The Experts below are selected from a list of 360 Experts worldwide ranked by ideXlab platform
Wolfgang Baumeister - One of the best experts on this subject based on the ideXlab platform.
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The promise and the challenges of cryo-Electron Tomography.
FEBS letters, 2020Co-Authors: Martin Turk, Wolfgang BaumeisterAbstract:Structural biologists have traditionally approached cellular complexity in a reductionist manner in which the cellular molecular components are fractionated and purified before being studied individually. This 'divide and conquer' approach has been highly successful. However, awareness has grown in recent years that biological functions can rarely be attributed to individual macromolecules. Most cellular functions arise from their concerted action, and there is thus a need for methods enabling structural studies performed in situ, ideally in unperturbed cellular environments. Cryo-Electron Tomography (Cryo-ET) combines the power of 3D molecular-level imaging with the best structural preservation that is physically possible to achieve. Thus, it has a unique potential to reveal the supramolecular architecture or 'molecular sociology' of cells and to discover the unexpected. Here, we review state-of-the-art Cryo-ET workflows, provide examples of biological applications, and discuss what is needed to realize the full potential of Cryo-ET.
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reliable estimation of membrane curvature for cryo Electron Tomography
PLOS Computational Biology, 2020Co-Authors: Maria Salfer, Wolfgang Baumeister, Javier Collado, Ruben Fernandezbusnadiego, Antonio MartinezsanchezAbstract:Curvature is a fundamental morphological descriptor of cellular membranes. Cryo-Electron Tomography (cryo-ET) is particularly well-suited to visualize and analyze membrane morphology in a close-to-native state and molecular resolution. However, current curvature estimation methods cannot be applied directly to membrane segmentations in cryo-ET, as these methods cannot cope with some of the artifacts introduced during image acquisition and membrane segmentation, such as quantization noise and open borders. Here, we developed and implemented a Python package for membrane curvature estimation from tomogram segmentations, which we named PyCurv. From a membrane segmentation, a signed surface (triangle mesh) is first extracted. The triangle mesh is then represented by a graph, which facilitates finding neighboring triangles and the calculation of geodesic distances necessary for local curvature estimation. PyCurv estimates curvature based on tensor voting. Beside curvatures, this algorithm also provides robust estimations of surface normals and principal directions. We tested PyCurv and three well-established methods on benchmark surfaces and biological data. This revealed the superior performance of PyCurv not only for cryo-ET, but also for data generated by other techniques such as light microscopy and magnetic resonance imaging. Altogether, PyCurv is a versatile open-source software to reliably estimate curvature of membranes and other surfaces in a wide variety of applications.
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The Architecture of Traveling Actin Waves Revealed by Cryo-Electron Tomography.
Structure, 2019Co-Authors: Marion Jasnin, Wolfgang Baumeister, Antonio Martinez-sanchez, Florian Beck, Mary Ecke, Yoshiyuki Fukuda, Günther GerischAbstract:Summary Actin waves are dynamic supramolecular structures involved in cell migration, cytokinesis, adhesion, and neurogenesis. Although wave-like propagation of actin networks is a widespread phenomenon, the actin architecture underlying wave propagation remained unknown. In situ cryo-Electron Tomography of Dictyostelium cells unveils the wave architecture and provides evidence for wave progression by de novo actin nucleation. Subtomogram averaging reveals the structure of Arp2/3 complex-mediated branch junctions in their native state, and enables quantitative analysis of the 3D organization of branching within the waves. We find an excess of branches directed toward the substrate-attached membrane, and tent-like structures at sites of branch clustering. Fluorescence imaging shows that Arp2/3 clusters follow accumulation of the elongation factor VASP. We propose that filament growth toward the membrane lifts up the actin network as the wave propagates, until depolymerization of oblique filaments at the back causes the collapse of horizontal filaments into a compact layer.
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Cryo-Electron Tomography: Can it Reveal the Molecular Sociology of Cells in Atomic Detail?
