Showing papers on "Electron tomography published in 2012"

Electron tomography at 2.4-ångström resolution

Abstract: Transmission electron microscopy is a powerful imaging tool that has found broad application in materials science, nanoscience and biology. With the introduction of aberration-corrected electron lenses, both the spatial resolution and the image quality in transmission electron microscopy have been significantly improved and resolution below 0.5 angstroms has been demonstrated. To reveal the three-dimensional (3D) structure of thin samples, electron tomography is the method of choice, with cubic-nanometre resolution currently achievable. Discrete tomography has recently been used to generate a 3D atomic reconstruction of a silver nanoparticle two to three nanometres in diameter, but this statistical method assumes prior knowledge of the particle's lattice structure and requires that the atoms fit rigidly on that lattice. Here we report the experimental demonstration of a general electron tomography method that achieves atomic-scale resolution without initial assumptions about the sample structure. By combining a novel projection alignment and tomographic reconstruction method with scanning transmission electron microscopy, we have determined the 3D structure of an approximately ten-nanometre gold nanoparticle at 2.4-angstrom resolution. Although we cannot definitively locate all of the atoms inside the nanoparticle, individual atoms are observed in some regions of the particle and several grains are identified in three dimensions. The 3D surface morphology and internal lattice structure revealed are consistent with a distorted icosahedral multiply twinned particle. We anticipate that this general method can be applied not only to determine the 3D structure of nanomaterials at atomic-scale resolution, but also to improve the spatial resolution and image quality in other tomography fields.

Biological Electron Microscopy: Theory, Techniques, and Troubleshooting

Abstract: 1 Specimen Preparation for Electron Microscopy.- 1 Techniques.- 2 Cryotechniques.- 2 Techniques.- 3 Ultramicrotomy.- 3 Techniques.- 4.- 4 Techniques.- 5.- 5 Techniques.- 6.- 6 Techniques.- 7 Support Films.- 7 Techniques.- 8 Replicas, Shadowing, and Negative Staining.- 8 Techniques.- 9 Transmission Electron Microscopy.- 10 Vacuum Systems.- 11 High-Voltage Transmission Electron Microscopes (HVEM).- 12 Intermediate Voltage Electron Microscopes (IVEM), Electron Tomography, and Single-Particle Electron Microscopy.- 13 Scanning Electron Microscopy.- 13 Techniques.- 14.- 15 Photography.- 15 Techniques.- 16 Digital Imaging and Telemedicine.- 17 Morphometry and Stereology.- 18 Photomicroscopy.- 18 Techniques.- 19 Laboratory Safety.- 20 General Sources for Information Concerning Microscopy.- 21.- Appendix A Computing Micrometer Bar Sizes.- Appendix B Calibrating the TEM and the SEM.- I. Transmission Electron Microscope.- II. Scanning Electron Microscope.- Appendix C Materials and Methods Write-Up Suggestions for Standard TEM and SEM Preparations.- I. Materials and Methods for Routine TEM Preparation.- II. Materials and Methods for Routine SEM Preparation.

Electron tomography of cells.

Abstract: The 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.

Precise, correlated fluorescence microscopy and electron tomography of lowicryl sections using fluorescent fiducial markers.

Abstract: The application of fluorescence and electron microscopy to the same specimen allows the study of dynamic and rare cellular events at ultrastructural detail Here, we present a correlative microscopy approach, which combines high accuracy of correlation, high sensitivity for detecting faint fluorescent signals, as well as robustness and reproducibility to permit large dataset collections We provide a step-by-step protocol that allows direct mapping of fluorescent protein signals into electron tomograms A localization precision of <100 nm is achieved by using fluorescent fiducial markers which are visible both in fluorescence images and in electron tomograms We explain the critical details of the procedure, give background information on the individual steps, present results from test experiments carried out during establishment of the method, as well as information about possible modifications to the protocol, such as its application to 2D electron micrographs This simple, robust, and flexible method can be applied to a large variety of cellular systems, such as yeast cell pellets and mammalian cell monolayers, to answer a broad spectrum of structure-function related questions

