Showing papers on "Electron tomography published in 2005"

Structural studies by electron tomography: from cells to molecules.

Abstract: ▪ Abstract Electron tomography (ET) is uniquely suited to obtain three-dimensional reconstructions of pleomorphic structures, such as cells, organelles or supramolecular assemblies. Although the principles of ET have been known for decades, its use has gathered momentum only in recent years, thanks to technological advances and its combination with improved specimen preparation techniques. The rapid freezing/freeze-substitution preparation is applicable to whole cells and tissues, and it is the method of choice for ET investigations of cellular ultrastructure. The frozen-hydrated preparation provides the best possible structural preservation and allows the imaging of molecules, complexes, and supramolecular assemblies in their native state and their natural environment. Devoid of staining and chemical fixation artifacts, cryo-ET provides a faithful representation of both the surface and internal structure of molecules. In combination with advanced computational methods, such as molecular identification ba...

Four-dimensional ultrafast electron microscopy

Abstract: Electron microscopy is arguably the most powerful tool for spatial imaging of structures. As such, 2D and 3D microscopies provide static structures with subnanometer and increasingly with angstrom-scale spatial resolution. Here we report the development of 4D ultrafast electron microscopy, whose capability imparts another dimension to imaging in general and to dynamics in particular. We demonstrate its versatility by recording images and diffraction patterns of crystalline and amorphous materials and images of biological cells. The electron packets, which were generated with femtosecond laser pulses, have a de Broglie wavelength of 0.0335 A at 120 keV and have as low as one electron per pulse. With such few particles, doses of few electrons per square angstrom, and ultrafast temporal duration, the long sought after but hitherto unrealized quest for ultrafast electron microscopy has been realized. Ultrafast electron microscopy should have an impact on all areas of microscopy, including biological imaging.

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

Abstract: Electron tomograph tomography is a well y well-established technique for three-dimensional structure determination of (almost) amorphous specimens in life science applications. With the recent advances in nanotechnology and the semiconductor industry, there is also an increasing need for high-resolution 3D structural information in physical sciences. In this paper, we evaluate the capabilities and limitations of TEM and 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 3D by electron tomography. For partially crystalline materials with small single crystalline domains, 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.

Correlative microscopy and electron tomography of GFP through photooxidation

Abstract: We have developed a simple correlative photooxidation method that allows for the direct ultrastructural visualization of the green fluorescent protein (GFP) upon illumination. The method, termed GRAB for GFP recognition after bleaching, uses oxygen radicals generated during the GFP bleaching process to photooxidize 3,3'-diaminobenzidine (DAB) into an electron-dense precipitate that can be visualized by routine electron microscopy and electron tomography. The amount of DAB product produced by the GRAB method appears to be linear with the initial fluorescence, and the resulting images are of sufficient quality to reveal detailed spatial information. This is exemplified by the observed intra-Golgi stack and intracisternal distribution of a human Golgi resident glycosylation enzyme, N-acetylgalactosaminyltransferase-2 fused either to enhanced GFP or CFP.

Handbook of microscopy for nanotechnology

Abstract: Optical Microscopy, Scanning Probe Microscopy, Ion Microscopy and Nanofabrication.- Confocal Scanning Optical Microscopy and Nanotechnology.- Scanning Near-Field Optical Microscopy in Nanosciences.- Scanning Tunneling Microscopy.- Visualization of Nanostructures with Atomic Force Microscopy.- Scanning Probe Microscopy for Nanoscale Manipulation and Patterning.- Scanning Thermal and Thermoelectric Microscopy.- Imaging Secondary Ion Mass Spectrometry.- Atom Probe Tomography.- Focused Ion Beam System-a Multifunctional Tool for Nanotechnology.- Electron Beam Lithography.- Electron Microscopy.- High-Resolution Scanning Electron Microscopy.- High Spatial Resolution Quantitative Electron Beam Microanalysis for Nanoscale Materials.- Characterization of Nano-Crystalline Materials Using Electron Backscatter Diffraction in the Scanning Electron Microscope.- High Resolution Transmission Electron Microscopy.- Scanning Transmission Electron Microscopy.- In-Situ Electron Microscopy for Nanomeasurements.- Environmental Transmission Electron Microscopy in Nanotechnology.- Electron Nanocrystallography.- Tomography Using the Transmission Electron Microscope.- Off-Axis Electron Holography.- SUB-NM Spatially Resolved Electron Energy-Loss Spectroscopy.- Imaging Magnetic Structures Using TEM.