Trends in Cell Biology, 2016Co-Authors: Martin Beck, Wolfgang BaumeisterAbstract:Traditionally, macromolecular structure determination is performed ex situ, that is, with purified materials. But, there are strong incentives to develop approaches to study them in situ in their native functional context. In recent years, cryo-Electron Tomography (cryo-ET) has emerged as a powerful method for visualizing the molecular organization of unperturbed cellular landscapes with the potential to attain near-atomic resolution. Here, we review recent work on several macromolecular assemblies, demonstrating the power of in situ studies. We also highlight technical challenges and discuss ways to meet them.
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In Situ Cryo-Electron Tomography: A Post-Reductionist Approach to Structural Biology.
Journal of molecular biology, 2015Co-Authors: Shoh Asano, Benjamin D. Engel, Wolfgang BaumeisterAbstract:Cryo-Electron Tomography is a powerful technique that can faithfully image the native cellular environment at nanometer resolution. Unlike many other imaging approaches, cryo-Electron Tomography provides a label-free method of detecting biological structures, relying on the intrinsic contrast of frozen cellular material for direct identification of macromolecules. Recent advances in sample preparation, detector technology, and phase plate imaging have enabled the structural characterization of protein complexes within intact cells. Here, we review these technical developments and outline a detailed computational workflow for in situ structural analysis. Two recent studies are described to illustrate how this workflow can be adapted to examine both known and unknown cellular complexes. The stage is now set to realize the promise of visual proteomics--a complete structural description of the cell's native molecular landscape.
Paul A Midgley - One of the best experts on this subject based on the ideXlab platform.
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scanning precession Electron Tomography for three dimensional nanoscale orientation imaging and crystallographic analysis
Nature Communications, 2015Co-Authors: Alexander S Eggeman, Robert Krakow, Paul A MidgleyAbstract:Three-dimensional (3D) reconstructions from Electron Tomography provide important morphological, compositional, optical and electro-magnetic information across a wide range of materials and devices. Precession Electron diffraction, in combination with scanning transmission Electron microscopy, can be used to elucidate the local orientation of crystalline materials. Here we show, using the example of a Ni-base superalloy, that combining these techniques and extending them to three dimensions, to produce scanning precession Electron Tomography, enables the 3D orientation of nanoscale sub-volumes to be determined and provides a one-to-one correspondence between 3D real space and 3D reciprocal space for almost any polycrystalline or multi-phase material.
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compressed sensing Electron Tomography
Ultramicroscopy, 2013Co-Authors: Rowan K Leary, Paul A Midgley, Zineb Saghi, Daniel J HollandAbstract:Abstract The recent mathematical concept of compressed sensing (CS) asserts that a small number of well-chosen measurements can suffice to reconstruct signals that are amenable to sparse or compressible representation. In addition to powerful theoretical results, the principles of CS are being exploited increasingly across a range of experiments to yield substantial performance gains relative to conventional approaches. In this work we describe the application of CS to Electron Tomography (ET) reconstruction and demonstrate the efficacy of CS–ET with several example studies. Artefacts present in conventional ET reconstructions such as streaking, blurring of object boundaries and elongation are markedly reduced, and robust reconstruction is shown to be possible from far fewer projections than are normally used. The CS–ET approach enables more reliable quantitative analysis of the reconstructions as well as novel 3D studies from extremely limited data.