‘Big Bang’ tomography as a new route to atomic-resolution electron tomography

Abstract: A tomography technique based on the idea that Fourier components of scattered electron waves obey a relationship analogous to that expressed in cosmology by Hubble’s law can be used to image at atomic resolution from a single viewing direction. State-of-the-art electron microscopy can readily resolve structures with subatomic resolution, but making three-dimensional images of similar resolution is a tougher challenge. Here, Dirk Van Dyck and Fu-Rong Chen describe an original image-reconstruction method that extracts information on the whereabouts of all atoms, in the plane as well as in the vertical direction, from just one projection. The concept is based on the assumption that each atom acts as a point source, scattering spherical waves that propagate to the detector, where they interfere with spherical waves from other atoms. The observed 'exit wave' contains information on all atoms in the sample, and can be retrieved using appropriate algorithms. The approach is demonstrated experimentally for a two-layered graphene sample. The authors note that their reconstruction technique resembles that used by cosmologists to construct a Hubble plot, hence the name 'Big Bang tomography'. Until now it has not been possible to image at atomic resolution using classical electron tomographic methods1, except when the target is a perfectly crystalline nano-object imaged along a few zone axes2. The main reasons are that mechanical tilting in an electron microscope with sub-angstrom precision over a very large angular range is difficult, that many real-life objects such as dielectric layers in microelectronic devices impose geometrical constraints and that many radiation-sensitive objects such as proteins limit the total electron dose. Hence, there is a need for a new tomographic scheme that is able to deduce three-dimensional information from only one or a few projections. Here we present an electron tomographic method that can be used to determine, from only one viewing direction and with sub-angstrom precision, both the position of individual atoms in the plane of observation and their vertical position. The concept is based on the fact that an experimentally reconstructed exit wave3,4 consists of the superposition of the spherical waves that have been scattered by the individual atoms of the object. Furthermore, the phase of a Fourier component of a spherical wave increases with the distance of propagation at a known ‘phase speed’. If we assume that an atom is a point-like object, the relationship between the phase and the phase speed of each Fourier component is linear, and the distance between the atom and the plane of observation can therefore be determined by linear fitting. This picture has similarities with Big Bang cosmology, in which the Universe expands from a point-like origin such that the distance of any galaxy from the origin is linearly proportional to the speed at which it moves away from the origin (Hubble expansion). The proof of concept of the method has been demonstrated experimentally for graphene with a two-layer structure and it will work optimally for similar layered materials, such as boron nitride and molybdenum disulphide.

Electron Microscopy of Biological Materials at the Nanometer Scale

Abstract: Electron microscopy of biological matter uses three different imaging modalities: (a) electron crystallography, (b) single-particle analysis, and (c) electron tomography. Ideally, these imaging modalities are applied to frozenhydrated samples to ensure an optimal preservation of the structures under scrutiny. Cryo-electron microscopy of biological matter has made important advances in the past decades. It has become a research tool that further expands the scope of structural research into unique areas of cell and molecular biology, and it could augment the materials research portfolio in the study of soft and hybrid materials. This review addresses how researchers using transmission electron microscopy can derive structural information at high spatial resolution from fully hydrated specimens, despite their sensitivity to ionizing radiation, despite the adverse conditions of high vacuum for samples that have to be kept in aqueous environments, and despite their low contrast resulting from weakly scattering building blocks.

Study of defect evolution by TEM with in situ ion irradiation and coordinated modeling

Abstract: The paper describes a novel transmission electron microscopy (TEM) experiment with in situ ion irradiation designed to improve and validate a computer model. TEM thin foils of molybdenum were irradiated in situ by 1 MeV Kr ions up to ∼0.045 displacements per atom (dpa) at 80°C at three dose rates −5 × 10−6, 5 × 10−5, and 5 × 10−4 dpa/s – at the Argonne IVEM-Tandem Facility. The low-dose experiments produced visible defect structure in dislocation loops, allowing accurate, quantitative measurements of defect number density and size distribution. Weak beam dark-field plane-view images were used to obtain defect density and size distribution as functions of foil thickness, dose, and dose rate. Diffraction contrast electron tomography was performed to image defect clusters through the foil thickness and measure their depth distribution. A spatially dependent cluster dynamic model was developed explicitly to model the damage by 1 MeV Kr ion irradiation in an Mo thin foil with temporal and spatial dependence of...

Mesoporosity of zeolite Y: quantitative three-dimensional study by image analysis of electron tomograms.

Abstract: Quantitative insight into the three-dimensional morphology of complex zeoliteY mesopore networks was achieved by combining electron tomography and image processing. Properties could be studied that are not measurable by other techniques, such as the size distribution of the intact microporous domains. This has great relevance in descriptions of the molecular diffusion through zeolite crystals and, hence, catalytic activity and selectivity.

