scholarly journals Electron tomography of cells

2011 ◽  
Vol 45 (1) ◽  
pp. 27-56 ◽  
Author(s):  
Lu Gan ◽  
Grant J. Jensen
Keyword(s):  
Electron Microscope ◽  
Cell Biology ◽  
3D Imaging ◽  
Imaging Technique ◽  
Electron Tomography ◽  
Three Dimensional ◽  
Molecular Organization ◽  
Biological Structure ◽  
Structural Cell ◽  
Projection Images

AbstractThe electron microscope has contributed deep insights into biological structure since its invention nearly 80 years ago. Advances in instrumentation and methodology in recent decades have now enabled electron tomography to become the highest resolution three-dimensional (3D) imaging technique available for unique objects such as cells. Cells can be imaged either plastic-embedded or frozen-hydrated. Then the series of projection images are aligned and back-projected to generate a 3D reconstruction or ‘tomogram’. Here, we review how electron tomography has begun to reveal the molecular organization of cells and how the existing and upcoming technologies promise even greater insights into structural cell biology.

scholarly journals Hyperspectral Three-Dimensional Fluorescence Imaging Using Snapshot Optical Tomography

Sensors ◽  
2021 ◽  
Vol 21 (11) ◽  
pp. 3652
Author(s):  
Cory Juntunen ◽  
Isabel M. Woller ◽  
Yongjin Sung
Keyword(s):  
Fourier Transform ◽  
3D Imaging ◽  
Optical Tomography ◽  
Three Dimensional ◽  
High Spectral Resolution ◽  
Functional Information ◽  
Fluorescent Beads ◽  
Different Depths ◽  
Projection Images ◽  
Imaging Performance

Hyperspectral three-dimensional (3D) imaging can provide both 3D structural and functional information of a specimen. The imaging throughput is typically very low due to the requirement of scanning mechanisms for different depths and wavelengths. Here we demonstrate hyperspectral 3D imaging using Snapshot projection optical tomography (SPOT) and Fourier-transform spectroscopy (FTS). SPOT allows us to instantaneously acquire the projection images corresponding to different viewing angles, while FTS allows us to perform hyperspectral imaging at high spectral resolution. Using fluorescent beads and sunflower pollens, we demonstrate the imaging performance of the developed system.

A Novel Method for Automated Acquisition of Tilt Series for Electron Tomography Based on Pre-Calibration of the Specimen Stage

2000 ◽  
Vol 6 (S2) ◽  
pp. 1148-1149
Author(s):  
U. Ziese ◽  
A.H. Janssen ◽  
T.P. van der Krift ◽  
A.G. van Balen ◽  
W.J. de Ruijter ◽  
...  
Keyword(s):  
High Resolution ◽  
3D Reconstruction ◽  
Cell Biology ◽  
Electron Tomography ◽  
Three Dimensional ◽  
Imaging Method ◽  
3D Images ◽  
Tilt Series ◽  
3D Information ◽  
Structural Arrangements

Electron tomography is a three-dimensional (3D) imaging method with transmission electron microscopy (TEM) that provides high-resolution 3D images of structural arrangements. Conventional TEM images are in first approximation mere 2D-projections of a 3D sample under investigation. With electron tomographya series of images is acquired of a sample that is tilted over a large angular range (±70°) with small angular tilt increments (so called tilt-series). For the subsequent 3D-reconstruction, the images of the tilt series are aligned relative to each other and the 3D-reconstruction is computed. Electron tomography is the only technique that can provide true 3D information with nm-scale resolution of individual and unique samples. For (cell) biology and material science applications the availability of high-resolution 3D images of structural arrangements within individual samples provides unique architectural information that cannot be obtained otherwise. Routine application of electron tomography will comprise a major revolutionary step forward in the characterization of complex materials and cellular arrangements.

scholarly journals Cellular Electron Cryo-Tomography to Study Virus-Host Interactions

2020 ◽  
Vol 7 (1) ◽  
pp. 239-262
Author(s):  
Emmanuelle R.J. Quemin ◽  
Emily A. Machala ◽  
Benjamin Vollmer ◽  
Vojtěch Pražák ◽  
Daven Vasishtan ◽  
...  
Keyword(s):  
Viral Entry ◽  
Cell Biology ◽  
Molecular Mechanisms ◽  
Virus Assembly ◽  
Three Dimensional ◽  
Surface Proteins ◽  
Host Cells ◽  
Phase Plate ◽  
Structural Cell ◽  
Host Interactions

Viruses are obligatory intracellular parasites that reprogram host cells upon infection to produce viral progeny. Here, we review recent structural insights into virus-host interactions in bacteria, archaea, and eukaryotes unveiled by cellular electron cryo-tomography (cryoET). This advanced three-dimensional imaging technique of vitreous samples in near-native state has matured over the past two decades and proven powerful in revealing molecular mechanisms underlying viral replication. Initial studies were restricted to cell peripheries and typically focused on early infection steps, analyzing surface proteins and viral entry. Recent developments including cryo-thinning techniques, phase-plate imaging, and correlative approaches have been instrumental in also targeting rare events inside infected cells. When combined with advances in dedicated image analyses and processing methods, details of virus assembly and egress at (sub)nanometer resolution were uncovered. Altogether, we provide a historical and technical perspective and discuss future directions and impacts of cryoET for integrative structural cell biology analyses of viruses.

