A new method for transmission electron microscope (TEM) specimen preparation using a focused ion beam (FIB) system that results in a lower rate of gallium (Ga) implantation has been developed. The method was applied to structural and analytical studies of composite materials such as silicon (Si)-devices and magneto-optical disk. To protect the specimens against Ga ion irradiation, amorphous tungsten (W) was deposited on the surface of the specimen prior to FIB milling. The deposition was quite effective in reducing the Ga implantation rate, and energy-dispersive X-ray (EDX) analysis of these specimens detected 0.3Ð1.5% Ga incorporated in the thinned area. FIB milling times for these specimens were 1.5Ð2 hr. Although the milling rate was high, all the materials were properly prepared for TEM study, and clear crystal lattice images were observed on all specimens.

Abstract: The atomic-scale structure of tilt grain boundaries has been studied by high-resolution electron microscopy (HREM) in several ceramics and metals such as NiO, yttria stabilized zirconia, Au, and Al. It is found that when grain boundaries are formed between two crystals a considerable variety of modes of relaxations, i.e., the rearrangements of atoms in and near the grain boundary core are possible, depending on grain boundary geometry and the interatomic interactions. The atomic relaxations within and near the grain boundary core often include relaxations that involve the formation of stacking faults in low-stacking-fault energy fcc materials. Grain boundary dissociations are also observed for several grain boundary geometries. Computer simulations of grain boundary structures in metals have in general been able to reproduce the relaxation features at least on a qualitative level. In contrast, ceramic grain boundaries typically are more complex and not always tractable by simple computer relaxation procedures, since the formation of point defects and partial occupancy of atomic columns may be involved in the relaxation processes. It is found that when a boundary is not allowed to relax to its lowest energy state due to geometrical constraints, a multitude of grain boundary core structures can exist. Such conditions are expected to be characteristic for nanocrystalline materials. Because of its close connection to grain boundary properties, quantification of the volume expansion by HREM techniques is an important goal of grain boundary research. Image simulations of grain boundaries suggest that for most materials of interest the 3-fold astigmatism must be corrected to better than 100 nm to achieve the desired accuracies.

Ultrafast electron diffraction (UED) enables studies of structural dynamics at atomic length and timescales, i.e., 0.1 nm and 0.1 ps, in single-shot mode. At present UED experiments are based on femtosecond laser photoemission from solid state cathodes. These photoemission sources perform excellently, but are not sufficiently bright for single-shot studies of, for example, biomolecular samples. We propose a new type of electron source, based on near-threshold photoionization of a laser-cooled and trapped atomic gas. The electron temperature of these sources can be as low as 10 K, implying an increase in brightness by orders of magnitude. We investigate a setup consisting of an ultracold electron source and standard radio-frequency acceleration techniques by GPT tracking simulations. The simulations use realistic fields and include all pairwise Coulomb interactions. We show that in this setup 120 keV, 0.1 pC electron bunches can be produced with a longitudinal emittance sufficiently small for enabling sub-100 fs bunch lengths at 1% relative energy spread. A transverse root-mean-square normalized emittance of εx = 10 nm is obtained, significantly better than from photoemission sources. Correlations in transverse phase-space indicate that the transverse emittance can be improved even further, enabling single-shot studies of biomolecular samples.

The mechanism of the contrast in ‘environmental’ or ‘gaseous’ secondary electron images in the environmental scanning electron microscope is at best poorly understood. The original theory suggested a simple gas amplification model in which emitted secondary electrons ionise the chamber gas, leading to signal amplification and finally measurement at a biased detector. This theory is being advanced but little attention has as yet been paid to the factors which influence the actual secondary emission, although unusual contrast effects have been noted in one case. The conven-tional view is that the positive ion product of the gas-electron interaction results in charge neu-tralisation at the sample surface.

The implantation and trapping of charge in non-conductive materials was recently described, in reference to electron range measurements. This work demonstrated that trapped charge influ-enced the secondary electron yield, with enhanced secondary electron emission above the region of trapped charge. The consequence is that the distribution of the trapped charge is seen as a bright circle on the surface of the specimen, centred on the point of beam exposure (Fig.l).

