Galectin-3 alters the lateral mobility and clustering of beta 1-integrin receptors

Glycoprotein receptors are influenced by myriad intermolecular interactions at the cell surface. Specific glycan structures may interact with endogenous lectins that enforce or disrupt receptor-receptor interactions. Glycoproteins bound by multivalent lectins may form extended oligomers or lattices, altering the lateral mobility of the receptor and influencing its function through endocytosis or changes in activation. In this study, we have examined the interaction of Galectin-3 (Gal-3), a human lectin, with adhesion receptors. We measured the effect of recombinant Gal-3 added exogenously on the lateral mobility of the alpha 5 beta 1 integrin on HeLa cells. Using single-particle tracking (SPT) we detected increased lateral mobility of the integrin in the presence of Gal-3, while its truncated C-terminal domain (Gal-3C) showed only minor reductions in lateral mobility. Treatment of cells with Gal-3 increased beta 1-integrin mediated migration with no apparent changes in viability. In contrast, Gal-3C decreased both cell migration and viability. Fluorescence microscopy allowed us to confirm that exogenous Gal-3 resulted in reorganization of the integrin into larger clusters. We used a proteomics analysis to confirm that cells expressed endogenous Gal-3, and found that addition of competitive oligosaccharide ligands for the lectin altered the lateral mobility of the integrin. Together, our results are consistent with a Gal-3-integrin lattice model of binding and confirm that the lateral mobility of integrins is natively regulated, in part, by galectins

Advancing the Characterization of Neuronal Cyto-Architecture by X-ray Phase-Contrast Tomography

Softcover, 17x24To bring physiology and pathology of the human brain into better micro-anatomical and histological context, studies with different methodologies are required. Established techniques such as electron microscopy or histology show limitations in view of invasiveness, labor-intense and artifact-prone sample preparation, as well as an adequate ratio between resolution and volume throughput. For this reason, X-ray phase-contrast tomography (PC-CT) has been proposed as a three-dimensional non-destructive imaging technique, which requires less effort in sample preparation and can assess larger volumes. Furthermore, it offers quantitative electron density based contrast even for unstained tissue. Up to now, however, PC-CT studies fell short in number of samples, so that structural alterations caused by neurodegenerative diseases cannot be distinguished from physiological inter-subject variations. In this thesis, the scalability of PC-CT with respect to the required number of samples and resolution-to-volume-throughput is demonstrated, and the methodology is advanced with respect to data acquisition, processing and segmentation. In addition to the human cerebellum, cortex and hippocampus are studied. Concerning quantification and analysis of PC-CT data, this work introduces optimal transport analysis to obtain quantitative metrics of the cyto-architecture and to identify changes due to neurodegenerative diseases. For the case of Alzheimer’s disease, this workflow reveals a yet undescribed compactification of granular cells in the human hippocampus. This thesis also provides optimized configurations to study neural tissues with laboratory instrumentation, and – finally – provides new correlative imaging approaches, in particular with scanning electron microscopy

Towards correlative imaging of neuronal tissue by phase-contrast x-ray tomography and SEM

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)

Evaluation of different heavy-metal stains and embedding media for phase contrast tomography of neuronal tissue

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)

Elemental quantification and analysis of structural abnormalities in neurons from Parkinson’s-diseased brains by X-ray fluorescence microscopy and diffraction

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3d virtual histology reveals pathological alterations of cerebellar granule cells in multiple sclerosis

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

Structure of the myenteric plexus in normal and diseased human ileum analyzed by X-ray virtual histology slices

BACKGROUND The 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. AIM To examine human ileum full-thickness biopsies using X-ray phase-contrast nanotomography without prior staining to visualize the ENS. METHODS Six 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. RESULTS In 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. CONCLUSION The 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

Structure of the myenteric plexus in normal and diseased human ileum analyzed by X-ray virtual histology slices

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

3d Virtual Histology Reveals Pathological Alterations of Cerebellar Granule Cells in Multiple Sclerosis

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

Nanoscale x-ray holotomography of human brain tissue with phase retrieval based on multienergy recordings

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