Cardiac fibroblasts are the most numerous cells in the heart and are critical in the formation and normal functioning of the organ. Cardiac fibroblasts are firmly attached to and surrounded by extracellular matrix (ECM). Mechanical forces transmitted through interaction with the ECM can result in changes of overall cellular shape, cytoskeletal organization, proliferation, and gene expression of cardiac fibroblasts. These responses may be different in the normally functioning heart, when compared with various pathological conditions, including inflammation or hypertrophy. It is apparent that cellular phenotype and physiology, in turn, are affected by multiple signal transduction pathways modulated directly by the state of polymerization of the actin cytoskeleton. Morphological changes in actin organization resulting from response to adverse conditions in fibroblasts and other cell types are basically descriptive. Some studies have approached quantifying changes in actin cytoskeletal morphology, but these have involved complex and difficult procedures. In this study, we apply image analysis and non-Euclidian geometrical fractal analysis to quantify and describe changes induced in the actin cytoskeleton of cardiac fibroblasts responding to mechanical stress. Characterization of these rapid responses of fibroblasts to mechanical stress may provide insight into the regulation of fibroblasts behavior and gene expression during heart development and disease.

Analysis and understanding of the role of hydrogen in metals is a significant challenge for the future of materials science, and this is a clear objective of recent work in the atom probe tomography (APT) community. Isotopic marking by deuteration has often been proposed as the preferred route to enable quantification of hydrogen by APT. Zircaloy-4 was charged electrochemically with hydrogen and deuterium under the same conditions to form large hydrides and deuterides. Our results from a Zr hydride and a Zr deuteride highlight the challenges associated with accurate quantification of hydrogen and deuterium, in particular associated with the overlap of peaks at a low mass-to-charge ratio and of hydrogen/deuterium containing molecular ions. We discuss possible ways to ensure that appropriate information is extracted from APT analysis of hydrogen in zirconium alloy systems that are important for nuclear power applications.

Cold spray is a thermo-mechanical process where the velocity of the sprayed particles affects the deformation, bonding, and mechanical properties of the deposited material, in the form of splats or coatings. At high strain rates, the impact stresses are converted into heat, a phenomenon known as adiabatic shear, which leads to grain re-crystallization. Grain re-crystallization and growth are shown to have a direct impact on the mechanical properties of the cold-sprayed material. The present study ties the microstructural features within the cold-sprayed Ti splats and the substrate to the bonding mechanism and mechanical properties. High-resolution electron channeling contrast imaging, electron backscatter diffraction mapping, and nanoindentation were used to correlate the microstructure to the mechanical properties distribution within the titanium cold-spray splats. The formation of nanograins was observed at the titanium splat/substrate interface and contributed to metallurgical bonding. An increase in grain re-crystallization within the splat and substrate materials was observed with pre-heating of the substrate. In the substrate material, the predominant mechanism of deformation was twinning. A good relationship was found between the hardness and distribution of the twins within the substrate and the size distribution of the re-crystallized grains within the splats.

Electron backscatter diffraction (EBSD) has become a common technique for measuring crystallographic orientations at spatial resolutions on the order of tens of nanometers and at angular resolutions <0.1°. In a recent search of EBSD papers using Google Scholar™, 60% were found to address some aspect of deformation. Generally, deformation manifests itself in EBSD measurements by small local misorientations. An increase in the local misorientation is often observed near grain boundaries in deformed microstructures. This may be indicative of dislocation pile-up at the boundaries but could also be due to a loss of orientation precision in the EBSD measurements. When the electron beam is positioned at or near a grain boundary, the diffraction volume contains the crystal lattices from the two grains separated by the boundary. Thus, the resulting pattern will contain contributions from both lattices. Such mixed patterns can pose some challenge to the EBSD pattern band detection and indexing algorithms. Through analysis of experimental local misorientation data and simulated pattern mixing, this work shows that some of the rise in local misorientation is an artifact due to the mixed patterns at the boundary but that the rise due to physical phenomena is also observed.

