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Добірка наукової літератури з теми "Cryo-electron microscopy and tomography"

Автор: Grafiati
Опубліковано: 29 березня 2025

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Статті в журналах з теми "Cryo-electron microscopy and tomography"

1

Stewart, Phoebe L. "Cryo-electron microscopy and cryo-electron tomography of nanoparticles." Wiley Interdisciplinary Reviews: Nanomedicine and Nanobiotechnology 9, no. 2 (June 23, 2016): e1417. http://dx.doi.org/10.1002/wnan.1417.

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2

Lyu, Cheng-An, Yao Shen, and Peijun Zhang. "Zooming in and out: Exploring RNA Viral Infections with Multiscale Microscopic Methods." Viruses 16, no. 9 (September 23, 2024): 1504. http://dx.doi.org/10.3390/v16091504.

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Анотація:
RNA viruses, being submicroscopic organisms, have intriguing biological makeups and substantially impact human health. Microscopic methods have been utilized for studying RNA viruses at a variety of scales. In order of observation scale from large to small, fluorescence microscopy, cryo-soft X-ray tomography (cryo-SXT), serial cryo-focused ion beam/scanning electron microscopy (cryo-FIB/SEM) volume imaging, cryo-electron tomography (cryo-ET), and cryo-electron microscopy (cryo-EM) single-particle analysis (SPA) have been employed, enabling researchers to explore the intricate world of RNA viruses, their ultrastructure, dynamics, and interactions with host cells. These methods evolve to be combined to achieve a wide resolution range from atomic to sub-nano resolutions, making correlative microscopy an emerging trend. The developments in microscopic methods provide multi-fold and spatial information, advancing our understanding of viral infections and providing critical tools for developing novel antiviral strategies and rapid responses to emerging viral threats.
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3

Carlson, David B., Jeff Gelb, Vadim Palshin, and James E. Evans. "Laboratory-Based Cryogenic Soft X-Ray Tomography with Correlative Cryo-Light and Electron Microscopy." Microscopy and Microanalysis 19, no. 1 (January 18, 2013): 22–29. http://dx.doi.org/10.1017/s1431927612013827.

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AbstractHere we present a novel laboratory-based cryogenic soft X-ray microscope for whole cell tomography of frozen hydrated samples. We demonstrate the capabilities of this compact cryogenic microscope by visualizing internal subcellular structures of Saccharomyces cerevisiae cells. The microscope is shown to achieve better than 50 nm half-pitch spatial resolution with a Siemens star test sample. For whole biological cells, the microscope can image specimens up to 5 μm thick. Structures as small as 90 nm can be detected in tomographic reconstructions following a low cumulative radiation dose of only 7.2 MGy. Furthermore, the design of the specimen chamber utilizes a standard sample support that permits multimodal correlative imaging of the exact same unstained yeast cell via cryo-fluorescence light microscopy, cryo-soft X-ray microscopy, and cryo-transmission electron microscopy. This completely laboratory-based cryogenic soft X-ray microscope will enable greater access to three-dimensional ultrastructure determination of biological whole cells without chemical fixation or physical sectioning.
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4

Weis, Felix, and Wim J. H. Hagen. "Combining high throughput and high quality for cryo-electron microscopy data collection." Acta Crystallographica Section D Structural Biology 76, no. 8 (July 27, 2020): 724–28. http://dx.doi.org/10.1107/s2059798320008347.

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Анотація:
Cryo-electron microscopy (cryo-EM) can be used to elucidate the 3D structure of macromolecular complexes. Driven by technological breakthroughs in electron-microscope and electron-detector development, coupled with improved image-processing procedures, it is now possible to reach high resolution both in single-particle analysis and in cryo-electron tomography and subtomogram-averaging approaches. As a consequence, the way in which cryo-EM data are collected has changed and new challenges have arisen in terms of microscope alignment, aberration correction and imaging parameters. This review describes how high-end data collection is performed at the EMBL Heidelberg cryo-EM platform, presenting recent microscope implementations that allow an increase in throughput while maintaining aberration-free imaging and the optimization of acquisition parameters to collect high-resolution data.
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5

Cyrklaff, M., M. Kudryashev, N. Kilian, P. Henrich, F. Frischknecht, and M. Lanzer. "Cryo-Electron Tomography of Malaria Parasites." Microscopy and Microanalysis 15, S2 (July 2009): 864–65. http://dx.doi.org/10.1017/s1431927609099267.

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6

Apkarian, Robert P. "Comments on Cryo High Resolution Scanning Electron Microscopy." Microscopy Today 12, no. 1 (January 2004): 45. http://dx.doi.org/10.1017/s1551929500051841.

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Анотація:
Stephen Carmichael wrote about Cryoelectron Tomography in the May 2003 issue of Microscopy Today. Citing new preparation methods, small cells can be vitrified, observed frozen in the TEM and a series of digital images captured while the specimen is being rotated around the axis perpendicular to the electron beam producing a 3-D tomogram. Gina Sosinski and Maryann Martone wrote about imaging big and messy biological structures using cryo-electron Tomography in the July issue of Microscopy Today. Cryo-HRSEM now also seeks to provide 3-D information approaching the molecular level from frozen hydrated cell and molecular systems. Vitrification procedures for small specimens such as platelets and biomolecules on grids are accomplished by plunge freezing in liquefied etiiane as is done with cryo-TEM procedures. Bulk specimens such as organic hydrogels and tissues are routinely high pressure frozen (HPF) in 3mm gold planchets. Employing an in-lens cryostage, identical to those used in cryo-TEM, cryo-HRSEM provides 3-D high-resolution images because secondary electrons are efficiently collected above the lens in a single scan thus minimizing specimen irradiation.
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7

Marko, M., C. Hsieh, A. Leith, and C. Mannella. "Requirements for Phase-Plate Cryo-Electron Tomography." Microscopy and Microanalysis 16, S2 (July 2010): 546–47. http://dx.doi.org/10.1017/s1431927610054048.

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8

Voorhout, W., F. De Haas, P. Frederik, R. Schoenmakers, W. Busing, and D. Hubert. "An Optimized Solution for Cryo Electron Tomography." Microscopy and Microanalysis 12, S02 (July 31, 2006): 1110–11. http://dx.doi.org/10.1017/s1431927606065822.

