scholarly journals Electron Tomography and Simulation of Baculovirus Actin Comet Tails Support a Tethered Filament Model of Pathogen Propulsion

PLoS Biology ◽  
2014 ◽  
Vol 12 (1) ◽  
pp. e1001765 ◽  
Author(s):  
Jan Mueller ◽  
Julia Pfanzelter ◽  
Christoph Winkler ◽  
Akihiro Narita ◽  
Christophe Le Clainche ◽  
...  
Keyword(s):  
Electron Tomography ◽  
Actin Comet Tails

Pushing with actin: from cells to pathogens

2015 ◽  
Vol 43 (1) ◽  
pp. 84-91 ◽  
Author(s):  
J. Victor Small
Keyword(s):  
Actin Filaments ◽  
Actin Polymerization ◽  
Electron Tomography ◽  
Function Analysis ◽  
Host Cells ◽  
Actin Networks ◽  
Resolution Model ◽  
Image Averaging ◽  
Actin Comet Tails

Actin polymerization is harnessed by cells to generate lamellipodia for movement and by a subclass of pathogens to facilitate invasion of their infected hosts. Using electron tomography (ET), we have shown that lamellipodia are formed via the generation of subsets of actin filaments joined by branch junctions. Image averaging produced a 2.9 nm resolution model of branch junctions in situ and revealed a close fit to the electron density map of the actin-related protein 2/3 (Arp2/3)–actin complex in vitro. Correlated live-cell imaging and ET was also used to determine how actin networks are created and remodelled during the initiation and inhibition of protrusion in lamellipodia. Listeria, Rickettsia and viruses, such as vaccinia virus and baculovirus, exploit the actin machinery of host cells to generate propulsive actin comet tails to disseminate their infection. By applying ET, we have shown that baculovirus generates at its rear a fishbone-like array of subsets of branched actin filaments, with an average of only four filaments engaged in pushing at any one time. In both of these studies, the application of ET of negatively stained cytoskeletons for higher filament resolution and cryo-ET for preserving overall 3D morphology was crucial for obtaining a complete structure–function analysis of actin-driven propulsion.

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.

Practical electron tomography

1995 ◽  
Vol 53 ◽  
pp. 742-743
Author(s):  
C.L. Woodcock
Keyword(s):  
Electron Tomography ◽  
Tomographic Reconstruction ◽  
Three Dimensional ◽  
3D Shape ◽  
Computational Resources ◽  
Shape Of Particles

Despite the potential of the technique, electron tomography has yet to be widely used by biologists. This is in part related to the rather daunting list of equipment and expertise that are required. Thanks to continuing advances in theory and instrumentation, tomography is now more feasible for the non-specialist. One barrier that has essentially disappeared is the expense of computational resources. In view of this progress, it is time to give more attention to practical issues that need to be considered when embarking on a tomographic project. The following recommendations and comments are derived from experience gained during two long-term collaborative projects.Tomographic reconstruction results in a three dimensional description of an individual EM specimen, most commonly a section, and is therefore applicable to problems in which ultrastructural details within the thickness of the specimen are obscured in single micrographs. Information that can be recovered using tomography includes the 3D shape of particles, and the arrangement and dispostion of overlapping fibrous and membranous structures.

Three-Dimensional reconstruction of isolated centrosomes by automated electron tomography

1994 ◽  
Vol 52 ◽  
pp. 310-311
Author(s):  
M.B. Braunfeld ◽  
M. Moritz ◽  
B.M. Alberts ◽  
J.W. Sedat ◽  
D.A. Agard
Keyword(s):  
Electron Tomography ◽  
Three Dimensional ◽  
Vital Role ◽  
Complex Nature ◽  
Animal Cells ◽  
Microtubule Organizing Center ◽  
Nucleation Sites ◽  
Dimensional Reconstruction ◽  
Organizing Center ◽  
Resolution Model