Three Dimensional imaging of biological macromolecules

1995 ◽  
Vol 53 ◽  
pp. 732-733
Author(s):  
A.J. Koster ◽  
J. Walz ◽  
D. Typke ◽  
M. Nitsch ◽  
W. Baumeister
Keyword(s):  
Data Collection ◽  
Structure Function ◽  
3D Imaging ◽  
Electron Tomography ◽  
Three Dimensional ◽  
Biological Molecules ◽  
Cellular Structures ◽  
Biological Macromolecules ◽  
Tilt Axis ◽  
Average Structure

3D imaging of both cellular structures as well as molecular assemblies of biological molecules has become an increasingly useful tool to study structure-function relationships of biological systems. In this paper instrumental and methodological developments are discussed towards automated 3D imaging, which will be illustrated by examples of structures studied in Martinsried. To image individual structures with dimensions in the range of 10-500 nm with a resolution of 1-5 nm, electron tomography is the only technique available. The strategy of choice depends on size and shape of the structure to be reconstructed. Single-tilt axis tomography is suitable for the reconstruction of unique structures (for example, irregularly shaped viruses or cellular structures). Random conical-tilt data collection, as well as angular reconstitution techniques, can be used to reconstruct the average structure of many copies of a particle, such as those present in suspension of one kind of protein. To reconstruct a unique structure with single-tilt axis tomography the tilt range and tilt increments are chosen to meet the resolution desired within the constraint of the allowable electron doses (Table 1).

Application of low-voltage, high-resolution SEM in the study of complex intracellular structures

1990 ◽  
Vol 48 (3) ◽  
pp. 18-19
Author(s):  
Hans Ris
Keyword(s):  
High Resolution ◽  
Cell Biology ◽  
Low Voltage ◽  
Three Dimensional ◽  
Nuclear Pore ◽  
Structural Cell ◽  
Macromolecular Assemblies ◽  
New Information ◽  
A Cell ◽  
Ultrathin Sections

Cellular architecture is a dynamic web of complex macromolecular assemblies accomplishing the diverse functions of a cell. Conventional electron microscopy on ultrathin sections or negatively stained preparations can provide little information on more extended three dimensional assemblies. High voltage and intermediate voltage TEM provide high resolution in much thicker specimens but are limited by problems of contrast and overlap of structures. In recent years new SEMs have become available that provide the high topographic contrast and three dimensionality of SEM at a resolution comparable to conventional TEM. I have used the low voltage high resolution SEM Hitachi S-900 at the Madison IMR and shall show some examples that illustrate the usefulness of LVSEM in structural cell biology. Most striking is the new information obtained about the nuclear pore complex (NPC). This structure is extremely important in controlling the selective and unidirectional transport of large molecules into and out of the nucleus.

scholarly journals Structural Cell Biology: Preparing Specimens for Cryo-Electron Tomography Using Focused-Ion-Beam Milling

2014 ◽  
Vol 20 (S3) ◽  
pp. 1222-1223
Author(s):  
Elizabeth Villa ◽  
Miroslava Schaffer ◽  
Ben Engel ◽  
Jürgen Plitzko ◽  
Wolfgang Baumeister
Keyword(s):  
Cell Biology ◽  
Focused Ion Beam ◽  
Electron Tomography ◽  
Ion Beam ◽  
Structural Cell ◽  
Cryo Electron Tomography ◽  
Focused Ion Beam Milling

scholarly journals Cryo-electron tomography: an ideal method to study membrane-associated proteins

2017 ◽  
Vol 372 (1726) ◽  
pp. 20160210 ◽  
Author(s):  
Michelle A. Dunstone ◽  
Alex de Marco
Keyword(s):  
Image Processing ◽  
Structural Information ◽  
Imaging Technique ◽  
Electron Tomography ◽  
Three Dimensional ◽  
Macromolecular Complexes ◽  
Membrane Pores ◽  
Cryo Electron Tomography ◽  
Associated Proteins ◽  
Ideal Method

Cryo-electron tomography (cryo-ET) is a three-dimensional imaging technique that makes it possible to analyse the structure of complex and dynamic biological assemblies in their native conditions. The latest technological and image processing developments demonstrate that it is possible to obtain structural information at nanometre resolution. The sample preparation required for the cryo-ET technique does not require the isolation of a protein and other macromolecular complexes from its native environment. Therefore, cryo-ET is emerging as an important tool to study the structure of membrane-associated proteins including pores. This article is part of the themed issue ‘Membrane pores: from structure and assembly, to medicine and technology’.