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9

Ziese, U., D. Typke, R. Hegerl, and W. Baumeister. "Cryo Electron Microscopy of SSV1 phage particles." Proceedings, annual meeting, Electron Microscopy Society of America 53 (August 13, 1995): 842–43. http://dx.doi.org/10.1017/s0424820100140580.

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Анотація:
SSVl phages are lemon-shaped particles, normally about 90 nm x 40 nm in size, with short tail fibres attached to one pole, produced by the thermophilic archaeon Sulfolobus shibate, isolate B12. They are made of 3 different proteins and DNA (15.5 kbp). Two proteins, together with host lipid, form the envelope, the third protein is associated with the DNA. Interestingly, this virus produces particles of varying size and shape. We have investigated the mass of the virions by STEM mass determination, the inner structure by cryo-electron microscopy, and the shape variability by electron tomography. Automatic electron tomography (AET) has been shown to be a useful technique for collecting 3D structural data of individual biological particles under low dose conditions, in negative stain as well as in frozen-hydrated preparations.Taking electron micrographs of vitrified samples we obtained images revealing some details of the inner structure and, on some particles, a periodic structure of the envelope. (Fig. 1a-b) The inner structure has periodicities of about 2.5 nm, which is in agreement with that found by Lepault et al on vitrified samples of the phages lambda and T4.
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10

Young, Lindsey N., and Elizabeth Villa. "Bringing Structure to Cell Biology with Cryo-Electron Tomography." Annual Review of Biophysics 52, no. 1 (May 9, 2023): 573–95. http://dx.doi.org/10.1146/annurev-biophys-111622-091327.

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Анотація:
Recent advances in cryo-electron microscopy have marked only the beginning of the potential of this technique. To bring structure into cell biology, the modality of cryo-electron tomography has fast developed into a bona fide in situ structural biology technique where structures are determined in their native environment, the cell. Nearly every step of the cryo-focused ion beam-assisted electron tomography (cryo-FIB-ET) workflow has been improved upon in the past decade, since the first windows were carved into cells, unveiling macromolecular networks in near-native conditions. By bridging structural and cell biology, cryo-FIB-ET is advancing our understanding of structure–function relationships in their native environment and becoming a tool for discovering new biology.
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Більше джерел

Дисертації з теми "Cryo-electron microscopy and tomography"

1

Sandin, Sara. "Cryo-electron tomography of individual protein molecules /." Stockholm, 2005. http://diss.kib.ki.se/2005/91-7140-462-7/.

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2

Fatmaoui, Fadwa. "Determination of pericentric heterochromatin structure by in situ cryo-electron tomography." Electronic Thesis or Diss., Strasbourg, 2024. http://www.theses.fr/2024STRAJ018.

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L'hétérochromatine constitutive est une forme condensée de chromatine, essentielle au maintien de la stabilité du génome et à la défense contre les rétrotransposons et les rétrovirus endogènes. À l'échelle moléculaire, elle se caractérise par des réseaux réguliers de nucléosomes, la méthylation de l'ADN et des histones et la liaison de protéines spécifiques associées à l'hétérochromatine (famille HP1). Cependant, la manière dont ces caractéristiques moléculaires conduisent à l'état condensé et définissent les propriétés fonctionnelles de l'hétérochromatine constitutive n'est pas encore claire. Le projet abordera cette question en déterminant la structure de l'hétérochromatine constitutive péricentrique directement à l'intérieur de son contenu cellulaire en utilisant la cryotomographie in situ de pointe. Les embryons de drosophile sont utilisés comme modèle expérimental, car dans leurs noyaux, les régions d'hétérochromatine péricentrique coalescent en chromocentres ronds de l'ordre du micron. Nous utilisons la cryosection avec des couteaux diamantés pour l'amincissement de l'échantillon, puis les tomogrammes des chromocentres, ainsi que d'autres domaines de la chromatine, seront enregistrés et reconstruits. Cela nous permettra de définir l'arrangement caractéristique des fibres de nucléosomes pour l'hétérochromatine péricentrique constitutive par comparaison avec l'empaquetage de la chromatine dans d'autres compartiments chromatiniens
Constitutive heterochromatin is a condensed form of chromatin, essential for the maintenance of genome stability and the defense against retrotransposons and endogenous retroviruses. At the molecular scale, it is characterized by regular nucleosome arrays, DNA and histone methylation and binding of specific heterochromatin-associated proteins (HP1 family). However, it remains unclear how these molecular features lead to the condensed state and define the functional properties of constitutive heterochromatin. The project will address this question by determining the structure of pericentric constitutive heterochromatin directly within its cellular content by using state-of-the-art in situ cryo-electron tomography. Drosophila embryos are used as the experimental model, because in their nuclei, the pericentric heterochromatin regions coalesce into round micron-scale chromocenters. We use cryo-sectioning with diamond knives for sample thinning, and then tomograms of chromocenters, as well as other chromatin domains will be recorded and reconstructed. This will enable us to define the characteristic nucleosome fiber arrangement for the constitutive pericentric heterochromatin by comparison with the chromatin packing in other chromatin compartments
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3

Moebel, Emmanuel. "New strategies for the identification and enumeration of macromolecules in 3D images of cryo electron tomography." Thesis, Rennes 1, 2019. http://www.theses.fr/2019REN1S007/document.

