Nevertheless, several pathological changes were observed, including vacuolar degeneration, autophagic activity, the appearance of sequestosomes, chromatolysis, and apoptosis. Furthermore, possible expulsion of pyknotic neurons and defects in the covering cellular network could be observed in serial slices. These changes partly corresponded to previous light microscopy findings.CONCLUSIONThe analysis of serial virtual slices could provide new information that cannot be obtained by classical light microscopy. The advantages, disadvantages, and future possibilities of this method are also discussed., Published by WJG Press, Beijing

Proceedings of the National Academy of Sciences of the United States of America 119(12), e2109717119 (2022). doi:10.1073/pnas.2109717119, Neural mechanisms behind stereopsis, which requires simultaneous disparity inputs from two eyes, have remained mysterious. Here we show how ultrafast mirror-symmetric photomechanical contractions in the frontal forward-facing left and right eye photoreceptors give Drosophila superresolution three-dimensional (3D) vision. By interlinking multiscale in vivo assays with multiscale simulations, we reveal how these photoreceptor microsaccades—by verging, diverging, and narrowing the eyes’ overlapping receptive fields—channel depth information, as phasic binocular image motion disparity signals in time. We further show how peripherally, outside stereopsis, microsaccadic sampling tracks a flying fly’s optic flow field to better resolve the world in motion. These results change our understanding of how insect compound eyes work and suggest a general dynamic stereo-information sampling strategy for animals, robots, and sensors., Published by National Acad. of Sciences, Washington, DC

SignificanceTo move efficiently, animals must continuously work out their x,y,z positions with respect to real-world objects, and many animals have a pair of eyes to achieve this. How photoreceptors actively sample the eyes' optical image disparity is not understood because this fundamental information-limiting step has not been investigated in vivo over the eyes' whole sampling matrix. This integrative multiscale study will advance our current understanding of stereopsis from static image disparity comparison to a morphodynamic active sampling theory. It shows how photomechanical photoreceptor microsaccades enable

Background: Enteric neuropathy is described in most patients with gastrointestinal dysmotility and may be found together with reduced intraepidermal nerve fiber density (IENFD). The aim of this pilot study was to assess whether three-dimensional (3d) imaging of skin biopsies could be used to examine various tissue components in patients with gastrointestinal dysmotility. Material and methods: Four dysmotility patients of different etiology and two healthy volunteers were included. From each subject, two 3-mm punch skin biopsies were stained with antibodies against protein gene product 9.5 or evaluated as a whole with two X-ray phase-contrast computed tomography (CT) setups, a laboratory µCT setup and a dedicated synchrotron radiation nanoCT end-station. Results: Two patients had reduced IENFD, and two normal IENFD, compared with controls. µCT and X-ray phase-contrast holographic nanotomography scanned whole tissue specimens, with optional high-resolution scans revealing delicate structures, without differentiation of various fibers and cells. Irregular architecture of dermal fibers was observed in the patient with Ehlers-Danlos syndrome and the patient with idiopathic dysmotility showed an abundance of mesenchymal ground substance. Conclusions: 3d phase-contrast tomographic imaging may be useful to illustrate traits of connective tissue dysfunction in various organs and to demonstrate whether disorganized dermal fibers could explain organ dysfunction. (Less)