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tem hrtem Electron holography and Electron Tomography studies of gamma and gamma nanoparticles in inconel 718 superalloy
Journal of Microscopy, 2009Co-Authors: Beata Dubiel, E Stepniowska, Dorin Geiger, Andrzej Kruk, Paul A Midgley, Pavel Formánek, J C Hernandez, Grzegorz Cempura, A CzyrskafilemonowiczAbstract:P>The aim of the study was the identification of gamma' and gamma ' strengthening precipitates in a commercial nickel-base superalloy Inconel 718 (Ni-19Fe-18Cr-5Nb-3Mo-1Ti-0.5Al-0.04C, wt %) using TEM dark-field, HRTEM, Electron holography and Electron Tomography imaging. To identify gamma' and gamma ' nanoparticles unambiguously, a systematic analysis of experimental and theoretical diffraction patterns were performed. Using HRTEM method it was possible to analyse small areas of precipitates appearance. Electron holography and Electron Tomography techniques show new possibilities of visualization of gamma' and gamma ' nanoparticles. The analysis by means of different complementary TEM methods showed that gamma ' particles exhibit a shape of thin plates, while gamma' phase precipitates are almost spherical.
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Electron Tomography and holography in materials science
Nature Materials, 2009Co-Authors: Paul A Midgley, Rafal E DuninborkowskiAbstract:The rapid development of Electron Tomography, in particular the introduction of novel tomographic imaging modes, has led to the visualization and analysis of three-dimensional structural and chemical information from materials at the nanometre level. In addition, the phase information revealed in Electron holograms allows electrostatic and magnetic potentials to be mapped quantitatively with high spatial resolution and, when combined with Tomography, in three dimensions. Here we present an overview of the techniques of Electron Tomography and Electron holography and demonstrate their capabilities with the aid of case studies that span materials science and the interface between the physical sciences and the life sciences.
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three dimensional real space crystallography of mcm 48 mesoporous silica revealed by scanning transmission Electron Tomography
Chemical Physics Letters, 2006Co-Authors: Timothy J V Yates, John Meurig Thomas, Josejesus Fernandez, Osamu Terasaki, Ryong Ryoo, Paul A MidgleyAbstract:High-angle annular dark-field scanning transmission Electron Tomography has been used to reveal the three-dimensional lattice structure of the mesoporous silica MCM-48. The incoherent nature of the dark-field signal leads to directly interpretable images and three-dimensional tomographic reconstructions. The ability to manipulate the three-dimensional reconstruction allows the structure to be visualised at any orientation, revealing detail in projection or as slices not hitherto seen by direct imaging techniques.
Lothar Houben - One of the best experts on this subject based on the ideXlab platform.
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Rapid low dose Electron Tomography using a direct Electron detection camera
Scientific Reports, 2015Co-Authors: Vadim Migunov, Lothar Houben, Henning Ryll, Xiaodong Zhuge, Martin Simson, Lothar Strüder, K. Joost Batenburg, Rafal E. Dunin-borkowskiAbstract:We demonstrate the ability to record a tomographic tilt series containing 3487 images in only 3.5 s by using a direct Electron detector in a transmission Electron microscope. The Electron dose is lower by at least one order of magnitude when compared with that used to record a conventional tilt series of fewer than 100 images in 15–60 minutes and the overall signal-to-noise ratio is greater than 4. Our results, which are illustrated for an inorganic nanotube, are important for ultra-low-dose Electron Tomography of Electron-beam-sensitive specimens and real-time dynamic Electron Tomography of nanoscale objects with sub-ms temporal resolution.
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cryo scanning transmission Electron Tomography of vitrified cells
Nature Methods, 2014Co-Authors: Sharon G Wolf, Lothar Houben, Michael ElbaumAbstract:Cryo-scanning transmission Electron Tomography (CSTET) of unstained, fully hydrated vitrified biological specimens is shown to have advantages over cryo-Electron Tomography (CET), notably at high sample tilts providing greater depth resolution for thick samples.