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Анотація:
La cryo-tomographie électronique (cryo-ET) est une technique d'imagerie capable de produire des vues 3D de spécimens biologiques. Cette technologie permet d’imager de larges portions de cellules vitrifiées à une résolution nanométrique. Elle permet de combiner plusieurs échelles de compréhension de la machinerie cellulaire, allant des interactions entre les groupes de protéines à leur structure atomique. La cryo-ET a donc le potentiel d'agir comme un lien entre l'imagerie cellulaire in vivo et les techniques atteignant la résolution atomique. Cependant, ces images sont corrompues par un niveau de bruit élevé et d'artefacts d'imagerie. Leur interprétabilité dépend fortement des méthodes de traitement d'image. Les méthodes computationelles existantes permettent actuellement d'identifier de larges macromolécules telles que les ribosomes, mais il est avéré que ces détections sont incomplètes. De plus, ces méthodes sont limitées lorsque les objets recherchés sont de très petite taille ou présentent une plus grande variabilité structurelle. L'objectif de cette thèse est de proposer de nouvelles méthodes d'analyse d'images, afin de permettre une identification plus robuste des macromolécules d'intérêt. Nous proposons deux méthodes computationelles pour atteindre cet objectif. La première vise à réduire le bruit et les artefacts d'imagerie, et fonctionne en ajoutant et en supprimant de façon itérative un bruit artificiel à l'image. Nous fournissons des preuves mathématiques et expérimentales de ce concept qui permet d'améliorer le signal dans les images de cryo-ET. La deuxième méthode s'appuie sur les progrès récents de l'apprentissage automatique et les méthodes convolutionelles pour améliorer la localisation des macromolécules. La méthode est basée sur un réseau neuronal convolutif et nous montrons comment l'adapter pour obtenir des taux de détection supérieur à l'état de l'art
Cryo electron tomography (cryo-ET) is an imaging technique capable of producing 3D views of biological specimens. This technology enables to capture large field of views of vitrified cells at nanometer resolution. These features allow to combine several scales of understanding of the cellular machinery, from the interactions between groups of proteins to their atomic structure. Cryo-ET therefore has the potential to act as a link between in vivo cell imaging and atomic resolution techniques. However, cryo-ET images suffer from a high amount of noise and imaging artifacts, and the interpretability of these images heavily depends on computational image analysis methods. Existing methods allow to identify large macromolecules such as ribosomes, but there is evidence that the detections are incomplete. In addition, these methods are limited when searched objects are smaller and have more structural variability. The purpose of this thesis is to propose new image analysis methods, in order to enable a more robust identification of macromolecules of interest. We propose two computational methods to achieve this goal. The first aims at reducing the noise and imaging artifacts, and operates by iteratively adding and removing artificial noise to the image. We provide both mathematical and experimental evidence that this concept allows to enhance signal in cryo-ET images. The second method builds on recent advances in machine learning to improve macromolecule localization. The method is based on a convolutional neural network, and we show how it can be adapted to achieve better detection rates than the current state-of- the-art
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4

Niehle, Michael. "Electron tomography and microscopy on semiconductor heterostructures." Doctoral thesis, Humboldt-Universität zu Berlin, Mathematisch-Naturwissenschaftliche Fakultät, 2016. http://dx.doi.org/10.18452/17607.

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Анотація:
Elektronentomographie erlaubt die dreidimensionale (3D) Charakterisierung von Kristalldefekten auf der Nanometerskala. Die Anwendung in der Forschung an epitaktischen Halbleiterheterostrukturen ist bisher nicht durchgesetzt worden, obwohl kleiner werdende Bauteile mit zunehmend dreidimensionaler Struktur entsprechende Untersuchungen verlangen, um die Beziehung von Struktur und physikalischen Eigenschaften in entsprechenden Materialsystemen zu verstehen. Die vorliegende Arbeit demonstriert die konsequente Anwendung der Elektronentomographie auf eine III-Sb basierte Laser- und eine 3D (In,Ga)N/GaN Nanosäulenheterostruktur. Die unerlässliche Zielpräparation von Proben mittels FIB-SEM-Zweistrahlmikroskops wird herausgestellt. Die kontrollierte Orientierung der Probe während der Präparation und die sorfältige Auswahl eines Abbildungsverfahrens im STEM werden detailliert beschrieben. Die umfassende räumliche Mikrostrukturanalyse einer antimonidbasierten Schichtstruktur folgt der Dimensionalität von Kristalldefekten. Die Facettierung und Lage einer Pore (3D Defekt), deren Auftreten in der MBE gewachsenen GaSb-Schicht untypisch ist, werden bestimmt. Das Zusammenspiel von anfänglich abgeschiedenen AlSb-Inseln auf dem Si-Substrat, der Ausbildung eines Fehlversetzungsnetzwerkes an der Grenzfläche der Heterostruktur (2D Defekt) und dem Auftreten von Durchstoßversetzungen wird mit Hilfe der Kombination tomographischer und komplementärer TEM-/STEM-Ergebnisse untersucht. Die räumliche Anordnung von Versetzungen (1D Defekte), die das ganze Schichtsystem durchziehen, wird mit Elektronentomographie offenbart. Die Wechselwirkung dieser Versetzungen mit Antiphasengrenzen und anderen Liniendefekten sind ein einzigartiges Ergebnis der Elektronentomographie. Abschließend sind Unterschiede im Indiumgehalt und in der Schichtdicke von (In,Ga)N-Einschlüssen auf verschiedenen Facetten schief aufgewachsener GaN-Nanosäulen einmalig per Elektronentomographie herausgearbeitet worden.
Electron tomography exhibits a very poor spread in the research field of epitaxial semiconductor heterostructures in spite of the ongoing miniaturization and increasing three-dimensional (3D) character of nano-structured devices. This necessitates a tomographic approach at the nanometre scale in order to characterize and understand the relation between structure and physical properties of respective material systems. The present work demonstrates the rigorous application of electron tomography to an III-Sb based laser and to an (In,Ga)N/GaN nanocolumn heterostructure. A specific target preparation using a versatile FIB-SEM dual-beam microscope is emphasized as indispensable. The purposeful orientation of the specimen during preparation and the careful selection of an imaging mode in the scanning-/transmission electron microscope (S/TEM) are regarded in great detail. The comprehensive spatial microstructure characterization of the antimonide based heterostructure follows the dimensionality of crystal defects. The facetting and position of a pore (3D defect) which is unexpected in the MBE grown GaSb layer, is determined. The interplay of the initially grown AlSb islands on Si, the formation of a misfit dislocation network at the heterostructure interface (2D defect) and the presence of threading dislocations is investigated by the correlation of tomographic and complementary S/TEM results. The spatial arrangement of dislocations (1D defects) penetrating the whole stack of antimonide layers is revealed by electron tomography. The interaction of these line defects with anti-phase boundaries and with other dislocations is exclusively observed in the 3D result. The insertion of (In,Ga)N into oblique GaN nanocolumns is uniquely accessed by electron tomography. The amount of incorporated indium and the (In,Ga)N layer thickness is shown to vary on the different facets of the GaN core.
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5

Sharp, Joanne. "Electron tomography of defects." Thesis, University of Cambridge, 2010. https://www.repository.cam.ac.uk/handle/1810/228638.