Electron-beam-induced soft-X-ray emission spectroscopy (SXES) that uses a grating spectrometer has been introduced to a conventional scanning electron microscope (SEM) for characterizing desired specimen areas of bulk materials. The spectrometer was designed as a grazing incidence flat-field optics by using aberration corrected (varied line spacing) gratings and a multichannel plate detector combined with a charge-coupled device camera, which has already been applied to a transmission electron microscope. The best resolution was confirmed as 0.13 eV at Mg L-emission (50 eV), which is comparable with that of recent dedicated electron energy-loss spectroscopy instruments. This SXES-SEM instrument presents density of states of simple metals of bulk Mg and Li. Apparent band-structure effects have been observed in Si L-emission of Si wafer, P L-emission of GaP wafer, and Al L-emissions of intermetallic compounds of AlCo, AlPd, Al2Pt, and Al2Au.

Through recent years, off-axis electron holography has helped us to understand and to overcome some experimental restrictions in transmission electron microscopy. With development of powerful electron microscopes, slow-scan CCD cameras, and computers, holography is not an academic technique anymore used by specialized laboratories. Holography has proven its wide range of applications in solving real-world problems in materials science and biology. At medium resolution, that is, on nanometer scale, holography allows access to large area phase contrast produced by magnetic fields and electric potentials. In the high-resolution domain, holography unveils its power by unscrambling amplitude and phase of the electron wave, resulting in an improved lateral resolution up to the information limit. Holography is a thoroughly quantitative method, and, in combination with the perfect zero-loss filtering inherent to this method, the interpretation of the reconstructed data is strongly simplified. After outlining the basics of holography, in this tutorial we focus on development of a step-by-step procedure for recording and reconstruction of holograms. At the end, some recent applications are discussed.

A procedure based on focused ion beam milling and in situ lift-out is introduced for the preparation of high-quality specimens for in situ annealing experiments in the transmission electron microscope. The procedure allows an electron-transparent lamella to be cleaned directly on a heating chip using a low ion energy and back-side milling in order to minimize redeposition and damage. The approach is illustrated through the preparation of an Al–Mn–Fe complex metallic alloy specimen.

Over the last few years there have been significant developments in the field of three-dimensional atom probe (3DAP) analysis. This article reviews some of the technical compromises that have led to different instrument designs and the recent improvements in performance. An instrument has now been developed, based around a novel reflectron configuration combining both energy compensation and focusing elements, that yields a large field of view and very high mass resolution. The use of laser pulsing in the 3DAP, together with developments in specimen preparation methods using a focused ion-beam instrument, have led to a significant widening in the range of materials science problems that can be addressed with the 3DAP. Recent studies of semiconductor materials and devices are described.

The ability to accurately reconstruct original spatial positions of field-evaporated ions emitted from a surface is fundamental to the success of atom probe tomography. As such, a clear understanding of the evolution of specimen shape and the resultant ions' trajectories during field evaporation plays an important role in improving reconstruction accuracy. To further this understanding, field-evaporation simulations of a bilayer specimen composed of two materials having an evaporation field difference of 20% were performed. The simulated field-evaporation patterns qualitatively compare favorably with experimental data, which provides confidence in the accuracy of specimen shapes predicted by the simulation. Correlations of known original atom positions with detector hit positions as a function of lateral detector position and evaporated depth were derived from the simulation. These correlations are contrasted with the current state-of-the-art reconstruction method thus outlining limitations of the current methodology. A pair of transformations are defined that take into account field-evaporated specimen shapes, and the resulting radial magnifications, to relate recorded ion positions in detector space to reconstructed atomic positions in specimen space. This novel process, when applied to simulated data, results in approximately a factor of 2 improvement in accuracy for reconstructions of interfaces with unequal fields (most general interfaces). This method is not constrained by the fundamental assumption of a hemispherical specimen shape.