Yeast cells represent a powerful model system in cell biology mainly due to their amenability to genetic manipulations. Increasingly, studies focus on mutant genes resulting in alterations of cellular structures and organelles. To ascertain the phenotypic changes involved, it is often desirable to use the resolving power of electron microscopy. In contrast to higher eukaryotic cells, yeast cells are particularly difficult to preserve mainly due to the presence of a thick cell wall that acts as a barrier against diffusion of fixatives. Although several procedures are targeted to overcome these difficulties, none of them have become established as a standard procedure. As a consequence, electron microscopy is still not used routinely as a tool in yeast cell biology. This prompted us to develop an easy-to-follow protocol for yeast transmission electron microscopy that should be useful in all cases where membrane integrity and organelle morphology is emphasized. One means of making the yeast cytoplasm more attainable to fixation and staining solutions is by enzymatic digestion of the cell wall. Following this approach, we were able to reliably preserve yeast cells and their cellular organelles. Enzymatic treatment with zymolyase 20T to partially remove the yeast cell wall allowed the fixation, preservation, and visualization of the yeast cytoplasm revealing detailed ultrastructure. The advancement of this technique is demonstrated with mitochondria as a model organelle. Our studies on various yeast mutants clearly show the power of the enzymatic digestion technique in visualizing subtle changes of membrane structure and organelle morphology.

The carbon contents in carburized steels were investigated by electron probe microanalysis (EPMA) for a range of carbon levels in the solid solution less than 1 wt%. This article describes the difficulties encountered with the classic analytical procedure using the k ratio of X-ray intensities and the φ(ρz) model. Here, a suitable calibration curve method is presented with emphasis on the metallographic study of standard specimens and on the carbon decontamination of samples.

Strain-induced selective growth was investigated in a 1.5% temper-rolled Fe∼1%Si alloy using the electron backscatter diffraction (EBSD) technique. The EBSD technique was used to quantify the presence of orientation spreads within grains and to show that this particular case of selective growth can be directly related to differences in stored energy as reflected in the geometrically necessary dislocation content. The differences in stored energy were sufficient to give rise to selective growth as evidenced by bi-modal grain sizes.

An alternative approach for simulating the field evaporation process in atom probe tomography is presented. The model uses the electrostatic Robin’s equation to directly calculate charge distribution over the tip apex conducting surface, without the need for a supporting mesh. The partial ionization state of the surface atoms is at the core of the method. Indeed, each surface atom is considered as a point charge, which is representative of its evaporation probability. The computational efficiency is ensured by an adapted version of the Barnes–Hut N-body problem algorithm. Standard desorption maps for cubic structures are presented in order to demonstrate the effectiveness of the method.

Identification and accurate characterization of platinum-group minerals (PGMs) is usually a very cumbersome procedure due to their small grain size (typically below 10 µm) and inconspicuous appearance under reflected light. A novel strategy for finding PGMs and quantifying their composition was developed. It combines a mineral liberation analyzer (MLA), a point logging system, and electron probe microanalysis (EPMA).

As a first step, the PGMs are identified using the MLA. Grains identified as PGMs are then marked and coordinates recorded and transferred to the EPMA. Case studies illustrate that the combination of MLA, point logging, and EPMA results in the identification of a significantly higher number of PGM grains than reflected light microscopy. Analysis of PGMs by EPMA requires considerable effort due to the often significant overlaps between the X-ray spectra of almost all platinum-group and associated elements. X-ray lines suitable for quantitative analysis need to be carefully selected. As peak overlaps cannot be avoided completely, an offline overlap correction based on weight proportions has been developed. Results obtained with the procedure proposed in this study attain acceptable totals and atomic proportions, indicating that the applied corrections are appropriate.