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Tomography of crystal defects in the electron microscope was first attempted in 2005 by the author and colleagues. This thesis further develops the technique, using a variety of samples and methods. Use of a more optimised, commercial tomographic reconstruction program on the original GaN weak beam dark-field (WBDF) tilt series gave a finer reconstruction with lower background, line width 10-20 nm. Four WBDF tilt series were obtained of a microcrack surrounded by dislocations in a sample of indented silicon, tilt axes parallel to g = 220, 220, 400 and 040. Moiré fringes in the defect impaired alignment and reconstruction. The effect on reconstruction of moiré fringe motion with tilt was simulated, resulting in an array of rods, not a flat plane. Dislocations in a TiAl alloy were reconstructed from WBDF images with no thickness contours, giving an exceptionally clear reconstruction. The effect of misalignment of the tilt axis with systematic row g(ng) was assessed by simulating tilt series with diffraction condition variation across the tilt range of Δn = 0, 1 and 2. Misalignment changed the inclination of the reconstructed dislocation with the foil surfaces, and elongated the reconstruction in the foil normal direction; this may explain elongation additional to the missing wedge effect in experiments. Tomography from annular dark-field (ADF) STEM dislocation images was also attempted. A tilt series was obtained from the GaN sample; the reconstructed dislocations had a core of bright intensity of comparable width to WBDF reconstructions, with a surrounding region of low intensity to 60 nm width. An ADF STEM reconstruction was obtained from the Si sample at the same microcrack as for WBDF; here automatic specimen drift correction in tomography acquisition software succeeded, a significant improvement. The microcrack surfaces in Si reconstructed as faint planes and dislocations were recovered as less fragmented lines than from the WBDF reconstruction. ADF STEM tomography was also carried out on the TiAl sample, using a detector inner angle (βin) that included the first order Bragg spots (in other series βin had been 4-6θ B). Extinctions occurred which were dependent on tilt; this produced only weak lines in the reconstruction. Bragg scattering in the ADF STEM image was estimated by summing simulated dark-field dislocation images from all Bragg beams at a zone axis; a double line was produced. It was hypothised that choosing the inner detector angle to omit these first Bragg peaks may preclude most dynamical image features. Additional thermal diffuse scattering (TDS) intensity due to dilatation around an edge dislocation was estimated and found to be insignificant. The Huang scattering cross section was estimated and found to be 9Å, ten times thinner than experimental ADF STEM dislocation images. The remaining intensity may be from changes to TDS from Bloch wave transitions at the dislocation; assessing this as a function of tilt is for further work. On simple assessment, only three possible axial channeling orientations were found over the tilt range for GaN; if this is typical, dechanneling contrast probably does not apply to defect tomography.
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6

Gedda, Magnus. "Contributions to 3D Image Analysis using Discrete Methods and Fuzzy Techniques : With Focus on Images from Cryo-Electron Tomography." Doctoral thesis, Uppsala universitet, Centrum för bildanalys, 2010. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-121579.

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Анотація:
With the emergence of new imaging techniques, researchers are always eager to push the boundaries by examining objects either smaller or further away than what was previously possible. The development of image analysis techniques has greatly helped to introduce objectivity and coherence in measurements and decision making. It has become an essential tool for facilitating both large-scale quantitative studies and qualitative research. In this Thesis, methods were developed for analysis of low-resolution (in respect to the size of the imaged objects) three-dimensional (3D) images with low signal-to-noise ratios (SNR) applied to images from cryo-electron tomography (cryo-ET) and fluorescence microscopy (FM). The main focus is on methods of low complexity, that take into account both grey-level and shape information, to facilitate large-scale studies. Methods were developed to localise and represent complex macromolecules in images from cryo-ET. The methods were applied to Immunoglobulin G (IgG) antibodies and MET proteins. The low resolution and low SNR required that grey-level information was utilised to create fuzzy representations of the macromolecules. To extract structural properties, a method was developed to use grey-level-based distance measures to facilitate decomposition of the fuzzy representations into sub-domains. The structural properties of the MET protein were analysed by developing a analytical curve representation of its stalk. To facilitate large-scale analysis of structural properties of nerve cells, a method for tracing neurites in FM images using local path-finding was developed. Both theoretical and implementational details of computationally heavy approaches were examined to keep the time complexity low in the developed methods. Grey-weighted distance definitions and various aspects of their implementations were examined in detail to form guidelines on which definition to use in which setting and which implementation is the fastest. Heuristics were developed to speed up computations when calculating grey-weighted distances between two points. The methods were evaluated on both real and synthetic data and the results show that the methods provide a step towards facilitating large-scale studies of images from both cryo-ET and FM.
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7

Xiao, Juan. "Development of electron tomography on liquid suspensions using environmental scanning electron microscopy." Thesis, Lyon, 2017. http://www.theses.fr/2017LYSEI050/document.