Cryo-fixation and associated developments in transmission electron microscopy: a cool future for nematology

Nematology ◽  
2016 ◽  
Vol 18 (1) ◽  
pp. 1-14 ◽  
Author(s):  
Wim Bert ◽  
Dieter Slos ◽  
Olivier Leroux ◽  
Myriam Claeys
Keyword(s):  
Electron Microscopy ◽  
Cell Biology ◽  
Electron Tomography ◽  
Light And Electron Microscopy ◽  
Three Dimensional ◽  
Freeze Substitution ◽  
Model Organisms ◽  
Fixation Method ◽  
Dimensional Reconstruction ◽  
Fixation Techniques

At present, the importance of sample preparation equipment for electron microscopy represents the driving force behind major breakthroughs in microscopy and cell biology. In this paper we present an introduction to the most commonly used cryo-fixation techniques, with special attention paid towards high-pressure freezing followed by freeze substitution. Techniques associated with cryo-fixation, such as immunolocalisation, cryo-sectioning, and correlative light and electron microscopy, are also highlighted. For studies that do not require high resolution, high quality results, or the immediate arrest of certain processes, conventional methods will provide answers to many questions. For some applications, such as immunocytochemistry, three-dimensional reconstruction of serial sections or electron tomography, improved preservation of the ultrastructure is required. This review of nematode cryo-fixation highlights that cryo-fixation not only results in a superior preservation of fine structural details, but also underlines the fact that some observations based on results solely obtained through conventional fixation approaches were either incorrect, or otherwise had severe limitations. Although the use of cryo-fixation has hitherto been largely restricted to model organisms, the advantages of cryo-fixation are sufficiently self-evident that we must conclude that the cryo-fixation method is highly likely to become the standard for nematode fixation in the near future.

scholarly journals Cryo-electron tomography: The challenge of doing structural biology in situ

2013 ◽  
Vol 202 (3) ◽  
pp. 407-419 ◽  
Author(s):  
Vladan Lučić ◽  
Alexander Rigort ◽  
Wolfgang Baumeister
Keyword(s):  
Cell Biology ◽  
Structural Information ◽  
Electron Tomography ◽  
Three Dimensional ◽  
Molecular Structures ◽  
New Techniques ◽  
Preparation Methods ◽  
Technical Advances ◽  
Macromolecular Organization

Electron microscopy played a key role in establishing cell biology as a discipline, by producing fundamental insights into cellular organization and ultrastructure. Many seminal discoveries were made possible by the development of new sample preparation methods and imaging modalities. Recent technical advances include sample vitrification that faithfully preserves molecular structures, three-dimensional imaging by electron tomography, and improved image-processing methods. These new techniques have enabled the extraction of high fidelity structural information and are beginning to reveal the macromolecular organization of unperturbed cellular environments.

scholarly journals Seeing gene expression in cells: the future of structural biology

2021 ◽  
Vol 10 ◽  
Author(s):  
Wei Dai ◽  
Seth A Darst ◽  
Christine M Dunham ◽  
Robert Landick ◽  
Gregory Petsco ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Biology ◽  
Electron Tomography ◽  
Molecular Assembly ◽  
Bacterial Cells ◽  
Major Step ◽  
Structural Cell ◽  
The Future ◽  
In Cells

Although much is known about the machinery that executes fundamental processes of gene expression in cells, much also remains to be learned about how that machinery works. A recent paper by O’Reilly et al. reports a major step forward in the direct visualization of central dogma processes at submolecular resolution inside bacterial cells frozen in a native state. The essential methodologies involved are cross-linking mass spectrometry (CLMS) and cryo-electron tomography (cryo-ET). In-cell CLMS provides in vivo protein interaction maps. Cryo-ET allows visualization of macromolecular complexes in their native environment. These methods have been integrated by O’Reilly et al. to describe a dynamic assembly in situ between a transcribing RNA polymerase (RNAP) and a translating ribosome – a complex known as the expressome – in the model bacterium Mycoplasma pneumoniae 1 . With the application of improved data processing and classification capabilities, this approach has allowed unprecedented insights into the architecture of this molecular assembly line, confirming the existence of a physical link between RNAP and the ribosome and identifying the transcription factor NusA as the linking molecule, as well as making it possible to see the structural effects of drugs that inhibit either transcription or translation. The work provides a glimpse into the future of integrative structural cell biology and can serve as a roadmap for the study of other molecular machineries in their native context.

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