FIB/SEM tomography with TEM-like resolution for 3D imaging of high-pressure frozen cells

Abstract: Focused ion beam/scanning electron microscopy (FIB/SEM) tomography is a novel powerful approach for three-dimensional (3D) imaging of biological samples. Thereby, a sample is repeatedly milled with the focused ion beam (FIB) and each newly produced block face is imaged with the scanning electron microscope (SEM). This process can be repeated ad libitum in arbitrarily small increments allowing 3D analysis of relatively large volumes such as eukaryotic cells. High-pressure freezing and freeze substitution, on the other hand, are the gold standards for electron microscopic preparation of whole cells. In this work, we combined these methods and substantially improved resolution by using the secondary electron signal for image formation. With this imaging mode, contrast is formed in a very small, well-defined area close to the newly produced surface. By using this approach, small features, so far only visible in transmission electron microscope (TEM) (e.g., the two leaflets of the membrane bi-layer, clathrin coats and cytoskeletal elements), can be resolved directly in the FIB/SEM in the 3D context of whole cells.

IPET and FETR: experimental approach for studying molecular structure dynamics by cryo-electron tomography of a single-molecule structure.

Abstract: The dynamic personalities and structural heterogeneity of proteins are essential for proper functioning. Structural determination of dynamic/heterogeneous proteins is limited by conventional approaches of X-ray and electron microscopy (EM) of single-particle reconstruction that require an average from thousands to millions different molecules. Cryo-electron tomography (cryoET) is an approach to determine three-dimensional (3D) reconstruction of a single and unique biological object such as bacteria and cells, by imaging the object from a series of tilting angles. However, cconventional reconstruction methods use large-size whole-micrographs that are limited by reconstruction resolution (lower than 20 A), especially for small and low-symmetric molecule (<400 kDa). In this study, we demonstrated the adverse effects from image distortion and the measuring tilt-errors (including tilt-axis and tilt-angle errors) both play a major role in limiting the reconstruction resolution. Therefore, we developed a “focused electron tomography reconstruction” (FETR) algorithm to improve the resolution by decreasing the reconstructing image size so that it contains only a single-instance protein. FETR can tolerate certain levels of image-distortion and measuring tilt-errors, and can also precisely determine the translational parameters via an iterative refinement process that contains a series of automatically generated dynamic filters and masks. To describe this method, a set of simulated cryoET images was employed; to validate this approach, the real experimental images from negative-staining and cryoET were used. Since this approach can obtain the structure of a single-instance molecule/particle, we named it individual-particle electron tomography (IPET) as a new robust strategy/approach that does not require a pre-given initial model, class averaging of multiple molecules or an extended ordered lattice, but can tolerate small tilt-errors for high-resolution single “snapshot” molecule structure determination. Thus, FETR/IPET provides a completely new opportunity for a single-molecule structure determination, and could be used to study the dynamic character and equilibrium fluctuation of macromolecules.

Scanning reciprocal space for solving unknown structures : energy filtered diffraction tomography and rotation diffraction tomography methods

Abstract: Structure solutions of CaFe2O4 from energy filtered and unfiltered precession electron diffraction tomography and rotation electron diffraction tomography data, collected on two different microscopes, are reported. The collected data are analysed with three available software packages (ADT3D, PETS and EDT-PROCESS) and the obtained results are compared. In all cases the structure solution is successfully achieved. Energy filtered precession electron diffraction tomography, performed here for the first time, gives sharper diffraction peaks and less background compared to the unfiltered data and the final recovered model is closer to the X-ray refinement. Simultaneously the first crystal structure solution obtained from the rotation electron diffraction tomography data is reported.

Recent advances in the application of electron tomography to materials chemistry.

Abstract: Nowadays, tomography plays a central role in pureand applied science, in medicine, and in many branches of engineering and technology. It entails reconstructing the three-dimensional (3D) structure of an object from a tilt series of two-dimensional (2D) images. Its origin goes back to 1917, when Radon showed mathematically how a series of 2D projection images could be converted to the 3D structural one. Tomographic X-ray and positron scanning for 3D medical imaging, with a resolution of ∼1 mm, is now ubiquitous in major hospitals. Electron tomography, a relatively new chemical tool, with a resolution of ∼1 nm, has been recently adopted by materials chemists as an invaluable aid for the 3D study of the morphologies, spatially-discriminating chemical compositions, and defect properties of nanostructured materials.In this Account, we review the advances that have been made in facilitating the recording of the required series of 2D electron microscopic images and the subsequent process of 3D reconstruction of...

