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).

Automated data collection for electron tomography

1992 ◽  
Vol 50 (2) ◽  
pp. 1044-1045
Author(s):  
D.A. Agard ◽  
A.J. Koster ◽  
M.B. Braunfeld ◽  
J.W. Sedat
Keyword(s):  
Data Collection ◽  
Electron Tomography ◽  
Three Dimensional ◽  
Ccd Camera ◽  
Higher Order ◽  
Projection Data ◽  
Voltage Electron ◽  
Digital Format ◽  
Central Interest ◽  
Focus Variation

Three-dimensional imaging has become an important addition to the variety of methods available for research on biological structures. Non-crystalline samples can be examined by high resolution electron tomography which requires that projection data be collected over a large range of specimen tilts. Practical limitations of tomography are set by the large number of micrographs to be processed, and by the required (and tedious) recentering and refocusing of the object during data collection; especially for dose sensitive specimens. With automated electron tomography a number of these problems can be overcome. First, the images are recorded directly in digital format, using a cooled slow scan CCD camera, and, with automatic tracking and correction for image shift and focus variation, a pre-aligned dataset is obtained, with every image recorded under well defined imaging conditions.At UCSF, we use intermediate voltage electron tomography to study higher-order chromatin structure. Of central interest is elucidating the higher-order arrangement of the 30nm chromatin fiber within condensed chromosomes through several phases of the cell cycle and, in collaboration with Chris Woodcock, the structure of the 30 nm fiber.

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 ETDB-Caltech: a blockchain-based distributed public database for electron tomography

2018 ◽  
Author(s):  
Davi R. Ortega ◽  
Catherine M. Oikonomou ◽  
H. Jane Ding ◽  
Prudence Rees-Lee ◽  
Alexandria ◽  
...  
Keyword(s):  
Data Storage ◽  
Electron Tomography ◽  
Three Dimensional ◽  
Cellular Structures ◽  
Public Database ◽  
Dimensional Electron ◽  
Advantages And Disadvantages ◽  
Novel Method ◽  
User Friendly ◽  
Microscopy Techniques

AbstractThree-dimensional electron microscopy techniques like electron tomography provide valuable insights into cellular structures, and present significant challenges for data storage and dissemination. Here we explored a novel method to publicly release more than 11,000 such datasets, more than 30 TB in total, collected by our group. Our method, based on a peer-to-peer file sharing network built around a blockchain ledger, offers a distributed solution to data storage. In addition, we offer a user-friendly browser-based interface, https://etdb.caltech.edu, for anyone interested to explore and download our data. We discuss the relative advantages and disadvantages of this system and provide tools for other groups to mine our data and/or use the same approach to share their own imaging datasets.

3D Imaging and Metrology of Yttria Dispersoids in INCOLOY MA956 by Electron Tomography

2012 ◽  
Vol 186 ◽  
pp. 37-40
Author(s):  
Adam Kruk ◽  
Beata Dubiel ◽  
Aleksandra Czyrska-Filemonowicz
Keyword(s):  
3D Reconstruction ◽  
Particle Shape ◽  
Quantitative Data ◽  
3D Imaging ◽  
Electron Tomography ◽  
Three Dimensional ◽  
Ods Alloy ◽  
Meso Scale ◽  
Stem Tomography

TEM, HAADF-STEM tomography and FIB/SEM tomography studies have been carried out to visualize three-dimensional morphology of the oxide dispersoids in ferritic ODS alloy INCOLOY MA956. Electron tomography results provided quantitative data about particle shape, size and distribution of the particles, complementary to those obtained by means of quantitative TEM metallography. It was shown that FIB/SEM, a meso-scale tomography technique, is suitable for 3D reconstruction of the objects of 100 nm in size or even smaller.