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correlating Electron Tomography and plasmon spectroscopy of single noble metal core shell nanoparticles
Nano Letters, 2012Co-Authors: Lev Chuntonov, Lothar Houben, Maya Barsadan, Gilad HaranAbstract:The 3D structure reconstruction of gold core-silver shell nanoparticles by Electron Tomography is combined with optical dark-field spectroscopy. Electron Tomography allows segmentation of the particles into core and shell subvolumes and facilitates avoiding Bragg diffraction artifacts inherent in 2D images. This advantage proves essential for accurate correlation of plasmon spectra and structure. We find that for the nanoparticles of near-spherical shape studied here the plasmon resonances depend on the relative size of the core and shell, rather than on their exact shapes and concentricity. A remarkable dependence of the spectral shape on the permittivity of the surrounding medium is also demonstrated, suggesting that core-shell nanoparticles can be used as ratiometric sensors with a very high dynamic range.
Richard D Leapman - One of the best experts on this subject based on the ideXlab platform.
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dual axis Electron Tomography of biological specimens extending the limits of specimen thickness with bright field stem imaging
Journal of Structural Biology, 2011Co-Authors: Alioscka A Sousa, Afrouz A Azari, Guofeng Zhang, Richard D LeapmanAbstract:Abstract The absence of imaging lenses after the specimen in the scanning transmission Electron microscope (STEM) enables Electron Tomography to be performed in the STEM mode on micrometer-thick plastic-embedded specimens without the deleterious effect of chromatic aberration, which limits spatial resolution and signal-to-noise ratio in conventional TEM. Using Monte Carlo calculations to simulate Electron scattering from gold nanoparticles situated at the top and bottom surfaces of a plastic section, we assess the optimal acquisition strategy for axial bright-field STEM Electron Tomography at a beam-energy of 300 keV. Dual tilt-axis STEM Tomography with optimized axial bight-field detector geometry is demonstrated by application to micrometer-thick sections of beta cells from mouse pancreatic islet. The quality of the resulting three-dimensional reconstructions is comparable to that obtained from much thinner (0.3-micrometer) sections using conventional TEM Tomography. The increased range of specimen thickness accessible to axial STEM Tomography without the need for serial sectioning enables the 3-D visualization of more complex and larger subcellular structures.
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nanoscale 3d cellular imaging by axial scanning transmission Electron Tomography
Nature Methods, 2009Co-Authors: Martin F Hohmannmarriott, Alioscka A Sousa, Afrouz A Azari, Svetlana Glushakova, Guofeng Zhang, Joshua Zimmerberg, Richard D LeapmanAbstract:Using an axial detector, scanning transmission Electron microscopy allows three-dimensional tomographic reconstruction of micrometer-thick sections of biological samples, at a resolution comparable to that obtained on thin sections. Electron Tomography provides three-dimensional structural information about supramolecular assemblies and organelles in a cellular context, but image degradation, caused by scattering of transmitted Electrons, limits applicability in specimens thicker than 300 nm. We found that scanning transmission Electron Tomography of 1,000-nm-thick samples using axial detection provided resolution comparable to that of conventional Electron Tomography. We demonstrated the method by reconstructing a human erythrocyte infected with the malaria parasite Plasmodium falciparum.
Martin F Hohmannmarriott - One of the best experts on this subject based on the ideXlab platform.
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nanoscale 3d cellular imaging by axial scanning transmission Electron Tomography
Nature Methods, 2009Co-Authors: Martin F Hohmannmarriott, Alioscka A Sousa, Afrouz A Azari, Svetlana Glushakova, Guofeng Zhang, Joshua Zimmerberg, Richard D LeapmanAbstract:Using an axial detector, scanning transmission Electron microscopy allows three-dimensional tomographic reconstruction of micrometer-thick sections of biological samples, at a resolution comparable to that obtained on thin sections. Electron Tomography provides three-dimensional structural information about supramolecular assemblies and organelles in a cellular context, but image degradation, caused by scattering of transmitted Electrons, limits applicability in specimens thicker than 300 nm. We found that scanning transmission Electron Tomography of 1,000-nm-thick samples using axial detection provided resolution comparable to that of conventional Electron Tomography. We demonstrated the method by reconstructing a human erythrocyte infected with the malaria parasite Plasmodium falciparum.