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La Microscopie Electronique à Balayage Environnementale permet l'observation de liquides dans certaines conditions de pression et température. En travaillant en transmission, i.e. en mode STEM (Scanning Transmission Electron Microscopy), des nano-objets présents au sein du liquide peuvent même être analysés (mode « Wet-STEM»). Dans les solutions concentrées, l'arrangement du soluté peut changer être un paramètre microstructural important, qu’il est alors nécessaire de caractériser. Dans ce contexte, le but de ce travail est de développer la tomographie électronique sur des suspensions liquides en utilisant le mode STEM en ESEM, de manière à obtenir la structure 3D de nano-objets dispersés dans un liquide. Dans une première partie, le contraste entre des nanoparticules et le film d’eau est étudié en combinant des images expérimentales Wet-STEM (en 2D) et des simulations Monte Carlo. Deux types de nano-matériaux sont choisis : des nanoparticules d’or sphériques, de diamètre environ 40 nm, dispersées dans l’eau, ainsi qu’une suspension aqueuse de latex SBA-PMMA, contenant 3% de PMMA utilisé comme tensioactif stérique. La comparaison entre les résultats simulés et expérimentaux permet d’estimer comment le contraste entre l’eau et les nanomatériaux est affecté par l’épaisseur du film d’eau. Dans une deuxième partie, des expériences de tomographie sont réalisées à sec sur des films de polyuréthane contenant des nanotubes de carbone multiparois greffés ou non, en utilisant une platine développée précédemment au laboratoire. Le volume a pu être reconstruit correctement. Cependant, en effectuant une acquisition 3D sur des suspensions de latex SBA-PMMA, le contrôle de la température de l’échantillon s’est révélé insuffisant. Nous proposons une amélioration à la fois de la platine et des conditions d’observations permettant de mieux contrôler l’évaporation et la condensation de l’eau sur des échantillons liquides. La troisième partie est dévolue à une analyse approfondie d’une suspension de latex SBA-PMMA, de différentes concentrations (d’un état dilué à très concentré), les acquisitions étant effectuées avec les conditions optimisées. L’arrangement des particules de latex est comparé à des modèles issus de la littérature, et avec des résultats expérimentaux obtenus par cryo-SEM sur suspensions congelées. Nous présentons ensuite une étude du même latex en présence de tensioactif. La couche de tensioactif peut être mise en évidence dans les volumes reconstruits et segmentés. En conclusion, nous résumons les potentialités de la tomographie wet-STEM pour la caractérisation de nanomatériaux solides et liquides. Des perspectives sont proposées pour continuer dans l’exploration de ces potentialités et des limites de la technique
ESEM (Environmental Scanning Electron Microscopy) allows the observation of liquids under specific conditions of pressure and temperature. When working in the transmission mode, i.e. in STEM (Scanning Transmission Electron Microscopy), nano-objects can even be analyzed inside the liquid (“wet-STEM” mode). Moreover, in situ evaporation of water can be performed to study the materials evolution from the wet to the dry state. This work aims at developing electron tomography on liquid suspensions using STEM-in-ESEM, to obtain the 3D structure of nano-objects dispersed in a liquid. In a first part, Monte Carlo simulations and 2D wet-STEM experimental images are combined to study the contrast. Two kinds of liquid nano-materials are chosen as the sample: spherical gold particles (diameter around 40 nm) in suspension in water; latex SBA-PMMA suspension, a copolymer derived from styrene and metacrylic acid esters in aqueous solution, 3% PMMA shell included as steric surfactant. The comparison between simulated and experimental results helps to determine how water can affect the contrast of hydrated nano-materials. Tomography experiments are then performed on dry PU-carbon nanotubes nanocomposites using a previously developed home-made tomography device, and the volume is well reconstructed. When performing tomography on latex suspension, limitations are found on the temperature control of samples. We propose an optimization of the device with new observations conditions to better control water evaporation and condensation of liquid samples. Afterwards, a full 3D analysis on SBA-PMMA latex from dilute suspension to very concentrated one is performed, and a further study is presented in presence of a surfactant. The encouraging reconstruction results are used to model the particles arrangement. This shows the potentialities of wet-STEM tomography for the characterization of both solid and liquid nano-materials
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Fogelqvist, Emelie. "Laboratory Soft X-Ray Cryo Microscopy: Source, System and Bio Applications." Doctoral thesis, KTH, Biomedicinsk fysik och röntgenfysik, 2017. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-206428.

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Soft x-ray microscopes routinely perform high-resolution 3D imaging of biological cells in their near-native environment with short exposure times at synchrotron radiation facilities. Some laboratory-sized microscopes are aiming to make this imaging technique more accessible to a wider scientific community. However, these systems have been hampered by source instabilities hindering routine imaging of biological samples with short exposure times. This Thesis presents work performed on the Stockholm laboratory x-ray microscope. A novel heat control system has been implemented, improving the stability of the laser-produced plasma source. In combination with recent upgrades to the imaging system and an improved cryofixation method, the microscope now has the capability to routinely produce images with 10-second exposure time of cryofixed biological samples. This has allowed for tomographic imaging of cell autophagy and cell-cell interactions. Furthermore, a numerical 3D image formation model is presented as well as a novel reconstruction approach dealing with the limited depth of focus in x-ray microscopes.

QC 20170505

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Le, Bihan Olivier. "Etude par microscopie électronique des mécanismes d'action de vecteurs synthétiques pour le transfert de gènes." Thesis, Bordeaux 1, 2009. http://www.theses.fr/2009BOR13972/document.

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La grande majorité des essais cliniques de transfert de gènes in vivo utilise des vecteurs viraux. Si ces derniers sont efficaces, ils présentent des risques immunogènes, toxiques, voire mutagènes avérés. Les vecteurs synthétiques (non viraux), par leur grande modularité et leur faible toxicité représentent une alternative très prometteuse. Le principal frein à leur utilisation est leur manque d’efficacité. L’objectif majeur de ce travail de thèse a été de comprendre le mécanisme de transfert de gènes associé à différents complexes vecteurs synthétiques/ADN plasmidique, ce qui est indispensable pour une conception rationnelle de nouveaux vecteurs. Nous avons étudié, sur cellules en culture, le mécanisme de transfert de gènes associé à deux lipides cationiques ; le BGTC (bis(guanidinium)-tren-cholesterol) et la DOSP (DiOleylamine A-Succinyl-Paromomycine) qui sont connus pour être des vecteurs efficaces in vitro. Nous avons ainsi pu visualiser par microscopie électronique leurs voies d’entrée, leurs remaniements structuraux ainsi que leur échappement endosomal qui représente une étape clé du processus de transfert de gènes. L’identification non ambigüe des lipoplexes tout au long de leur trafic intracellulaire a été rendue possible grâce au marquage de l’ADN par des nanoparticules de silice dotées d’un cœur de maghémite (Fe2O3) dense aux électrons. Cette stratégie de marquage a également été appliquée à l’étude du mécanisme d’action d’un autre vecteur synthétique de type polymère, le copolymère à blocs non ionique P188 ou Lutrol. Contrairement à la plupart des vecteurs synthétiques, celui-ci présente une efficacité de transfection in vivo chez la souris par injection in situ pour le tissu musculaire ou en intra trachéale dans le poumon. En revanche, il est totalement inefficace in vitro. Nous avons montré que le Lutrol permet une augmentation de l’internalisation d’ADN par les cellules mais n’induit pas son échappement endosomal, ce qui expliquerait son absence d’efficacité in vitro. D’autres voies d’entrée sont alors à envisager in vivo pour comprendre son mécanisme d’action
The vast majority of clinical trials of gene transfer in vivo use viral vectors. Although they are effective, they induce immunogenic, toxic or mutagenic risks. Due to their high modularity and low toxicity, synthetic vectors (non viral), represent a promising alternative despite their lack of effectiveness. The major objective of this work was to understand the mechanism of gene transfer using two prototypic synthetic vectors, in the context of a rational design of new vectors. We studied on cultured cells, the mechanism of action of two cationic lipids; BGTC (bis(guanidinium)-tren-cholesterol) and DOSP (DiOleylamine A-Succinyl-Paromomycine) formulated with plasmid DNA (lipoplexes) which are in vitro efficient vectors. We have been able to visualize by electron microscopy, their intracellular pathways, their structural alterations and their endosomal escape, the latter being a key step in the process of gene transfer. The unambiguous identification of lipoplexes throughout their intracellular trafficking has been made possible thanks to the labelling of DNA by core-shell silica nanoparticles with an electron dense maghemite core (Fe2O3). The labeling strategy has also been applied to study the mechanism of action of a nonionic block copolymer (P188 or Lutrol). Interestingly, these synthetic vectors have an in vivo transfection efficiency in mice lung and muscle tissue while they are totally inefficient in vitro. We have shown that Lutrol induces an increase of DNA internalization into cells and fails to trigger endosomal escape, which would explain the lack of in vitro efficacy. These findings suggest that the in vivo mechanism of action of Lutrol would involve other internalization pathways
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Krehl, Jonas. "Incorporating Fresnel-Propagation into Electron Holographic Tomography." Master's thesis, Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2017. http://nbn-resolving.de/urn:nbn:de:bsz:14-qucosa-217919.