Electron tomography of nanoparticle clusters: Implications for atmospheric lifetimes and radiative forcing of soot

Abstract: Nanoparticles are ubiquitous in nature. Their large surface areas and consequent chemical reactivity typically result in their aggregation into clusters. Their chemical and physical properties depend on cluster shapes, which are commonly complex and unknown. This is the first application of electron tomography with a transmission electron microscope to quantitatively determine the three-dimensional (3D) shapes, volumes, and surface areas of nanoparticle clusters. We use soot (black carbon, BC) nanoparticles as an example because it is a major contributor to environmental degradation and global climate change. To the extent that our samples are representative, we find that quantitative measurements of soot surface areas and volumes derived from electron tomograms differ from geometrically derived values by, respectively, almost one and two orders of magnitude. Global sensitivity studies suggest that the global burden and direct radiative forcing of fractal BC are only about 60% of the value if it is assumed that BC has a spherical shape.

Experimental imaging of silicon nanotubes

Abstract: Transmission electron microscopy (TEM), electron energy loss near edge structures (EELNES) and scanning tunneling microscopy (STM) were used to distinguish silicon nanotubes (SiNT) among the reaction products of a gas phase condensation synthesis. TEM images exhibit the tubular nature with a well-defined wall. The EELNES spectra performed on each single nanotube show that they are constituted by nonoxidized silicon atoms. STM images show that they have diameter ranging from 2 to 35 nm, have an atomic arrangement compatible with a puckered structure and different chiralities. Moreover, the I-V curves showed that SiNT can be semiconducting as well as metallic in character.

Scanning transmission electron microscopy and its application to the study of nanoparticles and nanoparticle systems

Abstract: Scanning transmission electron microscopy (STEM) techniques can provide imaging, diffraction and spectroscopic information, either simultaneously or in a serial manner, of the specimen with an atomic or a sub-nanometer spatial resolution. High-resolution STEM imaging, when combined with nanodiffraction, atomic resolution electron energy-loss spectroscopy and nanometer resolution X-ray energy dispersive spectroscopy techniques, is critical to the fundamental studies of importance to nanoscience and nanotechnology. The availability of sub-nanometer or sub-angstrom electron probes in a STEM instrument, due to the use of a field emission gun and aberration correctors, ensures the greatest capabilities for studies of sizes, shapes, defects, crystal and surface structures, and compositions and electronic states of nanometer-size regions of thin films, nanoparticles and nanoparticle systems. The various imaging, diffraction and spectroscopy modes available in a dedicated STEM or a field emission TEM/STEM instrument are reviewed and the application of these techniques to the study of nanoparticles and nanostructured catalysts is used as an example to illustrate the critical role of the various STEM techniques in nanotechnology and nanoscience research.

Spatial resolution limits in electron-beam-induced deposition

Abstract: Electron-beam-induced deposition (EBID) is a versatile micro- and nanofabrication technique based on electron-induced dissociation of metal-carrying gas molecules adsorbed on a target. EBID has the advantage of direct deposition of three-dimensional structures on almost any target geometry. This technique has occasionally been used in focused electron-beam instruments, such as scanning electron microscopes, scanning transmission electron microscopes (STEM), or lithography machines. Experiments showed that the EBID spatial resolution, defined as the lateral size of a singular deposited dot or line, always exceeds the diameter of the electron beam. Until recently, no one has been able to fabricate EBID features smaller than 15–20?nm diameter, even if a 2-nm-diam electron-beam writer was used. Because of this, the prediction of EBID resolution is an intriguing problem. In this article, a procedure to theoretically estimate the EBID resolution for a given energetic electron beam, target, and gaseous precursor is described. This procedure offers the most complete approach to the EBID spatial resolution problem. An EBID model was developed based on electron interactions with the solid target and with the gaseous precursor. The spatial resolution of EBID can be influenced by many factors, of which two are quantified: the secondary electrons, suspected by almost all authors working in this field, and the delocalization of inelastic electron scattering, a poorly known effect. The results confirm the major influence played by the secondary electrons on the EBID resolution and show that the role of the delocalization of inelastic electron scattering is negligible. The model predicts that a 0.2-nm electron beam can deposit structures with minimum sizes between 0.2 and 2?nm, instead of the formerly assumed limit of 15–20?nm. The modeling results are compared with recent experimental results in which 1-nm?W dots from a W(CO)6 precursor were written in a 200-kV STEM on a 30-nm SiN membrane.