We have used field emission scanning electron microscopy (FESEM) to study the high-resolution organization of cellulose microfibrils in onion epidermal cell walls. We frequently found that conventional “rule of thumb” conditions for imaging of biological samples did not yield high-resolution images of cellulose organization and often resulted in artifacts or distortions of cell wall structure. Here we detail our method of one-step fixation and dehydration with 100% ethanol, followed by critical point drying, ultrathin iridium (Ir) sputter coating (3 s), and FESEM imaging at a moderate accelerating voltage (10 kV) with an In-lens detector. We compare results obtained with our improved protocol with images obtained with samples processed by conventional aldehyde fixation, graded dehydration, sputter coating with Au, Au/Pd, or carbon, and low-voltage FESEM imaging. The results demonstrated that our protocol is simpler, causes little artifact, and is more suitable for high-resolution imaging of cell wall cellulose microfibrils whereas such imaging is very challenging by conventional methods.

Extended abstract of a paper presented at Microscopy and Microanalysis 2010 in Portland, Oregon, USA, August 1 – August 5, 2010.

Neospora caninum is an apicomplexan parasite first mentioned in 1984 as a causative agent of neuromuscular disease in dogs. It is closely related to Toxoplasma gondii and Hammondia heydorni, and its subsequent description in 1988 has been, and still is, accompanied by discussions on the true phylogenetical status of the genus Neospora. N. caninum exhibits features that clearly distinguish this parasite from other members of the Apicomplexa, including distinct ultrastructural properties, genetic background, antigenic composition, host cell interactions, and the definition of the dog as a final host. Most importantly, N. caninum has a particular significance as a cause of abortion in cattle. In vitro culture has been indispensable for the isolation of this parasite and for investigations on the ultrastructural, cellular, and molecular characteristics of the different stages of N. caninum. Tissue culture systems include maintenance of N. caninum tachyzoites, which represent the rapidly proliferating stage in a large number of mammalian host cells, culture of parasites in organotypic brain slice cultures as a tool to investigate cerebral infection by N. caninum, and the use of techniques to induce the stage conversion from the tachyzoite stage to the slowly proliferating and tissue cyst-forming bradyzoite stage. This review will focus on the use of these tissue culture models as well as light- and electron-microscopical techniques for studies on N. caninum tachyzoites and bradyzoites, and on the physical interactions between parasites and host cells.

Yttria-stabilized tetragonal zirconia polycrystals (3Y-TZP) is a ceramic material used in indirect dental restorations. However, phase transformation at body temperature may compromise the material’s mechanical properties, affecting the clinical performance of the restoration. The effect of mastication on 3Y-TZP aging has not been investigated. 3Y-TZP specimens (IPS E-max ZirCAD and Z5) were aged in three different modes (n=13): no aging (control), hydrothermal aging (HA), or chewing simulation (CS). Mechanical properties and surface topography were analyzed. Analysis of variance showed that neither aging protocol (p=0.692) nor material (p=0.283) or the interaction between them (p=0.216) had a significant effect on flexural strength, values ranged from 928.8 MPa (IPSHA) to 1,080.6 MPa (Z5HA). Nanoindentation analysis showed that material, aging protocol, and the interaction between them had a significant effect (p<0.001) on surface hardness and reduced Young’s modulus. The compositional analysis revealed similar yttrium content for all the experimental conditions (aging: p=0.997; material: p=0.248; interaction material×aging: p=0.720). Atomic force microscopy showed an effect of aging protocols on phase transformation, with samples submitted to CS exhibiting features compatible with maximized phase transformation, such as increased volume of the material microstructure at the surface leading to an increase in surface roughness.

One of the fundamental challenges in understanding the early stages of corrosion pitting in metals protected with an oxide film is that there are relatively few techniques that can probe microstructure with sufficient resolution while maintaining a wet environment. Here, we demonstrate that microstructural changes in Al thin films caused by aqueous NaCl solutions of varying chloride concentrations can be directly observed using a liquid flow cell enclosed within a transmission electron microscope (TEM) holder. In the absence of chloride, Al thin films did not exhibit significant corrosion when immersed in de-ionized water for 2 days. However, introducing 0.01 M NaCl solutions led to extensive random formation of blisters over the sample surface, while 0.1 M NaCl solutions formed anomalous structures that were larger than the typical grain size. Immersion in 1.0 M NaCl solutions led to fractal corrosion consistent with previously reported studies of Al thin films using optical microscopy. These results show the potential of in situ liquid cell electron microscopy for probing the processes that take place before the onset of pitting and for correlating pit locations with the underlying microstructure of the material.