A three-dimensional (3D) reconstruction of the cytoskeleton and a clathrin-coated pit in mammalian cells has been achieved from a focal-series of images recorded in an aberration-corrected scanning transmission electron microscope (STEM). The specimen was a metallic replica of the biological structure comprising Pt nanoparticles 2–3 nm in diameter, with a high stability under electron beam radiation. The 3D dataset was processed by an automated deconvolution procedure. The lateral resolution was 1.1 nm, set by pixel size. Particles differing by only 10 nm in vertical position were identified as separate objects with greater than 20% dip in contrast between them. We refer to this value as the axial resolution of the deconvolution or reconstruction, the ability to recognize two objects, which were unresolved in the original dataset. The resolution of the reconstruction is comparable to that achieved by tilt-series transmission electron microscopy. However, the focal-series method does not require mechanical tilting and is therefore much faster. 3D STEM images were also recorded of the Golgi ribbon in conventional thin sections containing 3T3 cells with a comparable axial resolution in the deconvolved dataset.

Scanning transmission electron microscopy (STEM) allows atomic scale characterization of solid–solid interfaces, but has seen limited applications to solid–liquid interfaces due to the volatility of liquids in the microscope vacuum. Although cryo-electron microscopy is routinely used to characterize hydrated samples stabilized by rapid freezing, sample thinning is required to access the internal interfaces of thicker specimens. Here, we adapt cryo-focused ion beam (FIB) “lift-out,” a technique recently developed for biological specimens, to prepare intact internal solid–liquid interfaces for high-resolution structural and chemical analysis by cryo-STEM. To guide the milling process we introduce a label-free in situ method of localizing subsurface structures in suitable materials by energy dispersive X-ray spectroscopy (EDX). Monte Carlo simulations are performed to evaluate the depth-probing capability of the technique, and show good qualitative agreement with experiment. We also detail procedures to produce homogeneously thin lamellae, which enable nanoscale structural, elemental, and chemical analysis of intact solid–liquid interfaces by analytical cryo-STEM. This work demonstrates the potential of cryo-FIB lift-out and cryo-STEM for understanding physical and chemical processes at solid–liquid interfaces.

Environmental sampling (ES) is a powerful technique used by safeguards inspectors of the International Atomic Energy Agency and the European Safeguards Office for the detection of undeclared nuclear activities. Since its implementation in the 1990s, ES has proven to be very sensitive and effective. Considering the consequences, the measurements should be carried out under a quality management programme. At the Institute for Reference Materials and Measurements, a new production method is under development for the preparation of reference uranium particles from well-certified UF6, allowing uranium particles with certified isotopic abundances to be prepared that are representative of those found in uranium enrichment facilities. Using an aerosol deposition chamber designed and built for the purpose, particles are formed by the hydrolysis of UF6 and their morphology and (isotopic) composition measured using SEM-EDX and SIMS. The SEM measurements show that by varying the relative humidity of the air in the reaction chamber, the morphology of the particles can be changed. By making a distribution map of the chemical composition of the particles, the relationship between fluorine and uranium as main constituents of the particle could be established. The presence of fluorine is a valuable indicator for the occurrence of nondeclared enrichment activities.

Atom probe tomography (APT) provides three-dimensional analytical imaging of materials with near-atomic resolution using pulsed field evaporation. The processes of field evaporation can cause atoms to be placed at positions in the APT reconstruction that can deviate slightly from their original site in the material. Here, we describe and model one such process—that of preferential retention of solute atoms in multicomponent systems. Based on relative field evaporation probabilities, we calculate the point spread function for the solute atom distribution in the “z,” or in-depth direction, and use this to extract more accurate solute concentration profiles.

A Monte Carlo model was used to simulate specimen-electron beam interactions relevant to electron back-scattered diffraction (EBSD). Electron trajectories were calculated for a variety of likely experimental conditions to examine the interaction volume of the incident electrons as well as that of the subset of incident electrons that emerge from the specimen, i.e., back-scattered electrons (BSEs). The spatial resolution of EBSD was investigated as functions of both materials properties, such as atomic number, atomic weight, and density, and experimental parameters, such as specimen thickness, tilt, and incident beam accelerating voltage. These simulations reveal that the achievable spatial resolution in EBSD is determined by these intrinsic and extrinsic parameters.