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Tomographic electron holography combines tomography, the reconstruction of three-dimensionally resolved data from multiple measurements with different specimen orientations, with electron holography, an interferometrical method for measuring the complex wave function inside a transmission electron microscope (TEM). Due to multiple scattering and free wave propagation conventional, ray projection based, tomography does perform badly when approaching atomic resolution. This is remedied by incorporating propagation effects into the projection while maintaining linearity in the object potential. Using the Rytov approach an approximation is derived, where the logarithm of the complex wave is linear in the potential. The ray projection becomes a convolution with a Fresnel propagation kernel, which is considerably more computationally expensive. A framework for such calculations has been implemented in Python. So has a multislice electron scattering algorithm, optimised for large fields of view and high numbers of atoms for simulations of scattering at nanoparticles. The Rytov approximation gives a remarkable increase in resolution and signal quality over the conventional approach in the tested system of a tungsten disulfide nanotube. The response to noise seems to be similar as in conventional tomography, so rather benign. This comes at the downside of much longer calculation time per iteration
Tomographische Elektronenholographie kombiniert Tomographie, die Rekonstruktion dreidimensional aufgelößter Daten aus einem Satz von mehreren Messungen bei verschiedenen Objektorientierungen, mit Elektronenholographie, eine interferrometrische Messung der komplexen Elektronenwelle im Transmissionselektronenmikroskop (TEM). Wegen Mehrfachstreuung und Propagationseffekten erzeugt konventionelle, auf einer Strahlprojektion basierende, Tomography ernste Probleme bei Hochauflösung hin zu atomarer Auflösung. Diese sollen durch ein Modell, welches Fresnel-Propagation beinhaltet, aber weiterhin linear im Potential des Objektes ist, vermindert werden. Mit dem Rytov-Ansatz wird eine Näherung abgeleitet, wobei der Logarithmus der komplexen Welle linear im Potential ist. Die Strahlen-Projektion ist dann eine Faltung mit dem Fresnel-Propagations-Faltungskernel welche rechentechnisch wesentlich aufwendiger ist. Ein Programm-Paket für solche Rechnungen wurde in Python implementiert. Weiterhin wurde ein Multislice Algorithmus für große Gesichtsfelder und Objekte mit vielen Atomen wie Nanopartikel optimiert. Die Rytov-Näherung verbessert sowohl die Auflösung als auch die Signalqualität immens gegenüber konventioneller Tomographie, zumindest in dem getesteten System eines Wolframdisulfid-Nanoröhrchens. Das Rauschverhalten scheint ähnlich der konventionallen Tomographie zu sein, also eher gutmütig. Im Gegenzug braucht die Tomographie basierend auf der Rytov-Näherung wesentlich mehr Rechenzeit pro Iteration
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Книги з теми "Cryo-electron microscopy and tomography"

1

Förster, Friedrich, and Ariane Briegel, eds. Cryo-Electron Tomography. Cham: Springer International Publishing, 2024. http://dx.doi.org/10.1007/978-3-031-51171-4.

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Adam, Kruk. Tomografia elektronowa i jej zastosowanie w obrazowaniu i metrologii mikrostruktury materiałów: Electron tomography and its application in imaging and metrology of the microstructure of materials. Kraków: Wydawnictwa AGH, 2012.

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Appasani, Krishnarao. Cryo-Electron Microscopy in Structural Biology. Boca Raton: CRC Press, 2024. http://dx.doi.org/10.1201/9781003326106.

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4

service), ScienceDirect (Online. Cryo-EM: Analyses, interpretation, and case studies. San Diego, Calif: Academic Press/Elsevier, 2010.

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Strauss, Mike. Cryo-electron microscopy of membrane proteins; lipid bilayer supports and vacuum-cryo-transfer. Ottawa: National Library of Canada, 2003.

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service), ScienceDirect (Online, ed. Cryo-EM: Sample preparation and data collection. San Diego, Calif: Academic Press/Elsevier, 2010.

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7

Frank, Joachim. Electron Tomography: Three-Dimensional Imaging with the Transmission Electron Microscope. Boston, MA: Springer US, 1992.

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8

Gutierrez-Vargas, Cristina. Single-particle cryo-electron microscopy studies of ribosomes with fragmented 28S rRNA. [New York, N.Y.?]: [publisher not identified], 2020.

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Twomey, Edward Charles. Structural Determinants of Ionotropic Glutamate Receptor Function Revealed by Cryo- electron Microscopy. [New York, N.Y.?]: [publisher not identified], 2018.

