Improvements on marker-free images alignment for electron tomography

AbstractElectron tomography is a technique to obtain three-dimensional structural information of samples. However, the technique is limited by shifts occurring during acquisition that need to be corrected before the reconstruction process. In 2009, we proposed an approach for post-acquisition alignment of tilt series images. This approach was marker-free, based on patch tracking and integrated in free software. Here, we present improvements to the method to make it more reliable, stable and accurate. In addition, we modified the image formation model underlying the alignment procedure to include different deformations occurring during acquisition. We propose a new way to correct these computed deformations to obtain reconstructions with reduced artifacts. The new approach has demonstrated to improve the quality of the final 3D reconstruction, giving access to better defined structures for different transmission electron tomography methods: resin embedded STEM-tomography and cryo-TEM tomography. The method is freely available in TomoJ software.

Download Full-text

Recent Advances in Electron Tomography: TEM and HAADF-STEM Tomography for Materials Science and Semiconductor Applications

Microscopy and Microanalysis ◽

10.1017/s1431927605050361 ◽

2005 ◽

Vol 11 (5) ◽

pp. 378-400 ◽

Cited By ~ 160

Author(s):

Christian Kübel ◽

Andreas Voigt ◽

Remco Schoenmakers ◽

Max Otten ◽

David Su ◽

...

Keyword(s):

Electron Microscopy ◽

Transmission Electron Microscopy ◽

High Resolution ◽

Structural Information ◽

Electron Tomography ◽

Semiconductor Devices ◽

Three Dimensional ◽

Crystalline Materials ◽

Transmission Electron ◽

Stem Tomography

Electron tomography is a well-established technique for three-dimensional structure determination of (almost) amorphous specimens in life sciences applications. With the recent advances in nanotechnology and the semiconductor industry, there is also an increasing need for high-resolution three-dimensional (3D) structural information in physical sciences. In this article, we evaluate the capabilities and limitations of transmission electron microscopy (TEM) and high-angle-annular-dark-field scanning transmission electron microscopy (HAADF-STEM) tomography for the 3D structural characterization of partially crystalline to highly crystalline materials. Our analysis of catalysts, a hydrogen storage material, and different semiconductor devices shows that features with a diameter as small as 1–2 nm can be resolved in three dimensions by electron tomography. For partially crystalline materials with small single crystalline domains, bright-field TEM tomography provides reliable 3D structural information. HAADF-STEM tomography is more versatile and can also be used for high-resolution 3D imaging of highly crystalline materials such as semiconductor devices.

Download Full-text

Automated electron tomography: from data collection to image processing

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100136507 ◽

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.

Download Full-text

Automated cryo-lamella preparation for high-throughput in-situ structural biology

10.1101/797506 ◽

2019 ◽

Author(s):

Genevieve Buckley ◽

Gediminas Gervinskas ◽

Cyntia Taveneau ◽

Hari Venugopal ◽

James C. Whisstock ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Structural Biology ◽

Focused Ion Beam ◽

Electron Tomography ◽

Ion Beam ◽

Automated Method ◽

Cellular Environment ◽

Transmission Electron

AbstractCryo-transmission electron tomography (cryo-ET) in association with cryo-focused ion beam (cryo-FIB) milling enables structural biology studies to be performed directly within the cellular environment. Cryo-preserved cells are milled and a lamella with a thickness of 200-300 nm provides an electron transparent window suitable for cryo-ET imaging. Cryo-FIB milling is an effective method, but it is a tedious and time-consuming process, which typically results in ~10 lamellae per day. Here, we introduce an automated method to reproducibly prepare cryo-lamellae on a grid and reduce the amount of human supervision. We tested the routine on cryo-preserved Saccharomyces cerevisiae and demonstrate that this method allows an increased throughput, achieving a rate of 5 lamellae/hour without the need to supervise the FIB milling. We demonstrate that the quality of the lamellae is consistent throughout the preparation and their compatibility with cryo-ET analyses.

Download Full-text

Three-Dimensional Dispersion of Nano-Fillers in Soft Composite as Revealed by Transmission Electron Microscopy/Electron Tomography (3D-TEM)

Materials Science Forum ◽

10.4028/www.scientific.net/msf.514-516.353 ◽

2006 ◽

Vol 514-516 ◽

pp. 353-358 ◽

Cited By ~ 3

Author(s):

Shinzo Kohjiya

Keyword(s):

Electron Microscopy ◽

Transmission Electron Microscopy ◽

Carbon Black ◽

Natural Rubber ◽

Structural Information ◽

Electron Tomography ◽

Nano Composites ◽

Nano Structure ◽

Transmission Electron ◽

Particulate Silica

. Generally rubber products are a typical soft material, and a composite of a nano-filler (typically, carbon black or particulate silica) and a rubber (natural rubber and various synthetics are used). The properties of these soft nano-composites have been well known to depend on the dispersion of the nano-filler in the rubbery matrix. The most powerful tool for the elucidation of it has been transmission electron microscopy (TEM). The microscopic techniques are based on the projection of 3-dimensional (3D) body on a plane (x, y plane), thus the structural information along the thickness (z axis) direction of the sample is difficult to obtain. This paper describes our recent results on the dispersion of carbon black (CB) and particulate silica in natural rubber (NR) matrix observed by TEM combined with electron tomography (3D-TEM) technique, which enabled us to obtain images of 3D nano-structure of the sample. Thus, 3D images of CB and silica in NR matrix are visualized and analyzed in this communication. These results are precious ones for the design of soft nano-composites, and the technique will become an indispensable one in nanotechnology.