Purpose Modern neuroimaging lacks the tools necessary for whole-brain, anatomically dense neuronal damage screening. An ideal approach would include unbiased histopathologic identification of aging and neurodegenerative disease. Methods We report the postmortem application of multiscale X-ray phase-contrast computed tomography (X-PCI-CT) for the label-free and dissection-free organ-level to intracellular-level 3D visualization of distinct single neurons and glia. In deep neuronal populations in the brain of aged wild-type and of 3xTgAD mice (a triply-transgenic model of Alzheimer’s disease), we quantified intracellular hyperdensity, a manifestation of aging or neurodegeneration. Results In 3xTgAD mice, the observed hyperdensity was identified as amyloid-β and hyper-phosphorylated tau protein deposits with calcium and iron involvement, by correlating the X-PCI-CT data to immunohistochemistry, X-ray fluorescence microscopy, high-field MRI, and TEM. As a proof-of-concept, X-PCI-CT was used to analyze hippocampal and cortical brain regions of 3xTgAD mice treated with LY379268, selective agonist of group II metabotropic glutamate receptors (mGlu2/3 receptors). Chronic pharmacologic activation of mGlu2/3 receptors significantly reduced the hyperdensity particle load in the ventral cortical regions of 3xTgAD mice, suggesting a neuroprotective effect with locoregional efficacy. Conclusions This multiscale micro-to-nano 3D imaging method based on X-PCI-CT enabled identification and quantification of cellular and sub-cellular aging and neurodegeneration in deep neuronal and glial cell populations in a transgenic model of Alzheimer’s disease. This approach quantified the localized and intracellular neuroprotective effects of pharmacological activation of mGlu2/3 receptors.

The mammalian brain shows a complex and hierarchical architecture, whose assessment at all functionally relevant scales requires the establishment of multiomics approaches. In this work, we propose a correlative workflow, which is based on large-scale overview PC-CT scans using the extended beams offered by laboratory μCT sources and parallel beam synchrotron radiation (SR), with subsequent zooms into specific regions-of-interest using cone-beam recordings with nanofocused laboratory sources or SR, and finally SEM in controlled and wellidentified sub-volumes obtained before. We demonstrate the workflow at the example of rOTO-stained murine corpus callosum tissue, a brain region rich in myelinated nerve fibers. Based on two different and complementary techniques, PC-CT and scanning electron microscopy (SEM), we approach the establishment of a correlative imaging workflow. As we show here, the workflow can be applied (i) in a correlative study, in order to add further quantitative value, for instance, or (ii) in a multiscale approach, to which PC-CT can contribute volume throughput, while SEM can contribute resolution. The findings from this work demonstrate the complementary strength of each modality in terms of resolution (FIB-SEM) and FOV or volume throughput (PC-CT).

Neural mechanisms behind stereopsis, which requires simultaneous disparity inputs from two eyes, have remained mysterious. Here we show how ultrafast mirror-symmetric photomechanical contractions in the frontal forward-facing left and right eye photoreceptors giveDrosophilasuper-resolution 3D-vision. By interlinking multiscalein vivoassays with multiscale simulations, we reveal how these photoreceptor microsaccades - by verging, diverging and narrowing the eyes’ overlapping receptive fields - channel depth information, as phasic binocular image motion disparity signals in time. We further show how peripherally, outside stereopsis, microsaccadic sampling tracks a flying fly’s optic flow field to better resolve the world in motion. These results change our understanding of how insect compound eyes work and suggest a general dynamic stereo-information sampling strategy for animals, robots and sensors.Significance statementTo move efficiently, animals must continuously work out their x,y,z-positions in respect to real-world objects, and many animals have a pair of eyes to achieve this. How photoreceptors actively sample the eyes’ optical image disparity is not understood because this fundamental information-limiting step has not been investigatedin vivoover the eyes’ whole sampling matrix. This integrative multiscale study will advance our current understanding of stereopsis from static image disparity comparison to a new morphodynamic active sampling theory. It shows how photomechanical photoreceptor microsaccades enableDrosophilasuper-resolution 3D-vision and proposes neural computations for accurately predicting these flies’ depth-perception dynamics, limits, and visual behaviors.