Electron Tomography in the (S)TEM: From Nanoscale Morphological Analysis to 3D Atomic Imaging

Abstract: In this short review, we discuss recent developments in electron tomography with examples across a range of materials science and nanotechnology. Challenges related to extending the resolution of the technique from the nanoscale to the atomic level are addressed, and the different routes proposed to meet those challenges are considered. We illustrate improvements in electron tomography brought about by recent developments in hardware and the advent of aberration-corrected microscopes. We focus also on developments in new reconstruction algorithms designed to enable reliable and accurate reconstructions from very limited projection data. These recent technique developments provide a genuine promise of routine 3D atomic imaging.

Sub-0.1 nm-resolution quantitative scanning transmission electron microscopy without adjustable parameters

Abstract: Atomic-resolution imaging in the scanning transmission electron microscope (STEM) constitutes a powerful tool for nanostructure characterization. Here, we demonstrate the quantitative interpretation of atomic-resolution high-angle annular dark-field (ADF) STEM images using an approach that does not rely on adjustable parameters. We measure independently the instrumental parameters that affect sub-0.1 nm-resolution ADF images, quantify their individual and collective contributions to the image intensity, and show that knowledge of these parameters enables a quantitative interpretation of the absolute intensity and contrast across all accessible spatial frequencies. The analysis also provides a method for the in-situ measurement of the STEM’s effective source distribution.

Bridging Microscopes: 3D correlative light and scanning electron microscopy of complex biological structures

Abstract: The rationale of correlative light and electron microscopy (CLEM) is to collect data on different information levels--ideally from an identical area on the same sample--with the aim of combining datasets at different levels of resolution to achieve a more holistic view of the hierarchical structural organization of cells and tissues. Modern three-dimensional (3D) imaging techniques in light and electron microscopy opened up new possibilities to expand morphological studies into the third dimension at the nanometer scale and over various volume dimensions. Here, we present two alternative approaches to correlate 3D light microscopy (LM) data with scanning electron microscopy (SEM) volume data. An adapted sample preparation method based on high-pressure freezing for structure preservation, followed by freeze-substitution for multimodal en-bloc imaging or serial-section imaging is described. The advantages and potential applications are exemplarily shown on various biological samples, such as cells, individual organisms, human tissue, as well as plant tissue. The two CLEM approaches presented here are per se not mutually exclusive, but have their distinct advantages. Confocal laser scanning microscopy (CLSM) and focused ion beam-SEM (FIB-SEM) is most suitable for targeted 3D correlation of small volumes, whereas serial-section LM and SEM imaging has its strength in large-area or -volume screening and correlation. The second method can be combined with immunocytochemical methods. Both methods, however, have the potential to extract statistically relevant data of structural details for systems biology.

Tuning the Pore Size of Ink-Bottle Mesopores by Atomic Layer Deposition

Abstract: Atomic layer deposition is demonstrated to be greatly suitable to tune the pore size of ink-bottle mesopores at the atomic level. This is confirmed by quantitative electron tomography, which enable...

Synthesis, electron tomography and single-particle optical response of twisted gold nano-bipyramids.

Abstract: A great number of works focus their interest on the study of gold nanoparticle plasmonic properties Among those, sharp nanostructures appear to exhibit the more interesting features for further developments In this paper, a complete study on bipyramidal-like gold nanostructures is presented The nano-objects are prepared in high yield using an original method This chemical process enables a precise control of the shape and the size of the particles The specific photophysical properties of gold bipyramids in suspension are ripened by recording the plasmonic response of single and isolated objects Resulting extinction spectra are precisely correlated to their geometrical structure by mean of electron tomography at the single-particle level The interplay between the geometrical structure and the optical properties of twisted gold bipyramids is further discussed on the basis of numerical calculations The influence of several parameters is explored such as the structural aspect ratio or the tip truncation In the case of an incident excitation polarized along the particle long axis, this study shows how the plasmon resonance position can be sensitive to these parameters and how it can then be efficiently tuned on a large wavelength range

Scanning transmission electron microscopy strain measurement from millisecond frames of a direct electron charge coupled device

Abstract: A high-speed direct electron detection system is introduced to the field of transmission electron microscopy and applied to strain measurements in semiconductor nanostructures. In particular, a focused electron probe with a diameter of 0.5 nm was scanned over a fourfold quantum layer stack with alternating compressive and tensile strain and diffracted discs have been recorded on a scintillator-free direct electron detector with a frame time of 1 ms. We show that the applied algorithms can accurately detect Bragg beam positions despite a significant point spread each 300 kV electron causes during detection on the scintillator-free camera. For millisecond exposures, we find that strain can be measured with a precision of 1.3 × 10−3, enabling, e.g., strain mapping in a 100×100 nm2 region with 0.5 nm resolution in 40 s.