Electron backscatter diffraction (EBSD) is becoming a widely used technique to determine crystallographic orientation in biogenic carbonates. Despite this use, there is little information available on preparation for the analysis of biogenic carbonates. EBSD data are compared for biogenic aragonite and calcite in the common blue mussel, Mytilus edulis, using different types of resin and thicknesses of carbon coating. Results indicate that carbonate biomineral samples provide better EBSD results if they are embedded in resin, particularly epoxy resin. A uniform layer of carbon of 2.5 nm thickness provides sufficient conductivity for EBSD analyses of such insulators to avoid charging without masking the diffracted signal. Diffraction intensity decreases with carbon coating thickness of 5 nm or more. This study demonstrates the importance of optimizing sample preparation for EBSD analyses of insulators such as carbonate biominerals.

Although acidocalcisomes have been well characterized morphologically in other apicomplexan parasites, no such characterization has been done in Plasmodium spp. Here, we report that Plasmodium falciparum merozoites possess electron-dense organelles rich in phosphorus and calcium, as detected by X-ray microanalysis of intact cells, which are similar to the acidocalcisomes of other apicomplexans, but of more irregular form. In agreement with these results malaria parasites possess large amounts of short- and long-chain polyphosphate (polyP), which are associated with acidocalcisomes in other organisms. PolyP levels were highest in the trophozoite stage of the parasite. Treatment of isolated trophozoites with chloroquine resulted in a significant hydrolysis of polyP. Taken together, these results provide evidence that acidocalcisomes from Plasmodium falciparum do not differ significantly from acidocalcisomes of other apicomplexan parasites.

Crystals within volcanic rocks record geochemical and textural signatures during magmatic evolution before eruption. Clues to this magmatic history can be examined using crystal size distribution (CSD) studies. The analysis of CSDs is a standard petrological tool, but laborious due to manual hand-drawing of crystal margins. The trainable Weka segmentation (TWS) plugin in ImageJ is a promising alternative. It uses machine learning and image segmentation to classify an image. We recorded back-scattered electron (BSE) images of three volcanic samples with different crystallinity (35, 50 and ≥85 vol. %), using scanning electron microscopes (SEM) of variable image resolutions, which we then tested using TWS. Crystal measurements obtained from the automatically segmented images are compared with those of the manual segmentation. Samples up to 50 vol. % crystallinity are successfully segmented using TWS. Segmentation at significantly higher crystallinities fails, as crystal boundaries cannot be distinguished. Accuracy performance tests for the TWS classifiers yield high F-scores (>0.930), hence, TWS is a successful and fast computing tool for outlining crystals from BSE images of glassy rocks. Finally, reliable CSD’s can be derived using a low-cost desktop SEM, paving the way for a wide range of research to take advantage of this new petrological method.

Pluripotent cells are unique because of their ability to differentiate into the cell lineages forming the entire organism. True pluripotent stem cells with germ line contribution have been reported for mice and rats. Human pluripotent cells share numerous features of pluripotentiality, but confirmation of their in vivo capacity for germ line contribution is impossible due to ethical and legal restrictions. Progress toward derivation of embryonic stem cells from domestic species has been made, but the derived cells were not able to produce germ line chimeras and thus are termed embryonic stem-like cells. However, domestic animals, in particular the domestic pig (Sus scrofa), are excellent large animals models, in which the clinical potential of stem cell therapies can be studied. Reprogramming technologies for somatic cells, including somatic cell nuclear transfer, cell fusion, in vitro culture in the presence of cell extracts, in vitro conversion of adult unipotent spermatogonial stem cells into germ line derived pluripotent stem cells, and transduction with reprogramming factors have been developed with the goal of obtaining pluripotent, germ line competent stem cells from domestic animals. This review summarizes the present state of the art in the derivation and maintenance of pluripotent stem cells in domestic animals.