Proceedings of the National Academy of Sciences of the United States of America 118(48), e2113835118 (2021). doi:10.1073/pnas.2113835118, We have studied the three-dimensional (3D) cytoarchitecture of the human hippocampus in neuropathologically healthy and Alzheimer’s disease (AD) individuals, based on phase-contrast X-ray computed tomography of postmortem human tissue punch biopsies. In view of recent findings suggesting a nuclear origin of AD, we target in particular the nuclear structure of the dentate gyrus (DG) granule cells. Tissue samples of 20 individuals were scanned and evaluated using a highly automated approach of measurement and analysis, combining multiscale recordings, optimized phase retrieval, segmentation by machine learning, representation of structural properties in a feature space, and classification based on the theory of optimal transport. Accordingly, we find that the prototypical transformation between a structure representing healthy granule cells and the pathological state involves a decrease in the volume of granule cell nuclei, as well as an increase in the electron density and its spatial heterogeneity. The latter can be explained by a higher ratio of heterochromatin to euchromatin. Similarly, many other structural properties can be derived from the data, reflecting both the natural polydispersity of the hippocampal cytoarchitecture between different individuals in the physiological context and the structural effects associated with AD pathology., Published by National Acad. of Sciences, Washington, DC

Biomedical optics express 12(12), 7582 - 7598 (2021). doi:10.1364/BOE.434885, In this work, we optimize the setups and experimental parameters of X-ray phase-contrast computed-tomography for the three-dimensional imaging of the cyto- and myeloarchitecture of cerebral cortex, including both human and murine tissue. We present examples for different optical configurations using state-of-the art synchrotron instruments for holographic tomography, as well as compact laboratory setups for phase-contrast tomography in the direct contrast (edge-enhancement) regime. Apart from unstained and paraffin-embedded tissue, we tested hydrated tissue, as well as heavy metal stained and resin-embedded tissue using two different protocols. Further, we show that the image quality achieved allows to assess the neuropathology of multiple sclerosis in a biopsy sample collected during surgery., Published by Optica, Washington, DC

In this work we use scanning X-ray microscopy to study the structure and elemental composition of neuromelanin-positive neurons in substantia nigra tissue of Parkinson patients (PD) and controls. A total of 53 neurons were analyzed with X-ray fluorescence (XRF) and diffraction using sub-µm-focused synchrotron radiation. A statistical evaluation identified copper as the most group-discriminating element and indicated that interindividual and intraindividual variations are of great relevance in tissue measurements of diseased patients and prevent from automated group clustering. XRF analyses of two Lewy bodies (LBs) highlight a heterogeneity in elemental distributions in these LBs, whereas an innovative X-ray diffraction-based method approach was used to reveal β-sheet-rich crystalline structures in LBs. Overall, sub-µm-focus X-ray microscopy highlighted the elemental heterogeneity in PD pathology.

We present a three-dimensional (3D) approach for virtual histology and histopathology based on multi-scale phase contrast x-ray tomography, and use this to investigate the parenchymal architecture of unstained lung tissue from patients who succumbed to Covid-19. Based on this first proof-of-concept study, we propose multi-scale phase contrast x-ray tomography as a tool to unravel the pathophysiology of Covid-19, extending conventional histology by a third dimension and allowing for full quantification of tissue remodeling. By combining parallel and cone beam geometry, autopsy samples with a maximum cross section of 8 mm are scanned and reconstructed at a resolution and image quality, which allows for the segmentation of individual cells. Using the zoom capability of the cone beam geometry, regions-of-interest are reconstructed with a minimum voxel size of 167 nm. We exemplify the capability of this approach by 3D visualization of diffuse alveolar damage (DAD) with its prominent hyaline membrane formation, by mapping the 3D distribution and density of lymphocytes infiltrating the tissue, and by providing histograms of characteristic distances from tissue interior to the closest air compartment. Open-Access-Publikationsfonds 2020 peerReviewed

