Energy-filtered transmission electron microscopy based on inner-shell ionization

We demonstrate that energy-filtered transmission electron microscopy (EFTEM) based on inner-shell ionization can contain atomic resolution information. We present a comparison between experimental data and simulation for the EFTEM image of the _N4,5${\mathrm{N}}_{4,5}$ edge (threshold energy 99 eV) of lanthanum in _LaB6${\mathrm{LaB}}_6$ in which direct interpretation of the location of the lanthanum columns is possible. Our first principles approach is based on calculating transition potentials for inelastic scattering. For our case study, the localization of the transition potentials is such that elastic contrast is only weakly preserved in the EFTEM image. This is not always the case, but we show how the approach based on calculating the elastic wave function and the transition potentials can provide insight about when direct interpretation may and may not be possible. In our test specimen, the direct interpretation fails for thicker specimens when the long tails of the transition potential from multiple adjacent sites leads to significant image features other than at the sites of the element of interest. We can thus anticipate instances where direct interpretation may be more reliable, such as looking for a single impurity in an otherwise well known sample.     

Specimen thickness dependence of hydrogen evolution during cryo‐transmission electron microscopy of hydrated soft materials

The evolution of hydrogen from many hydrated cryo‐preserved soft materials under electron irradiation in the transmission electron microscope can be observed at doses of the order of 1000 e nm^?2 and above. Such hydrogen causes artefacts in conventional transmission electron microscope or scanning transmission electron microscopy (STEM) imaging as well as in analyses by electron energy‐loss spectroscopy. Here we show that the evolution of hydrogen depends on specimen thickness. Using wedge‐shaped specimens of frozen‐hydrated Nafion, a perfluorinated ionomer, saturated with the organic solvent DMMP together with both thin and thick sections of frozen‐hydrated porcine skin, we show that there is a thickness below which hydrogen evolution is not detected either by bubble observation in transmission electron microscope image mode or by spectroscopic analysis in STEM electron energy‐loss spectroscopy mode. We suggest that this effect is due to the diffusion of hydrogen, whose diffusivity remains significant even at liquid nitrogen temperature over the length scales and time scales relevant to transmission electron microscopy analysis of thin specimens. In short, we speculate that sufficient hydrogen can diffuse to the specimen surface in thin sections so that concentrations are too low for bubbling or for spectroscopic detection. Significantly, this finding indicates that higher electron doses can be used during the imaging of radiation‐sensitive hydrated soft materials and, consequently, higher spatial resolution can be achieved, if sufficiently thin specimens are used in order to avoid the evolution of hydrogen‐based artefacts.     

Development of a new bimodal imaging methodology: a combination of fluorescence microscopy and high-resolution secondary ion mass spectrometry

In this paper, we present a new experimental methodology to combine mass spectrometry (NanoSIMS) with fluorescence microscopy to provide subcellular information on the location of small molecules in cultured cells. We demonstrate this by comparing the distribution of 5-bromo-2-deoxyuridine in the same cells given by both NanoSIMS analysis and by fluorescence immunohistochemistry. Fiducial markers in the substrates ensured that the images formed by SIMS mapping of bromine ions could be co-registered exactly with images from fluorescence microscopy. The NanoSIMS was shown to faithfully reproduce the information from fluorescence microscopy, but at a much higher spatial resolution. We then show preliminary SIMS images on the distribution of ATN-224, a therapeutic copper chelator for which there is no fluorescent marker, co-registered with conventional Lysotracker and Hoechst stains on the same cells.     

Texture of cellulose microfibrils of root hair cell walls of Arabidopsis thaliana, Medicago truncatula, and Vicia sativa

Cellulose is the most abundant biopolymer on earth, and has qualities that make it suitable for biofuel. There are new tools for the visualisation of the cellulose synthase complexes in living cells, but those do not show their product, the cellulose microfibrils (CMFs). In this study we report the characteristics of cell wall textures, i.e. the architectures of the CMFs in the wall, of root hairs of Arabidopsis thaliana, Medicago truncatula and Vicia sativa and compare the different techniques we used to study them. Root hairs of these species have a random primary cell wall deposited at the root hair tip, which covers the outside of the growing and fully grown hair. The secondary wall starts between 10 (Arabidopsis) and 40 (Vicia) μm from the hair tip and the CMFs make a small angle, Z as well as S direction, with the long axis of the root hair. CMFs are 3-4 nm wide in thin sections, indicating that single cellulose synthase complexes make them. Thin sections after extraction of cell wall matrix, leaving only the CMFs, reveal the type of wall texture and the orientation and width of CMFs, but CMF density within a lamella cannot be quantified, and CMF length is always underestimated by this technique. Field emission scanning electron microscopy and surface preparations for transmission electron microscopy reveal the type of wall texture and the orientation of individual CMFs. Only when the orientation of CMFs in subsequent deposited lamellae is different, their density per lamella can be determined. It is impossible to measure CMF length with any of the EM techniques.     

Element specific imaging with high lateral resolution: an experimental study on layer structures

Planar defects and individual layers in ceramic material are chemically imaged by high resolution energy-filtering TEM using a post-column imaging electron energy filter. Objects are barium layers in the cuprate superconductor NdBa₂Cu₃O_7−δ (isostructual to YBa₂Cu₃O_7−δ) as well as planar defects and precipitates of β-WB in tungsten- and chromium-doped TiB₂. The barium layers with a spacing of 0.42 nm in the cuprate are resolved in jump-ratio images using the Ba_N edge. In the boride system the β-WB precipitates with thickness of 0.8 nm can be chemically imaged in elemental maps of B_K, Ti_L ,Cr_L and W_M. The B as well as the Ti map show a decrease in intensity at the precipitates, whereas in the W map an increase in intensity is observed. The boron-deficient layers with a spacing of 0.38 nm in the β-WB precipitate can be resolved in boron jump-ratio images. Additionally, defects containing single boron-deficient layers are chemically imaged. Hence structures in the dimension of interatomic distances can be imaged with respect to their elemental constituents. Although high resolution electron spectroscopic images contain strong interference contrast from elastic scattering, after normalization or background subtraction the element specific images are dominated by chemical contrast.     

Scanning electron microscopy and transmission electron microscopy study of hot-deformed γ-TiAl-based alloy microstructure

The aim of this work was to assess the changes in the microstructure of hot‐deformed specimens made of alloys containing 46–50 at.% Al, 2 at.% Cr and 2 at.% Nb (and alloying additions such as carbon and boron) with the aid of scanning electron microscopy and transmission electron microscopy techniques. After homogenization and heat treatment performed in order to make diverse lamellae thickness, the specimens were compressed at 1000 °C. Transmission electron microscopy examinations of specimens after the compression test revealed the presence of heavily deformed areas with a high density of dislocation. Deformation twins were also observed. Dynamically recrystallized grains were revealed. For alloys no. 2 and no. 3, the recovery and recrystallization processes were more extensive than for alloy no. 1.