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.

Design and performance of instrumentation for automated single-tilt-axis electron tomography

1993 ◽  
Vol 51 ◽  
pp. 448-449
Author(s):  
A.J. Koster ◽  
H. Chen ◽  
W. Clyborne ◽  
J.W. Sedat ◽  
D.A. Agard
Keyword(s):  
Data Collection ◽  
Electron Tomography ◽  
Limiting Factors ◽  
Projection Data ◽  
Reconstruction Algorithms ◽  
Native Form ◽  
Voltage Electron ◽  
Digital Format ◽  
High Resolution Analysis ◽  
And Performance

One of the driving questions in our group is into understanding how chromosomes are constructed from fibers of DNA wrapped around histones in their native form. To permit high resolution analysis of these highly complex fibers, we use intermediate voltage electron tomography. To obtain 50Å resolutions, we incorporate new approaches to overcome the resolution limiting factors determined by specimen fixation and staining techniques, data collection and 3D reconstruction algorithms.With our present instrumentation we can automatically collect a series of projection data of large, radiation sensitive objects with only a minimum of manual operation, with high accuracy and consistency. The images are recorded directly in digital format to overcome the time consuming task of digitizing negatives. Furthermore, the system offers automated eucentricity setting, automatic tracking of image shifts, and automatic focusing during data collection. Highly reliable data collection is ensured by closely monitoring the variation in image shift, defocus, average image intensity, and exposure time throughout the tilt series.

A Novel Method of Data Collection for Automated Electron Tomography Based upon Pre-cal1bration of Image Shifts and Defocus Changes

2001 ◽  
Vol 7 (S2) ◽  
pp. 78-79
Author(s):  
Ulrike Ziese ◽  
Ries Janssen ◽  
Willie Geerts ◽  
Theo van der Krift ◽  
Auke van Balen ◽  
...  
Keyword(s):  
Data Collection ◽  
3D Reconstruction ◽  
Electron Tomography ◽  
Ccd Camera ◽  
Current Status ◽  
Imaging Method ◽  
Major Step ◽  
Experimental Conditions ◽  
Digital Format ◽  
Tilt Series

Electron tomography is a three-dimensional (3D) imaging method with transmission electron microscopy (TEM) that provides high-resolution 3D images of structural arrangements. with electron tomography a series of images is acquired of a sample that is tilted over a large angular range (±70°) with small angular tilt increments. For the 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 3D information with nm-scale resolution of individual and unique samples. Routine application of electron tomography will comprise a major step forward in the characterization of complex materials and cellular arrangements. When collecting tilt series for electron tomography image shifts and defocus changes have to be corrected for by the human operator. The repetitive correction of these changes is highly time consuming, error prone and very hard to carry out under low-dose imaging conditions.Many practical problems are overcome when electron tomography data collection is performed in an automated fashion. Automation includes the (a) image acquisition on a (digital) CCD camera, which implies that (b) changes in image position and defocus can be detected by on-line image processing and (c) immediately be corrected for by computer control of the microscope, (d) Finally, tilt series are directly available in digital format for subsequent processing. Typically, carrying out such an experiment would take a day, and the actual data collection 2-4 hours. in spite of the enormous progress made in terms of data collection speed during the last few years, the current status of automated tomography still does not meet the requirements that would make it a routinely applicable tool. For a great number of biological assays and research projects, results obtained under different experimental conditions have to be compared, and series of experiments have to be carried out. Therefore, we propose a novel approach for recording a tilt series that significantly increases data collection speed, and widens the applicability of the technique.

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.

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.

Three-dimensional tomography using a soft X-ray holographic microscope and CCD camera

1998 ◽  
Vol 5 (3) ◽  
pp. 1088-1089 ◽  
Author(s):  
Norio Watanabe ◽  
Sadao Aoki
Keyword(s):  
Tungsten Wire ◽  
Three Dimensional ◽  
Numerical Aperture ◽  
Ccd Camera ◽  
Depth Resolution ◽  
Projection Data ◽  
Back Projection ◽  
X Ray ◽  
Dimensional Reconstruction ◽  
Transverse Resolution

The depth resolution of a soft X-ray hologram is much worse than its transverse resolution because a single soft X-ray hologram has a small numerical aperture. To obtain a three-dimensional image, in-line holograms of a specimen were recorded from various directions and reconstructed to obtain two-dimensional projection data. Then, a three-dimensional reconstruction was performed by back-projection of these reconstructed holograms. Three-dimensional images of a tungsten wire of diameter 10 µm and a fossil of a diatom were obtained.