X-ray cone-beam holotomography of unstained tissue from the human central nervous system reveals details down to subcellular length scales. This visualization of variations in the electron density of the sample is based on phase-contrast techniques using intensities formed by self-interference of the beam between object and detector. Phase retrieval inverts diffraction and overcomes the phase problem by constraints such as several measurements at different Fresnel numbers for a single projection. Therefore, the object-to-detector distance (defocus) can be varied. However, for cone-beam geometry, changing defocus changes magnification, which can be problematic in view of image processing and resolution. Alternatively, the photon energy can be altered (multi-E). Far from absorption edges, multi-E data yield the wavelength-independent electron density. We present the multi-E holotomography at the Göttingen Instrument for Nano-Imaging with X-Rays (GINIX) setup of the P10 beamline at Deutsches Elektronen-Synchrotron. The instrument is based on a combined optics of elliptical mirrors and an x-ray waveguide positioned in the focal plane for further coherence, spatial filtering, and high numerical aperture. Previous results showed the suitability of this instrument for nanoscale tomography of unstained brain tissue. We demonstrate that upon energy variation, the focal spot is stable enough for imaging. To this end, a double-crystal monochromator and automated alignment routines are required. Three tomograms of human brain tissue were recorded and jointly analyzed using phase retrieval based on the contrast transfer function formalism generalized to multiple photon energies. Variations of the electron density of the sample are successfully reconstructed.

Objectives: Light microscopical analysis in two dimensions, combined with immunohistochemistry, is presently the gold standard to describe the enteric nervous system (ENS). Our aim was to assess the usefulness of three-dimensional (3D) imaging by X-ray phase-contrast tomography in evaluating the ENS of the human bowel. Material and methods: Myenteric ganglia were identified in full-thickness biopsies of the ileum and colon by hematoxylin & eosin staining. A1-mm biopsy punch was taken from the paraffin blocks and placed into a Kapton® tube for subsequent tomographic investigation. The samples were scanned, without further preparation, using phase-contrast tomography at two different scales: overview scans (performed with laboratory setups), which allowed localization of the nervous tissue (∼1µm effective voxel size); and high-resolution scans (performed with a synchrotron endstation), which imaged localized regions of 320x320x320 µm3 (176 nm effective voxel size). Results: The contrast allowed us to follow the shape and the size changes of the ganglia, as well as to study their cellular components together with the cells and cellular projections of the periganglional space. Furthermore, it was possible to show the 3D network of the myenteric plexus and to quantify its volume within the samples. Conclusions: Phase-contrast X-ray tomography can be applied for volume analyses of the human ENS and to study tissue components in unstained paraffin-embedded tissue biopsies. This technique could potentially be used to study disease mechanisms, and to compare healthy and diseased tissues in clinical research.

X-ray cone-beam holo-tomography of unstained tissue from the human central nervous system reveals details down to sub-cellular length scales.1 This visualization of variations in the electron density of the sample is based on phase contrast techniques using intensities formed by self-interference of the beam between object and detector. Phase retrieval inverts diffraction and overcomes the phase problem by constraints such as several measurements at different Fresnel numbers for a single projection. Therefore, the object-to-detector distance (defocus) can be varied. However, for cone beam geometry, changing defocus changes magnification, which can be problematic in view of image processing and resolution. Alternatively, the photon energy can be altered (multi-E). Far from absorption edges, multi-E data yield the wavelength independent electron density. In this contribution we present multi-E holo-tomography at the GINIX setup of the P10 beamline at DESY. The instrument is based on a combined optics of elliptical mirrors and an x-ray waveguide positioned in the focal plane for further coherence, spatial Filtering and high numerical aperture.2 Previous results showed the suitability of this instrument for nanoscale tomography of unstained brain tissue.1 We demonstrate that upon energy variation, the focal spot is stable enough for imaging. To this end, a double crystal monochromator and automated alignment routines are required. Three tomograms of human brain tissue were recorded and jointly analyzed using phase retrieval based on the contrast transfer function formalism generalized to multiple photon energies. Variations of the electron density of the sample are successfully reconstructed.