Download Full-text

Cryomesh™: A New Substrate for Cryo-Electron Microscopy

Microscopy and Microanalysis ◽

10.1017/s1431927609991310 ◽

2010 ◽

Vol 16 (1) ◽

pp. 43-53 ◽

Cited By ~ 31

Author(s):

Craig Yoshioka ◽

Bridget Carragher ◽

Clinton S. Potter

Keyword(s):

Electron Microscopy ◽

Semiconductor Industry ◽

Three Dimensional ◽

Quality Of Data ◽

Temperature Conductivity ◽

Cryo Electron Microscopy ◽

Transmission Electron ◽

Using Data ◽

Collection Methods

AbstractHere we evaluate a new grid substrate developed by ProtoChips Inc. (Raleigh, NC) for cryo-transmission electron microscopy. The new grids are fabricated from doped silicon carbide using processes adapted from the semiconductor industry. A major motivating purpose in the development of these grids was to increase the low-temperature conductivity of the substrate, a characteristic that is thought to affect the appearance of beam-induced movement (BIM) in transmission electron microscope (TEM) images of biological specimens. BIM degrades the quality of data and is especially severe when frozen biological specimens are tilted in the microscope. Our results show that this new substrate does indeed have a significant impact on reducing the appearance and severity of beam-induced movement in TEM images of tilted cryo-preserved samples. Furthermore, while we have not been able to ascertain the exact causes underlying the BIM phenomenon, we have evidence that the rigidity and flatness of these grids may play a major role in its reduction. This improvement in the reliability of imaging at tilt has a significant impact on using data collection methods such as random conical tilt or orthogonal tilt reconstruction with cryo-preserved samples. Reduction in BIM also has the potential for improving the resolution of three-dimensional cryo-reconstructions in general.

Download Full-text

Primary ciliary dyskinesia with normal ultrastructure: three-dimensional tomography detects absence of DNAH11

European Respiratory Journal ◽

10.1183/13993003.01809-2017 ◽

2018 ◽

Vol 51 (2) ◽

pp. 1701809 ◽

Cited By ~ 17

Author(s):

Amelia Shoemark ◽

Thomas Burgoyne ◽

Robert Kwan ◽

Mellisa Dixon ◽

Mitali P. Patel ◽

...

Keyword(s):

Primary Ciliary Dyskinesia ◽

Electron Tomography ◽

Three Dimensional ◽

Structural Abnormality ◽

Healthy Controls ◽

Ultrastructural Studies ◽

Transmission Electron ◽

Causal Variants ◽

Diagnostic Samples ◽

Ciliary Dyskinesia

In primary ciliary dyskinesia (PCD), motile ciliary dysfunction arises from ciliary defects usually confirmed by transmission electron microscopy (TEM). In 30% of patients, such as those with DNAH11 mutations, apparently normal ultrastructure makes diagnosis difficult. Genetic analysis supports diagnosis, but may not identify definitive causal variants. Electron tomography, an extension of TEM, produces three-dimensional ultrastructural ciliary models with superior resolution to TEM. Our hypothesis is that tomography using existing patient samples will enable visualisation of DNAH11-associated ultrastructural defects. Dual axis tomograms from araldite-embedded nasal cilia were collected in 13 PCD patients with normal ultrastructure (DNAH11 n=7, HYDIN n=2, CCDC65 n=3 and DRC1 n=1) and six healthy controls, then analysed using IMOD and Chimera software.DNAH11 protein is localised to the proximal ciliary region. Within this region, electron tomography indicated a deficiency of >25% of proximal outer dynein arm volume in all patients with DNAH11 mutations (n=7) compared to other patients with PCD and normal ultrastructure (n=6) and healthy controls (n=6). DNAH11 mutations cause a shared abnormality in ciliary ultrastructure previously undetectable by TEM. Advantageously, electron tomography can be used on existing diagnostic samples and establishes a structural abnormality where ultrastructural studies were previously normal.