Three Dimensional Energy Filtered Transmission Electron Microscopy (3D-EFTEM)

2001 ◽  
Vol 7 (S2) ◽  
pp. 1162-1163
Author(s):  
M Weyland ◽  
P.A. Midgley
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Electron Tomography ◽  
3D Structure ◽  
Three Dimensional ◽  
Biological Macromolecules ◽  
Physical Sciences ◽  
Flexible Tool ◽  
Spatially Resolved ◽  
Transmission Electron

Energy filtered transmission electron microscopy (EFTEM) has been developed in recent years to be a highly flexible tool for spatially resolved microanalysis. The basic technique has been extended to allow fully quantitative mapping of single elements, or a large range of elements using an image-spectroscopy approach . All these techniques are still limited by the nature of the TEM as a structure projector; any elemental map is only a 2D projection of what is in reality a 3D structure. A fully 3D approach to EFTEM analysis would offer additional insights into many materials problems and make possible meaningful analysis of certain systems for the first time. Three-dimensional electron microscopy (electron tomography) offers one path for the reconstruction of 3D structures from 2D projections. Although this has been well developed for solving the structure of biological macromolecules and viruses its use in the physical sciences is quite new.

Approaches for High Resolution Cryo-Electron Tomography of Biological Specimens

1997 ◽  
Vol 3 (S2) ◽  
pp. 1111-1112
Author(s):  
D.A. Agard ◽  
M.B. Braunfeld ◽  
Hans Chen ◽  
Rebecca McQuitty ◽  
John Sedat
Keyword(s):  
Data Collection ◽  
Structural Information ◽  
Electron Tomography ◽  
Three Dimensional ◽  
Electron Microscopic ◽  
Subcellular Organelles ◽  
Voltage Electron ◽  
Cell Components ◽  
Electron Microscopes ◽  
Biological Complexes

Electron tomography is a powerful tool for elucidating the three-dimensional architecture of large biological complexes and subcellular organelles. Use of intermediate voltage electron microscopes extended the technique by providing the means to examine very large and non-symmetrical subcellular organelles, at resolutions beyond what would be possible using light microscopy. Recent studies using electron tomography on a variety cellular organelles and assemblies such as centrosomes (Moritz et al.,1995a,b), kinetochores (McEwen, 1993) and chromatin (Woodcock, 1994), have clearly demonstrated the power of this method for obtaining 3D structural information on non-symmetric cell components. When combined with biochemical and molecular observations, these 3D reconstructions have provided significant new insights into biological function.Although the information that tomography provides is unique, its use as a general tool in the biological community has been limited due to the complexities involved in data collection and processing.We are simultaneously trying to make this approach accessible through automation as well as trying to extend the resolution and accuracy of the reconstructions. Significant, has been the use of low-dose cryo-electron microscopic automated data collection methods.

3-D reconstruction of molecular shape from microcrystal thin sections

1983 ◽  
Vol 41 ◽  
pp. 748-749
Author(s):  
S. Cusack ◽  
J.-C. Jésior
Keyword(s):  
Three Dimensional ◽  
Molecular Shape ◽  
Biological Molecules ◽  
Fourier Space ◽  
Single Particles ◽  
Thin Sections ◽  
Standard Methods ◽  
X Ray ◽  
X Ray Crystallography ◽  
Dimensional Reconstruction

Three-dimensional reconstruction techniques using electron microscopy have been principally developed for application to 2-D arrays (i.e. monolayers) of biological molecules and symmetrical single particles (e.g. helical viruses). However many biological molecules that crystallise form multilayered microcrystals which are unsuitable for study by either the standard methods of 3-D reconstruction or, because of their size, by X-ray crystallography. The grid sectioning technique enables a number of different projections of such microcrystals to be obtained in well defined directions (e.g. parallel to crystal axes) and poses the problem of how best these projections can be used to reconstruct the packing and shape of the molecules forming the microcrystal.Given sufficient projections there may be enough information to do a crystallographic reconstruction in Fourier space. We however have considered the situation where only a limited number of projections are available, as for example in the case of catalase platelets where three orthogonal and two diagonal projections have been obtained (Fig. 1).