Using an X-ray waveguide, the focal spot size and coherence properties of the NanoMAX Kirkpatrick–Baez mirror system have been measured., The focusing and coherence properties of the NanoMAX Kirkpatrick–Baez mirror system at the fourth-generation MAX IV synchrotron in Lund have been characterized. The direct measurement of nano-focused X-ray beams is possible by scanning of an X-ray waveguide, serving basically as an ultra-thin slit. In quasi-coherent operation, beam sizes of down to 56 nm (FWHM, horizontal direction) can be achieved. Comparing measured Airy-like fringe patterns with simulations, the degree of coherence |μ| has been quantified as a function of the secondary source aperture (SSA); the coherence is larger than 50% for SSA sizes below 11 µm at hard X-ray energies of 14 keV. For an SSA size of 5 µm, the degree of coherence has been determined to be 87%.

Developments in X-Ray Tomography XII, San Diego, United States, 11 Aug 2019 - 15 Aug 2019; Proceedings (2019). doi:10.1117/12.2528432, In the present work, we evaluate and compare the contrast and resolution obtained on different neuronal tissues with propagation-based x-ray phase contrast computed tomography (PB-CT). At our laboratory-based liquid metal-jet setup, we obtain overview datasets at sub-micron resolution of mm3 -sized volumes. In order to evaluate these parameters down to the sub-cellular level, we utilize the synchrotron endstation GINIX at P10, DESY. At this dedicated endstation1 developed and operated by our group, we utilize x-ray waveguide optics for highresolution cone-beam scans at strong geometrical magnification M. Exploiting this multi-scale approach, we investigate the image quality of cerebellum tissue treated by different heavy-metal stains. In addition, we study the electron density contrast in unstained tissues. Different embedding media are utilized depending on the stain, which also significantly affects contrast and image quality. With this work, we want to contribute to an optimized sample preparation to study the neuronal architecture of the brain tissue in greater detail in three dimensions (3d)., Published by VDE-Verl., Berlin

Purpose: Recently, progress has been achieved in implementing phase-contrast tomography of soft biological tissues at laboratory sources. This opens up opportunities for three-dimensional (3-D) histology based on x-ray computed tomography (μ- and nanoCT) in the direct vicinity of hospitals and biomedical research institutions. Combining advanced x-ray generation and detection techniques with phase reconstruction algorithms, 3-D histology can be obtained even of unstained tissue of the central nervous system, as shown, for example, for biopsies and autopsies of human cerebellum. Depending on the setup, i.e., source, detector, and geometric parameters, laboratory-based tomography can be implemented at very different sizes and length scales. We investigate the extent to which 3-D histology of neuronal tissue can exploit the cone-beam geometry at high magnification M using a nanofocus transmission x-ray tube (nanotube) with a 300 nm minimal spot size (Excillum), combined with a single-photon counting camera. Tightly approaching the source spot with the biopsy punch, we achieve high M≈101−102, high flux density, and exploit the superior efficiency of this detector technology. Approach: Different nanotube configurations such as spot size and flux, M, as well as exposure time, Fresnel number, and coherence are varied and selected in view of resolution, field of view, and/or phase-contrast requirements. Results: The data show that the information content for the cytoarchitecture is enhanced by the phase effect. Comparison of results to those obtained at a microfocus rotating-anode x-ray tomography setup with a high-resolution detector, i.e., in low-M geometry, reveals similar to slightly superior data quality for the nanotube setup. In addition to its compactness, reduced power consumption by a factor of 103, and shorter scan duration, the particular advantage of the nanotube setup also lies in its suitability for pixel detector technology, enabling an increased range of opportunities for applications in laboratory phase-contrast x-ray tomography. Conclusions: The phase retrieval scheme utilized mixes amplitude and phase contrast, with results being robust with respect to reconstruction parameters. Structural information content is comparable to slightly superior to previous results achieved with a microfocus rotating-anode setup but can be obtained in shorter scan time. Beyond advantages as compactness, lowered power consumption, and flexibility, the nanotube setup’s scalability in view of the progress in pixel detector technology is particularly beneficial. Further progress is thus likely to bring 3-D virtual histology to the performance in scan time and throughput required for clinical practice in neuropathology.