Inhomogeneity of Nacre Lamellae on the Nanometer Length Scale

Abstract: We present experimental evidence of inhomogeneous distribution of organic inclusions within individual aragonitic lamellae forming the nacre layer of mollusk shells. This nanoscale-inhomogeneity is visualized in the Perna canaliculus (green mussel) shells by the aid of high-angle annular dark field scanning transmission electron microscopy (HAADF-STEM) in the tomography mode. Electron tomography reconstructions of the three-dimensional distribution of intracrystalline organic inclusions, ranging in size between 2 to 25 nm, clearly reveal 50 nm wide depletion zones adjacent to the organic sheets between lamellae. The interrelation between the observed fine-scale inhomogeneity of nacre layer and its improved mechanical properties is discussed.

Visualization of yeast cells by electron microscopy

Abstract: In the 1970s, hydrocarbon or methanol utilizable yeasts were considered as a material for foods and ethanol production. During the course of studies into the physiology of yeasts, we found that these systems provide a suitable model for the biogenesis and ultrastructure research of microbodies (peroxisomes). Microbodies of hydrocarbon utilizing Candida tropicalis multiply profusely from the preexisting microbody. β oxidation enzymes in the microbody were determined by means of immunoelectron microscopy. We examined the ultrastructure of Candida boidinii microbodies grown on methanol, and found a composite crystalloid of two enzymes, alcohol oxidase and catalase, by analyzing using the optical diffraction and filtering technique and computer simulation. We established methods for preparing the protoplasts of Schizosaccharomyces pombe and conditions for the complete regeneration of the cell wall. The dynamic process of cell wall formation was clarified through our study of the protoplasts, using an improved ultra high resolution (UHR) FESEM S-900 and an S-900LV. It was found that β-1,3-glucan, β-1,6-glucan and α-1,3-glucan, as well as α-galactomannan, are ingredients of the cell wall. The process of septum formation during cell division was examined after cryo-fixation by high pressure freezing (HPF). It was also found that α-1,3- and β-1,3-glucans were located in the invaginating nascent septum, and later, highly branched β-1,6-glucan also appeared on the second septum. The micro-sampling method, using a focused ion beam (FIB), has been applied to our yeast cell wall research. A combination of FIB and scanning transmission electron microscopy is useful in constructing 3D images and analyzing the molecular architecture of cells, as well as for electron tomography of thick sections of biological specimens.

From the physics of secondary electron emission to image contrasts in scanning electron microscopy

Abstract: Image formation in scanning electron microscopy (SEM) is a combination of physical processes, electron emissions from the sample, and of a technical process related to the detection of a fraction of these electrons. For the present survey of image contrasts in SEM, simplified considerations in the physics of the secondary electron emission yield, δ, are combined with the effects of a partial collection of the emitted secondary electrons. Although some consideration is initially given to the architecture of modern SEM, the main attention is devoted to the material contrasts with the respective roles of the sub-surface and surface compositions of the sample, as well as with the roles of the field effects in the vacuum gap. The recent trends of energy filtering in normal SEM and the reduction of the incident energy to a few electron volts in very low-energy electron microscopy are also considered. For an understanding by the SEM community, the mathematical expressions are explained with simple physical arguments.

Environmental transmission electron microscopy in an aberration-corrected environment.

Abstract: The increasing use of environmental transmission electron microscopy (ETEM) in materials science provides exciting new possibilities for investigating chemical reactions and understanding both the interaction of fast electrons with gas molecules and the effect of the presence of gas on high-resolution imaging. A gaseous atmosphere in the pole-piece gap of the objective lens of the microscope alters both the incoming electron wave prior to interaction with the sample and the outgoing wave below the sample. Whereas conventional TEM samples are usually thin (below 100 nm), the gas in the environmental cell fills the entire gap between the pole pieces and is thus not spatially localized. By using an FEI Titan environmental transmission electron microscope equipped with a monochromator and an aberration corrector on the objective lens, we have investigated the effects on imaging and spectroscopy caused by the presence of the gas.

Monte Carlo simulation of secondary electron images for real sample structures in scanning electron microscopy

Abstract: Monte Carlo simulation methods for the study of electron beam interaction with solids have been mostly concerned with specimens of simple geometry. In this article, we propose a simulation algorithm for treating arbitrary complex structures in a real sample. The method is based on a finite element triangular mesh modeling of sample geometry and a space subdivision for accelerating simulation. Simulation of secondary electron image in scanning electron microscopy has been performed for gold particles on a carbon substrate. Comparison of the simulation result with an experiment image confirms that this method is effective to model complex morphology of a real sample.