Design and calibration of an IVEM for general 3-dimensional imaging of non-diffracting materials

1992 ◽  
Vol 50 (2) ◽  
pp. 1040-1041
Author(s):  
Neil Rowlands ◽  
Jeff Price ◽  
Michael Kersker ◽  
Seichi Suzuki ◽  
Steve Young ◽  
...  
Keyword(s):  
3D Imaging ◽  
Tomographic Reconstruction ◽  
Three Dimensional ◽  
3 Dimensional ◽  
3D Microstructure ◽  
Image Translation ◽  
Digital Correlation ◽  
On Line ◽  
Mechanical Stage ◽  
Projection Angle

Three-dimensional (3D) microstructure visualization on the electron microscope requires that the sample be tilted to different positions to collect a series of projections. This tilting should be performed rapidly for on-line stereo viewing and precisely for off-line tomographic reconstruction. Usually a projection series is collected using mechanical stage tilt alone. The stereo pairs must be viewed off-line and the 60 to 120 tomographic projections must be aligned with fiduciary markers or digital correlation methods. The delay in viewing stereo pairs and the alignment problems in tomographic reconstruction could be eliminated or improved by tilting the beam if such tilt could be accomplished without image translation.A microscope capable of beam tilt with simultaneous image shift to eliminate tilt-induced translation has been investigated for 3D imaging of thick (1 μm) biologic specimens. By tilting the beam above and through the specimen and bringing it back below the specimen, a brightfield image with a projection angle corresponding to the beam tilt angle can be recorded (Fig. 1a).

Automated electron tomography: from data collection to image processing

1995 ◽  
Vol 53 ◽  
pp. 26-27
Author(s):  
Weiping Liu ◽  
Jennifer Fung ◽  
W.J. de Ruijter ◽  
Hans Chen ◽  
John W. Sedat ◽  
...  
Keyword(s):  
Image Processing ◽  
Data Collection ◽  
Structural Information ◽  
Imaging Technique ◽  
Electron Tomography ◽  
Ccd Camera ◽  
Automated Data Collection ◽  
Full Control ◽  
Transmission Electron ◽  
Amorphous Structures

Electron tomography is a technique where many projections of an object are collected from the transmission electron microscope (TEM), and are then used to reconstruct the object in its entirety, allowing internal structure to be viewed. As vital as is the 3-D structural information and with no other 3-D imaging technique to compete in its resolution range, electron tomography of amorphous structures has been exercised only sporadically over the last ten years. Its general lack of popularity can be attributed to the tediousness of the entire process starting from the data collection, image processing for reconstruction, and extending to the 3-D image analysis. We have been investing effort to automate all aspects of electron tomography. Our systems of data collection and tomographic image processing will be briefly described.To date, we have developed a second generation automated data collection system based on an SGI workstation (Fig. 1) (The previous version used a micro VAX). The computer takes full control of the microscope operations with its graphical menu driven environment. This is made possible by the direct digital recording of images using the CCD camera.

Defocus ramp correction in spot-scan imaging

1992 ◽  
Vol 50 (1) ◽  
pp. 130-131
Author(s):  
Kenneth H. Downing
Keyword(s):  
Computer Control ◽  
Three Dimensional ◽  
Sufficient Accuracy ◽  
Objective Lens ◽  
Electron Crystallography ◽  
Contrast Transfer Function ◽  
Tilt Axis ◽  
Specimen Height ◽  
The Difference ◽  
Envelope Functions

Three-dimensional structures of a number of samples have been determined by electron crystallography. The procedures used in this work include recording images of fairly large areas of a specimen at high tilt angles. There is then a large defocus ramp across the image, and parts of the image are far out of focus. In the regions where the defocus is large, the contrast transfer function (CTF) varies rapidly across the image, especially at high resolution. Not only is the CTF then difficult to determine with sufficient accuracy to correct properly, but the image contrast is reduced by envelope functions which tend toward a low value at high defocus.We have combined computer control of the electron microscope with spot-scan imaging in order to eliminate most of the defocus ramp and its effects in the images of tilted specimens. In recording the spot-scan image, the beam is scanned along rows that are parallel to the tilt axis, so that along each row of spots the focus is constant. Between scan rows, the objective lens current is changed to correct for the difference in specimen height from one scan to the next.

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