Download Full-text

Three-Dimensional Imaging of Shear Band Produced by ECAP Process in Al-Ag Alloy

Materials Science Forum ◽

10.4028/www.scientific.net/msf.503-504.603 ◽

2006 ◽

Vol 503-504 ◽

pp. 603-608

Author(s):

Koji Inoke ◽

Kenji Kaneko ◽

Z. Horita

Keyword(s):

Electron Microscopy ◽

Transmission Electron Microscopy ◽

Electron Tomography ◽

Shear Bands ◽

Three Dimensional ◽

Angular Pressing ◽

Ecap Process ◽

Transmission Electron ◽

Scanning Transmission ◽

The Matrix

A significant change in microstructure occurs during the application of severe plastic deformation (SPD) such as by equal-channel angular pressing (ECAP). In this study, intense plastic strain was imposed on an Al-10.8wt%Ag alloy by the ECAP process. The amount of strain was controlled by the numbers of passes. After 1 pass of ECAP, shear bands became visible within the matrix. With increasing numbers of ECAP passes, the fraction of shear bands was increased. In this study, the change in microstructures was examined by three-dimensional electron tomography (3D-ET) in transmission electron microscopy (TEM) or scanning transmission electron microscopy (STEM). With this 3D-ET method, it was possible to conduct a precise analysis of the sizes, widths and distributions of the shear bands produced by the ECAP process. It is demonstrated that the 3D-ET method is promising to understand mechanisms of microstructural refinement using the ECAP process.

Download Full-text

Three-Dimensional Chemistry of Multiphase Nanomaterials by Energy-Filtered Transmission Electron Microscopy Tomography

Microscopy and Microanalysis ◽

10.1017/s143192761200116x ◽

2012 ◽

Vol 18 (5) ◽

pp. 1118-1128 ◽

Cited By ~ 12

Author(s):

Lucian Roiban ◽

Loïc Sorbier ◽

Christophe Pichon ◽

Pascale Bayle-Guillemaud ◽

Jacques Werckmann ◽

...

Keyword(s):

Electron Microscopy ◽

Transmission Electron Microscopy ◽

Electron Tomography ◽

Three Dimensional ◽

Point Of View ◽

Analysis Tool ◽

Shell Nanoparticles ◽

3D Nanostructures ◽

Transmission Electron ◽

Silica Alumina

AbstractA three-dimensional (3D) study of multiphase nanostructures by chemically selective electron tomography combining tomographic approach and energy-filtered imaging is reported. The implementation of this technique at the nanometer scale requires careful procedures for data acquisition, computing, and analysis. Based on the performances of modern transmission electron microscopy equipment and on developments in data processing, electron tomography in the energy-filtered imaging mode is shown to be a very appropriate analysis tool to provide 3D chemical maps at the nanoscale. Two examples highlight the usefulness of analytical electron tomography to investigate inhomogeneous 3D nanostructures, such as multiphase specimens or core-shell nanoparticles. The capability of discerning in a silica-alumina porous particle the two different components is illustrated. A quantitative analysis in the whole specimen and toward the pore surface is reported. This tool is shown to open new perspectives in catalysis by providing a way to characterize precisely 3D nanostructures from a chemical point of view.

Download Full-text

Three-dimensional real-space crystallography of MCM-48 mesoporous silica revealed by scanning transmission electron tomography

Chemical Physics Letters ◽

10.1016/j.cplett.2005.11.031 ◽

2006 ◽

Vol 418 (4-6) ◽

pp. 540-543 ◽

Cited By ~ 40

Author(s):

Timothy J.V. Yates ◽

John Meurig Thomas ◽

Jose-Jesus Fernandez ◽

Osamu Terasaki ◽

Ryong Ryoo ◽

...

Keyword(s):

Mesoporous Silica ◽

Electron Tomography ◽

Three Dimensional ◽

Real Space ◽

Scanning Transmission Electron ◽

Transmission Electron ◽

Scanning Transmission

Download Full-text

Electron Tomography

Microscopy Today ◽

10.1017/s1551929500057771 ◽

2002 ◽

Vol 10 (2) ◽

pp. 3-5

Author(s):

Stephen W. Carmichael

Keyword(s):

Electron Tomography ◽

Three Dimensional ◽

Two Dimensions ◽

Three Dimensions ◽

Specimen Preparation ◽

Two Dimensional Image ◽

Transmission Electron ◽

Different Depths ◽

Depth Dimension ◽

Pass Through

The transmission electron microscope (TEM) was invented in the 1930's, and developments in specimen preparation in the 1950's led to its widespread use as a tool to study structure in biologic systems. Similar in principle to the light microscope, but utilizing a much shorter wavelength for better resolution, the TEM has the image-forming beam pass through the specimen. This results in a two-dimensional image which can be difficult to interpret because features from different depths of the three dimensional specimen are superimposed. Traditionally this was dealt with by cutting sections of plastic-embedded specimens so thin (in the 40 to SO nanometer range) that they effectively had only two dimensions. To allow biologists to examine structures in three dimensions, serial sections are stacked and structures reconstructed. Even though computers have made reconstruction easier, the reality is that resolution in the depth dimension is limited by the section thickness. The technique of electron tomography is emerging as a way to overcome this limitation.

Download Full-text