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Ho, Danny Nam. Structure Characterization of the 70S-BipA Complex Using Novel Methods of Single-Particle Cryo-Electron Microscopy. [New York, N.Y.?]: [publisher not identified], 2014.

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Частини книг з теми "Cryo-electron microscopy and tomography"

1

Plitzko, Jürgen, and Wolfgang P. Baumeister. "Cryo-Electron Tomography." In Springer Handbook of Microscopy, 189–228. Cham: Springer International Publishing, 2019. http://dx.doi.org/10.1007/978-3-030-00069-1_4.

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Zheng, Shawn, Axel Brilot, Yifan Cheng, and David A. Agard. "Beam-Induced Motion Mechanism and Correction for Improved Cryo-Electron Microscopy and Cryo-Electron Tomography." In Cryo-Electron Tomography, 293–314. Cham: Springer International Publishing, 2024. http://dx.doi.org/10.1007/978-3-031-51171-4_10.

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3

Oikonomou, Catherine M., and Grant J. Jensen. "After the Microscope: Long-Term Care of Electron Tomography Data." In Cryo-Electron Tomography, 379–89. Cham: Springer International Publishing, 2024. http://dx.doi.org/10.1007/978-3-031-51171-4_13.

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Castón, José R. "Conventional Electron Microscopy, Cryo-Electron Microscopy and Cryo-Electron Tomography of Viruses." In Subcellular Biochemistry, 79–115. Dordrecht: Springer Netherlands, 2013. http://dx.doi.org/10.1007/978-94-007-6552-8_3.

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5

Luque, Daniel, and José R. Castón. "Cryo-Electron Microscopy and Cryo-Electron Tomography of Viruses." In Physical Virology, 283–306. Cham: Springer International Publishing, 2023. http://dx.doi.org/10.1007/978-3-031-36815-8_12.

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6

Khanna, Kanika. "Emerging Technologies in Cryo-Electron Tomography." In Cryo-Electron Microscopy in Structural Biology, 395–406. Boca Raton: CRC Press, 2024. http://dx.doi.org/10.1201/9781003326106-37.

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Lučić, Vladan, and Wolfgang P. Baumeister. "3D Electron Microscopy Based on Cryo-Electron Tomography." In Encyclopedia of Biophysics, 7–10. Berlin, Heidelberg: Springer Berlin Heidelberg, 2013. http://dx.doi.org/10.1007/978-3-642-16712-6_618.

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Hanein, Dorit, and Niels Volkmann. "Functional Studies of the Actin Cytoskeleton by Cryogenic Electron Tomography." In Cryo-Electron Microscopy in Structural Biology, 418–28. Boca Raton: CRC Press, 2024. http://dx.doi.org/10.1201/9781003326106-39.

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9

Weyland, Matthew, and Paul Midgley. "Electron Tomography." In Transmission Electron Microscopy, 343–76. Cham: Springer International Publishing, 2016. http://dx.doi.org/10.1007/978-3-319-26651-0_12.

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Midgley, Paul A., and Matthew Weyland. "STEM Tomography." In Scanning Transmission Electron Microscopy, 353–92. New York, NY: Springer New York, 2010. http://dx.doi.org/10.1007/978-1-4419-7200-2_8.

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Тези доповідей конференцій з теми "Cryo-electron microscopy and tomography"

1

Zhang, Yu, Kevin Davidson, Po-Cheng Lu, Frieder Baumann, and Travis Mitchell. "Unlocking Insights into 3D Transistor Defects: The Power of Supplementing TEM with Elemental Electron Tomography." In ISTFA 2024, 374–76. ASM International, 2024. http://dx.doi.org/10.31399/asm.cp.istfa2024p0374.

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Abstract This study investigates the application of 3D electron tomography to enhance transmission electron microscopy (TEM)-based failure analysis of 3D FinFET transistors. Traditional TEM analysis is challenged by projection effects due to the thickness of the sample, complicating accurate defect characterization in miniaturized semiconductor structures. The defects seen by conventional (2D projection) TEM imaging are unclear and difficult to interpret. Leveraging scanning transmission electron microscopy (STEM) and energy dispersive X-ray spectroscopy (EDS) tomography techniques, the study presents detailed examinations of two semiconductor samples exhibiting high leakage currents. Results reveal etched-out epitaxial regions subsequently filled with gate materials, critical for understanding device failure. By digitally reconstructing TEM lamellae in three dimensions, this approach overcomes projection artifacts and precisely localizes defects. The findings underscore the efficacy of 3D electron tomography in semiconductor failure analysis, offering insights crucial for improving device reliability and manufacturing processes in advanced semiconductor technologies.
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2

Cognigni, Flavio, Giulio Lamedica, Domenico Mello, Philippe Von Gunten, Guillaume Fiannaca, Heiko Stegmann, Anton du Plessis, and Marco Rossi. "Integrating Multimodal Microscopy and Artificial Intelligence Solutions for Laser Dicing Process Induced Defect Identification." In ISTFA 2024, 273–81. ASM International, 2024. http://dx.doi.org/10.31399/asm.cp.istfa2024p0273.

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Abstract In semiconductor manufacturing, the process of laser dicing can result in a loss of yield due to defects associated to the laser interaction with the sample. These defects can be difficult to identify, especially before a proper tuning of the process. Traditional investigation methods, like infrared (IR) inspection and focused-ion beam scanning electron microscopy (FIB-SEM) analysis, are labor-intensive and lack comprehensive insights. Here, we propose a robust correlative microscopy (CM) workflow integrating IR, X-ray Microscopy (XRM), and FIB-SEM tomography analyses, leveraging artificial intelligence (AI) driven algorithm for time- and quality-improved dataset reconstruction, automatic segmentation and defect site identification. Our approach streamlines defect identification, preparation, and characterization. Through AI-enhanced methodologies, as well as femtosecond (fs) laser, we optimize investigation efficiency and extract crucial information about defects properties and evolution. Our research aims to advance semiconductor failure analysis by integrating AI for enhanced defect localization and high-quality 3D dataset acquisition in the realm of laser dicing processes.
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3

Zhang, Pengcheng, and Fei Zhou. "A novel particle picking approach for cryo-electron microscopy images." In International Conference on Cloud Computing, Performance Computing, and Deep Learning, edited by Wanyang Dai and Xiangjie Kong, 8. SPIE, 2024. http://dx.doi.org/10.1117/12.3050643.