Mechanisms governing secretion of proteins underlie the biologic activities and functions of human eosinophils, leukocytes of the innate immune system, involved in allergic, inflammatory, and immunoregulatory responses. In response to varied stimuli, eosinophils are recruited from the circulation into inflammatory foci, where they modulate immune responses through the release of granule-derived products. Transmission electron microscopy (TEM) is the only technique that can clearly identify and distinguish between different modes of cell secretion. In this review, we highlight the advances in understanding mechanisms of eosinophil secretion, based on TEM findings, that have been made over the past years and that have provided unprecedented insights into the functional capabilities of these cells.

The relation between image resolution and information transfer is explored. It is shown that the existence of higher frequency transfer in the image is just a necessary but not sufficient condition for the achievement of higher resolution. Adopting a two-point resolution criterion, we suggest that a 10% contrast level between two features in an image should be used as a practical definition of resolution. In the context of scanning transmission electron microscopy, it is shown that the channeling effect does not have a direct connection with image resolution because sharp channeling peaks do not move with the scanning probe. Through a quantitative comparison between experimental image and simulation, a Fourier-space approach is proposed to estimate defocus and sample thickness. The effective atom size in Z-contrast imaging depends on the annular detector's inner angle. Therefore, an optimum angle exists for the highest resolution as a trade-off between reduced atom size and reduced signal with limited information transfer due to noise.

Structures of Pt-nanowires, synthesized in channels of silica mesoporous materials MCM-41, SBA-15 and MCM-48, were investigated by transmission electron microscopy. One dimensional (1D) Pt-nanowires were formed inside the channels of the MCM-41, and were single crystals with a length of several tens to several hundreds nanometers and a diameter of ca. 3 nm Pt-nanowires synthesized in SBA-15 formed a new 3D-network following 3D-pore geometry of SBA-15; that is, the main 1D-channels are interconnected to each other through randomly distributed tunnels. These Pt-nanowires showed a well single crystalline. MCM-48 has two non-intersecting chiral channels, and Pt-networks were mostly formed in one of the two channels. Therefore the networks were also chiral; however, the chirality of Pt-networks remained to be determined. It was shown that all Pt-nanowires were formed following the channel geometries of silica mesoporous materials used.

The micro- and nanoarchitectures of water-swollen hydrogels were routinely analyzed in three dimensions at very high resolution by two cryopreparation methods that provide stable low-temperature specimens for in-lens high magnification recordings. Gemini surfactants (gS), poly-N-isopropylacrylamides (p-NIP Am), and elastin-mimetic di- (db-E) and triblock (tb-E) copolymer proteins that form hydrogels have been routinely analyzed to the sub-10-nm level in a single day. After they were quench or high pressure frozen, samples in bulk planchets were subsequently chromium coated and observed at low temperature in an in-lens field emission SEM. Pre-equilibrated planchets (4–40°C) that hold 5–10 μl of hydrogel facilitate dynamic morphological studies above and below their transition temperatures. Rapidly frozen samples were fractured under liquid nitrogen, low-temperature metal coated, and observed in-lens to assess the dispersion characteristics of micelles and fragile colloidal assemblies within bulk frozen water. Utilizing the same planchet freezing system, the cryoetch-HRSEM technique removed bulk frozen water from the hydrogel matrix by low-temperature, high-vacuum sublimation. The remaining frozen solid-state sample faithfully represented the hydrogel matrix. Cryo- and cryoetch-HRSEM provided vast vistas of hydrogels at low and intermediate magnifications whereas high magnification recordings and anaglyphs (stereo images) provided a three-dimensional prospective and measurements on a molecular level.