Structured-Light Based Rapid 3D Measurement of Plant Canopy Structure

2014 ◽  
Vol 701-702 ◽  
pp. 549-553 ◽  
Author(s):  
Zhe Chen ◽  
Xu Dong Li ◽  
Shao Guang Shi ◽  
Hong Zhi Jiang ◽  
Hui Jie Zhao
Keyword(s):  
Data Collection ◽  
Structure Data ◽  
Measurement System ◽  
Structured Light ◽  
Canopy Structure ◽  
Three Dimensional ◽  
Ccd Camera ◽  
Plant Canopy ◽  
Laser Stripe ◽  
3D Data

Density three dimensional plant canopy structure data has numerous applications in agriculture, but many existing 3D data collection approaches are time-consuming. In this paper, we present a measurement system based on structured-light for plant canopy structure data collection. The structured-light projector projects laser beam reflected by dual-oscillating mirror, arrives to the plant canopy, which is captured by a camera. We propose a new scanning mode, that is, during one exposure time of CCD camera, one mirror keeps moving in high frequency and small angle, while the other one maintains the same position, so that we can get a laser stripe rather than a spot in each image, from which about 100 sub-pixel centers of laser stripe can be extracted. Experiments show that the measurement system can rapid collect three dimensional information of the plant.

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).

Properties and dose dependence of embedding media for cryo-automated electron tomography

1993 ◽  
Vol 51 ◽  
pp. 436-437
Author(s):  
M.B. Braunfeld ◽  
A.J. Koster ◽  
J.W. Sedat ◽  
D.A. Agard
Keyword(s):  
Data Collection ◽  
Radiation Damage ◽  
Electron Tomography ◽  
Dose Dependence ◽  
Electron Microscopic ◽  
Voltage Electron ◽  
High Doses ◽  
Beam Dose ◽  
Stability And Consistency ◽  
Imaging Conditions

Electron tomography is well suited to the study of complicated, non symmetric biological structures. In our laboratory, we use intermediate voltage electron microscopic tomography to follow complex paths of chromatin fibers within intact sections of Hela telophase chromosomes. In order to accurately reconstruct these features at resolutions beyond 50Å, precise imaging conditions and data collection schemes have been developed and employed.To obtain useful high resolution information, the specimen needs to be well preserved. Data collection must also be accurate and self-consistent. However, a serious limitation has been radiation damage to the specimen during scanning, and data collection. Because of the high doses required for tomography, the standard approach has been to accept the inevitability of serious radiation damage, and to heavily pre-irradiate the sample in an attempt to provide stability and consistency during data collection.The use of fully-automated data collection methods allows a substantial decrease in beam dose, suggesting that the entire approach should be reevaluated.

scholarly journals The in situ structures of mono-, di-, and trinucleosomes in human heterochromatin

2018 ◽  
Vol 29 (20) ◽  
pp. 2450-2457 ◽  
Author(s):  
Shujun Cai ◽  
Désirée Böck ◽  
Martin Pilhofer ◽  
Lu Gan
Keyword(s):  
Basic Principle ◽  
Electron Tomography ◽  
Three Dimensional ◽  
Higher Order ◽  
Conformational Variability ◽  
Repressive Chromatin ◽  
Cryo Electron Tomography ◽  
Chromatin Folding

The in situ three-dimensional organization of chromatin at the nucleosome and oligonucleosome levels is unknown. Here we use cryo-electron tomography to determine the in situ structures of HeLa nucleosomes, which have canonical core structures and asymmetric, flexible linker DNA. Subtomogram remapping suggests that sequential nucleosomes in heterochromatin follow irregular paths at the oligonucleosome level. This basic principle of higher-order repressive chromatin folding is compatible with the conformational variability of the two linker DNAs at the single-nucleosome level.

scholarly journals High-Voltage Electron Tomography of Spindle Pole Bodies and Early Mitotic Spindles in the YeastSaccharomyces cerevisiae

1999 ◽  
Vol 10 (6) ◽  
pp. 2017-2031 ◽  
Author(s):  
Eileen T. O’Toole ◽  
Mark Winey ◽  
J. Richard McIntosh
Keyword(s):  
High Voltage ◽  
Electron Tomography ◽  
Three Dimensional ◽  
Spindle Pole ◽  
Dimensional Structure ◽  
Microtubule Organizing Center ◽  
Median Length ◽  
Intact Cells ◽  
Voltage Electron ◽  
High Voltage Electron

The spindle pole body (SPB) is the major microtubule-organizing center of budding yeast and is the functional equivalent of the centrosome in higher eukaryotic cells. We used fast-frozen, freeze-substituted cells in conjunction with high-voltage electron tomography to study the fine structure of the SPB and the events of early spindle formation. Individual structures were imaged at 5–10 nm resolution in three dimensions, significantly better than can be achieved by serial section electron microscopy. The SPB is organized in distinct but coupled layers, two of which show ordered two-dimensional packing. The SPB central plaque is anchored in the nuclear envelope with hook-like structures. The minus ends of nuclear microtubules (MTs) are capped and are tethered to the SPB inner plaque, whereas the majority of MT plus ends show a distinct flaring. Unbudded cells containing a single SPB retain 16 MTs, enough to attach to each of the expected 16 chromosomes. Their median length is ∼150 nm. MTs growing from duplicated but not separated SPBs have a median length of ∼130 nm and interdigitate over the bridge that connects the SPBs. As a bipolar spindle is formed, the median MT length increases to ∼300 nm and then decreases to ∼30 nm in late anaphase. Three-dimensional models confirm that there is no conventional metaphase and that anaphase A occurs. These studies complement and extend what is known about the three-dimensional structure of the yeast mitotic spindle and further our understanding of the organization of the SPB in intact cells.

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