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4

"Versatile Cryo-FIB Lamella Lift-out for Cryo-electron Tomography and Material Analysis." In Microscience Microscopy Congress 2023 incorporating EMAG 2023. Royal Microscopical Society, 2023. http://dx.doi.org/10.22443/rms.mmc2023.273.

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Brown, Hamish G., Manasi Mudaliyar, Matthew D. Johnson, Bronte A. Johnstone, Debnath Ghosal, and Eric Hanssen. "Montage electron cryo-tomography with square and rectangular beams." In 13th Asia Pacific Microscopy Congress 2025. ScienceOpen, 2025. https://doi.org/10.14293/apmc13-2025-0299.

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6

Chen, Muyuan. "Computational methods for in situ structure determination with cryo-electron tomography." In European Microscopy Congress 2020. Royal Microscopical Society, 2021. http://dx.doi.org/10.22443/rms.emc2020.196.

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Obr, Martin, and Marc Storms. "Towards Visual Proteomics: Cell Biology at High Resolution Using Cryo-Electron Tomography." In 13th Asia Pacific Microscopy Congress 2025. ScienceOpen, 2025. https://doi.org/10.14293/apmc13-2025-0248.

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"Compressive Cryo FIB-SEM Tomography." In Microscience Microscopy Congress 2023 incorporating EMAG 2023. Royal Microscopical Society, 2023. http://dx.doi.org/10.22443/rms.mmc2023.161.

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Fahy, Kenneth. "Laboratory-scale cryo soft X-ray tomography." In European Light Microscopy Initiative 2021. Royal Microscopical Society, 2021. http://dx.doi.org/10.22443/rms.elmi2021.185.

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"Laboratory-based cryo-Soft X-ray Tomography." In Microscience Microscopy Congress 2023 incorporating EMAG 2023. Royal Microscopical Society, 2023. http://dx.doi.org/10.22443/rms.mmc2023.123.

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Звіти організацій з теми "Cryo-electron microscopy and tomography"

1

Edmondson, Philip D. An On-Axis Tomography Holder for Correlative Electron and Atom Probe Microscopy. Office of Scientific and Technical Information (OSTI), October 2018. http://dx.doi.org/10.2172/1479802.

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2

Elbaum, Michael, and Peter J. Christie. Type IV Secretion System of Agrobacterium tumefaciens: Components and Structures. United States Department of Agriculture, March 2013. http://dx.doi.org/10.32747/2013.7699848.bard.

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Objectives: The overall goal of the project was to build an ultrastructural model of the Agrobacterium tumefaciens type IV secretion system (T4SS) based on electron microscopy, genetics, and immunolocalization of its components. There were four original aims: Aim 1: Define the contributions of contact-dependent and -independent plant signals to formation of novel morphological changes at the A. tumefaciens polar membrane. Aim 2: Genetic basis for morphological changes at the A. tumefaciens polar membrane. Aim 3: Immuno-localization of VirB proteins Aim 4: Structural definition of the substrate translocation route. There were no major revisions to the aims, and the work focused on the above questions. Background: Agrobacterium presents a unique example of inter-kingdom gene transfer. The process involves cell to cell transfer of both protein and DNA substrates via a contact-dependent mechanism akin to bacterial conjugation. Transfer is mediated by a T4SS. Intensive study of the Agrobacterium T4SS has made it an archetypal model for the genetics and biochemistry. The channel is assembled from eleven protein components encoded on the B operon in the virulence region of the tumor-inducing plasmid, plus an additional coupling protein, VirD4. During the course of our project two structural studies were published presenting X-ray crystallography and three-dimensional reconstruction from electron microscopy of a core complex of the channel assembled in vitro from homologous proteins of E. coli, representing VirB7, VirB9, and VirB10. Another study was published claiming that the secretion channels in Agrobacterium appear on helical arrays around the membrane perimeter and along the entire length of the bacterium. Helical arrangements in bacterial membranes have since fallen from favor however, and that finding was partially retracted in a second publication. Overall, the localization of the T4SS within the bacterial membranes remains enigmatic in the literature, and we believe that our results from this project make a significant advance. Summary of achievements : We found that polar inflations and other membrane disturbances relate to the activation conditions rather than to virulence protein expression. Activation requires low pH and nutrient-poor medium. These stress conditions are also reflected in DNA condensation to varying degrees. Nonetheless, they must be considered in modeling the T4SS as they represent the relevant conditions for its expression and activity. We identified the T4SS core component VirB7 at native expression levels using state of the art super-resolution light microscopy. This marker of the secretion system was found almost exclusively at the cell poles, and typically one pole. Immuno-electron microscopy identified the protein at the inner membrane, rather than at bridges across the inner and outer membranes. This suggests a rare or transient assembly of the secretion-competent channel, or alternatively a two-step secretion involving an intermediate step in the periplasmic space. We followed the expression of the major secreted effector, VirE2. This is a single-stranded DNA binding protein that forms a capsid around the transferred oligonucleotide, adapting the bacterial conjugation to the eukaryotic host. We found that over-expressed VirE2 forms filamentous complexes in the bacterial cytoplasm that could be observed both by conventional fluorescence microscopy and by correlative electron cryo-tomography. Using a non-retentive mutant we observed secretion of VirE2 from bacterial poles. We labeled the secreted substrates in vivo in order detect their secretion and appearance in the plant cells. However the low transfer efficiency and significant background signal have so far hampered this approach.
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Kim, Doo Nam, Andrew August, Henry Kvinge, and James Evans. Structures via Reasoning - Applying AI to Cryo Electron Microscopy to Reveal Structural Variability. Office of Scientific and Technical Information (OSTI), January 2022. http://dx.doi.org/10.2172/1989048.

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Powell, Samantha, Mowei Zhou, James Evans, Grant Johnson, and Ljiljana Pasa-Tolic. Developing High-Flux Ion Soft Landing with Mass-Selection for Improved Cryo-Electron Microscopy. Office of Scientific and Technical Information (OSTI), September 2022. http://dx.doi.org/10.2172/1984695.

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