Dual-Axis Electron Tomography for Three-Dimensional Observations of Polymeric Nanostructures

Abstract: Transmission electron microtomography (TEMT) has proven to be useful for investigating three-dimensional (3D) nanostructures in polymeric systems. However, the conventional TEMT that uses a tilt series around an axis occasionally fails to reconstruct nanostructures, especially when the nanostructure is highly anisotropic. Namely, if a certain geometrical relationship between orientation of the nanostructure and the tilt axis is fulfilled, the nanostructure does not appear in the 3D reconstruction. This problem can be solved by tilting the specimen around two (orthogonal) tilt axes and combining the resulting two 3D reconstructions after precise alignment between them. The strategy, called “dual-axis tomography”, is used to observe one of the commonly observed anisotropic polymer nanostructures, cylindrical morphology, of poly(styrene-block-isoprene) block copolymer. It was experimentally observed that the cylindrical nanodomains that were “missing” in one of the two tomograms were complementary reconstruc...

Assessment of the 3D Localization of Metallic Nanoparticles in Pd/SiO2 Cogelled Catalysts by Electron Tomography

Abstract: The purpose of this study is to analyze the localization of palladium nanoparticles within their silica support, in two heterogeneous catalysts synthesized by the sol-gel process, with different metal loadings. Electron tomography demonstrates that the palladium particles are localized deep inside the silica skeleton. The use of digital image analysis further shows that the dispersion of palladium is optimal in the sample with the lowest loading. The particles are regularly spaced in the middle of the silica skeleton, with a distance between them comparable to the diameter of the struts of silica.

An energy-based three-dimensional segmentation approach for the quantitative interpretation of electron tomograms

Abstract: Electron tomography allows for the determination of the three-dimensional structures of cells and tissues at resolutions significantly higher than that which is possible with optical microscopy. Electron tomograms contain, in principle, vast amounts of information on the locations and architectures of large numbers of subcellular assemblies and organelles. The development of reliable quantitative approaches for the analysis of features in tomograms is an important problem, and a challenging prospect due to the low signal-to-noise ratios that are inherent to biological electron microscopic images. This is, in part, a consequence of the tremendous complexity of biological specimens. We report on a new method for the automated segmentation of HIV particles and selected cellular compartments in electron tomograms recorded from fixed, plastic-embedded sections derived from HIV-infected human macrophages. Individual features in the tomogram are segmented using a novel robust algorithm that finds their boundaries as global minimal surfaces in a metric space defined by image features. The optimization is carried out in a transformed spherical domain with the center an interior point of the particle of interest, providing a proper setting for the fast and accurate minimization of the segmentation energy. This method provides tools for the semi-automated detection and statistical evaluation of HIV particles at different stages of assembly in the cells and presents opportunities for correlation with biochemical markers of HIV infection. The segmentation algorithm developed here forms the basis of the automated analysis of electron tomograms and will be especially useful given the rapid increases in the rate of data acquisition. It could also enable studies of much larger data sets, such as those which might be obtained from the tomographic analysis of HIV-infected cells from studies of large populations.

Nucleocapsid-like structures of Ebola virus reconstructed using electron tomography.

Abstract: Electron tomography (ET) is a new technique for high resolution, three-dimensional (3D) reconstruction of pleiomorphic macromolecular complexes, such as virus components. By employing this technique, we resolved the 3D structure of Ebola virus nucleocapsid-like (NC-like) structures in the cytoplasm of cells expressing NP, VP24, and VP35: the minimum components required to form these NC-like structures. Reconstruction of these tubular NC-like structures of Ebola virus showed them to be composed of left-handed helices spaced at short intervals, which is structurally consistent with other non-segmented negative-strand RNA viruses.