In animal cells, the centrosome functions as the primary microtubule organizing center (MTOC). As such the centrosome plays a vital role in determining a cell's shape, migration, and perhaps most importantly, its division. Despite the obvious importance of this organelle little is known about centrosomal regulation, duplication, or how it nucleates microtubules. Furthermore, no high resolution model for centrosomal structure exists.We have used automated electron tomography, and reconstruction techniques in an attempt to better understand the complex nature of the centrosome. Additionally we hope to identify nucleation sites for microtubule growth.Centrosomes were isolated from early Drosophila embryos. Briefly, after large organelles and debris from homogenized embryos were pelleted, the resulting supernatant was separated on a sucrose velocity gradient. Fractions were collected and assayed for centrosome-mediated microtubule -nucleating activity by incubating with fluorescently-labeled tubulin subunits. The resulting microtubule asters were then spun onto coverslips and viewed by fluorescence microscopy.

On the Use of POCS for Restoring the “Missing Wedge” in Electron Tomography

1990 ◽  
Vol 48 (1) ◽  
pp. 520-521
Author(s):  
Neng-Yu Zhang ◽  
Bruce F. McEwen ◽  
Joachim Frank
Keyword(s):  
Function Space ◽  
Convex Sets ◽  
Electron Tomography ◽  
Spinal Ganglion ◽  
Fourier Space ◽  
Missing Information ◽  
Voltage Electron ◽  
High Voltage Electron Microscope ◽  
Energy Bound ◽  
Spatial Boundedness

Reconstructions of asymmetric objects computed by electron tomography are distorted due to the absence of information, usually in an angular range from 60 to 90°, which produces a “missing wedge” in Fourier space. These distortions often interfere with the interpretation of results and thus limit biological ultrastructural information which can be obtained. We have attempted to use the Method of Projections Onto Convex Sets (POCS) for restoring the missing information. In POCS, use is made of the fact that known constraints such as positivity, spatial boundedness or an upper energy bound define convex sets in function space. Enforcement of such constraints takes place by iterating a sequence of function-space projections, starting from the original reconstruction, onto the convex sets, until a function in the intersection of all sets is found. First applications of this technique in the field of electron microscopy have been promising.To test POCS on experimental data, we have artificially reduced the range of an existing projection set of a selectively stained Golgi apparatus from ±60° to ±50°, and computed the reconstruction from the reduced set (51 projections). The specimen was prepared from a bull frog spinal ganglion as described by Lindsey and Ellisman and imaged in the high-voltage electron microscope.

Electron tomography of microelectronic device interconnects

2006 ◽  
Vol 97 (7) ◽  
pp. 880-884
Author(s):  
Qing Yang ◽  
John Mardinly ◽  
Christian Kübel ◽  
Chris Nelson ◽  
Christian Kisielowski
Keyword(s):  
Electron Tomography ◽  
Microelectronic Device

Embedded Gold Markers for Improved TEM/STEM Tomography Reconstruction

2008 ◽  
Author(s):  
Jian-Shing Luo ◽  
Chia-Chi Huang ◽  
Jeremy D. Russell
Keyword(s):  
3D Reconstruction ◽  
Feature Tracking ◽  
Semiconductor Device ◽  
Electron Tomography ◽  
Tracking Process ◽  
Carbon Coated ◽  
Novel Method ◽  
Tomography Reconstruction ◽  
Tilt Series ◽  
20 Nm

Abstract Electron tomography includes four main steps: tomography data acquisition, image processing, 3D reconstruction, and visualization. After acquisition, tilt-series alignments are performed. Two methods are used to align the tilt-series: cross-correlation and feature tracking. Normally, about 10-20 nm of fiducial markers, such as gold beads, are deposited onto one side of 100 mesh carbon-coated grids during the feature-tracking process. This paper presents a novel method for preparing electron tomography samples with gold beads inside to improve the feature tracking process and quality of 3D reconstruction. Results show that the novel electron tomography sample preparation method improves image alignment, which is essential for successful tomography in many contemporary semiconductor device structures.

Sign in / Sign up

Export Citation Format

Share Document