Conical tomography II: A method for the study of cellular

Abstract: 13 We have used conical electron tomography in order to reconstruct neuronal organelles in thin sections of plastic embedded rat 14 somato-sensory cortical tissue. The conical tilt series were collected at a 55 tilt and at 5 rotations, aligned using gold particles as 15 fiduciary markers, and reconstructed using the weighted back projection algorithm. After a refinement process based on projection 16 matching, the 3D maps showed the ‘‘unit membrane pattern’’ along the entire reconstructed volume. This pattern is indicative of the 17 bilayer arrangement of phospholipids in biological membranes. Based on Fourier correlation methods as well as the visualization of 18 the ‘‘unit membrane’’ pattern, we estimated resolutions of 4 nm. To illustrate the prospective advantages of conical tomography, 19 we segmented ‘‘coated’’ vesicles in the reconstructed volumes. These vesicles were comprised of a central core enclosing a small 20 lumen, and a protein ‘‘coating’’ extending into the cytoplasm. The ‘‘coated’’ vesicle was attached to the plasma membrane through

Dark-field transmission electron microscopy for a tilt series of ordering alloys: toward electron tomography.

Abstract: Here we show a technique to obtain a tilt series of dark-field (DF) transmission electron microscopy (TEM) images in ordering alloys for tomographic three-dimensional (3D) observations. A tilt series of DF TEM images of D1a-ordered Ni4Mo precipitates in a Ni-Mo alloy was successfully obtained by adjusting a diffraction condition for a superlattice reflection from the Ni4Mo precipitates. Since the superlattice reflection usually has a long extinction distance, dynamic diffraction effects such as thickness fringes can be suppressed to some extent with precise realignment of the diffraction condition. By using the tilt series of the DF TEM images, we attempted a computed TEM tomography to visualize 3D shapes and positions of the precipitates.

Reduction of anisotropic image resolution in transmission electron microtomography by use of quadrangular prism-shaped section.

Abstract: A quadrangular prism specimen (a 'prism-shaped' section) was observed in three-dimension (3-D) by transmission electron microtomography (TEMT) over as wide a tilt range as possible. Two types of specimens were prepared for the TEMT experiments: (i) a prism-shaped section of a block copolymer nanostructure, whose cross section was 200 nm on each side and (ii) a conventional ultrathin section having the same thickness (approximately 200 nm) as the prism-shaped section. Image quality of the projections taken at high tilt angles, e.g. 60 degrees, of the prism-shaped section was considerably better than that of the ultrathin section. This was because the path length of electron beam of the prism-shaped section was shorter (and hence the transmission was higher) than that of the ultrathin section at the same tilt angle. Thus, although the projections of the ultrathin section at the tilt angle larger than approximately 40 degrees did not effectively contribute to the 3-D reconstruction, those of the prism-shaped section at very high tilt angles, e.g. +/-75 degrees, can still be used for the 3-D reconstruction. Three orthogonal cross-sectional views of the 3-D reconstruction were extensively compared between the two sections. It was found that (i) the image contrast of the 3-D reconstruction was significantly enhanced and (ii) an elongation of structural object due to limitation of the angular range in the electron tomography was considerably reduced using the prism-shaped section.

In situ Observation of Solid–liquid Interfaces by Transmission Electron Microscopy

Abstract: Recent progress in in situ observation of solid–liquid interfaces by means of transmission electron microscopy, carried out by the Nagoya group, was reviewed. The results obtained on pure materials are discussed based on Jackson’s theory. The structure of the solid–liquid interfaces of eutectic alloys was also observed. The in situ observation technique of solid–liquid interface is applied to industrially important reactions which include liquid phases.

Element mapping device, scanning transmission electron microscope, and element mapping method

Abstract: There is provided an element mapping unit, scanning transmission electron microscope, and element mapping method that enable to acquire an element mapping image very easily. On the scanning transmission electron microscope, the electron beam transmitted through an object to be analyzed enters into the element mapping unit. The electron beam is analyzed of its energy into spectrum by an electron spectrometer and an electron energy loss spectrum is acquired. Because the acceleration voltage data for each element and window data for 2-window method, 3-window method or contrast tuning method are already stored in a database and accordingly the spectrum measurement is carried out immediately even when an element to be analyzed is changed to another, the operator can confirm a two-dimensional element distribution map immediately. Besides, because every electron beam that enters into an energy filter passes through the object point, aberration strain in the electron spectrometer can be minimized and higher energy stability can be achieved. As a result, drift of the electron energy loss spectrum acquired by analyzing the electron beam into spectrum can be minimized and element distribution with higher accuracy can be acquired.