Quantitative atomic resolution mapping using high-angle annular dark field scanning transmission electron microscopy

A model-based method is proposed to relatively quantify the chemical composition of atomic columns using high angle annular dark field (HAADF) scanning transmission electron microscopy (STEM) images. The method is based on a quantification of the total intensity of the scattered electrons for the individual atomic columns using statistical parameter estimation theory. In order to apply this theory, a model is required describing the image contrast of the HAADF STEM images. Therefore, a simple, effective incoherent model has been assumed which takes the probe intensity profile into account. The scattered intensities can then be estimated by fitting this model to an experimental HAADF STEM image. These estimates are used as a performance measure to distinguish between different atomic column types and to identify the nature of unknown columns with good accuracy and precision using statistical hypothesis testing. The reliability of the method is supported by means of simulated HAADF STEM images as well as a combination of experimental images and electron energy-loss spectra. It is experimentally shown that statistically meaningful information on the composition of individual columns can be obtained even if the difference in averaged atomic number Z is only 3. Using this method, quantitative mapping at atomic resolution using HAADF STEM images only has become possible without the need of simultaneously recorded electron energy loss spectra.     

Dynamics of annular bright field imaging in scanning transmission electron microscopy

We explore the dynamics of image formation in the so-called annular bright field mode in scanning transmission electron microscopy, whereby an annular detector is used with detector collection range lying within the cone of illumination, i.e. the bright field region. We show that this imaging mode allows us to reliably image both light and heavy columns over a range of thickness and defocus values, and we explain the contrast mechanisms involved. The role of probe and detector aperture sizes is considered, as is the sensitivity of the method to intercolumn spacing and local disorder.     

Prospects for lithium imaging using annular bright field scanning transmission electron microscopy: a theoretical study

There is strong interest in lithium imaging, particularly because of its significance in battery materials. However, light atoms only scatter electrons weakly and atomic resolution direct imaging of lithium has proven difficult. This paper explores theoretically the conditions under which lithium columns can be expected to be directly visible using annular bright field scanning transmission electron microscopy. A detailed discussion is given of the controllable parameters and the conditions most favourable for lithium imaging.     

Cation segregation in Nb16W18O94 using high angle annular dark field scanning transmission electron microscopy and image processing

We report the characterization of the complex oxide Nb₁₆W₁₈O₉₄ using high angle annular dark field imaging at 200 kV in a scanning transmission electron microscope. The results of this study suggest that the W and Nb cations are not uniformly distributed among the cation columns projected along [001] but that there is preferential segregation of the heavier species to certain column sites. In order to analyse the experimental data obtained, an image processing methodology has been developed which may also find application in locating specific motifs within a generally distorted image field.     

Nanoscale scanning transmission electron tomography

Electron tomography enables the study of complex three‐dimensional objects with nanometre resolution. In materials science, scanning transmission electron microscopy provides images with minimal coherent diffraction effects and with high atomic number contrast that makes them ideal for electron tomographic reconstruction. In this study, we reviewed the topic of scanning transmission electron microscopy‐based tomography and illustrated the power of the technique with a number of examples with critical dimensions at the nanoscale.     

The fine structure of fenestrated adrenocortical capillaries revealed by in-lens field-emission scanning electron microscopy and scanning transmission electron microscopy

Cell biologists probing the physiologic movement of macromolecules and solutes across the fenestrated microvascular endothelial cell have used electron microscopy to locate the postulated pore within the fenestrae. Prior to the advent of in-lens field-emission high-resolution scanning electron microscopy (HRSEM) and ultrathin m et al coating technology, quick-freeze, platinum-carbon replica and grazing thin-section transmission electron microscopy (TEM) methods provided two-dimensional or indirect imaging methods. Wedge-shaped octagonal channels composed of fibrils interwoven in a central mesh were depicted as the filtering structures of fenestral diaphragms in images of platinum replicas enhanced by photographic augmentation. However, image accuracy was limited to replication of the cell surface. Subsequent to this, HRSEM technology was developed and provided a high-fidelity, three-dimensional topographic image of the fenestral surface directly from a fixed and dried bulk adrenal specimen coated with a 1 nm chromium film. First described from TEM replicas, the “flower-like” structure comprising the fenestral pores was readily visualized by HRSEM. High-resolution images contained particulate ectodomains on the lumenal surface of the endothelial cell membrane. Particles arranged in a rough octagonal shape formed the fenestral rim. Digital acquisition of analog photographic recordings revealed a filamentous meshwork in the diaphragm, thus confirming and extending observations from replica and grazing section TEM preparations. Endothelial cell pockets, first described in murine renal peritubular capillaries, were observed in rhesus and rabbit adrenocortical capillaries. This report features recent observations of fenestral diaphragms and endothelial pockets fitted with multiple diaphragms utilizing a Schottky field-emission electron microscope. In-lens staging of bulk and thin section specimens allowed tandem imaging in HRSEM and scanning TEM modes at 25 kV.     

Local crystal structure analysis with several picometer precision using scanning transmission electron microscopy

We report a local crystal structure analysis with a high precision of several picometers on the basis of scanning transmission electron microscopy (STEM). Advanced annular dark-field (ADF) imaging has been demonstrated using software-based experimental and data-processing techniques, such as the improvement of signal-to-noise ratio, the reduction of image distortion, the quantification of experimental parameters (e.g., thickness and defocus) and the resolution enhancement by maximum-entropy deconvolution. The accuracy in the atom position measurement depends on the validity of the incoherent imaging approximation, in which an ADF image is described as the convolution between the incident probe profile and scattering objects. Although the qualitative interpretation of ADF image contrast is possible for a wide range of specimen thicknesses, the direct observation of a crystal structure with deep-sub-angstrom accuracy requires a thin specimen (e.g., 10 nm), as well as observation of the structure image by conventional high-resolution transmission electron microscopy.     

Characterization of an oil-degrading Microcoleus consortium by means of confocal scanning microscopy, scanning electron microscopy and transmission electron microscopy

A consortium of microorganisms with the capacity to degrade crude oil has been characterized by means of confocal laser scanning microscopy (CLSM), transmission electron microscopy (TEM), and scanning electron microscopy (SEM). The analysis using CLSM shows that Microcoleus chthonoplastes is the dominant organism in the consortium. This cyanobacterium forms long filaments that group together in bundles inside a mucopolysaccharide sheath. Scanning electron microscopy and transmission electron microscopy have allowed us to demonstrate that this cyanobacterium forms a consortium primarily with three morphotypes of the heterotrophic microorganisms found in the Microcoleus chthonoplastes sheath. The optimal growth of Microcoleus consortium was obtained in presence of light and crude oil, and under anaerobic conditions. When grown in agar plate, only one type of colony (green and filamentous) was observed.     

Imaging of semiconducting polymer blend systems using environmental scanning electron microscopy and environmental scanning transmission electron microscopy

This study has investigated the potential of environmental electron microscopy techniques for studying the structure of polymer‐based electronic devices. Polymer blend systems composed of F8BT and PFB were examined. Excellent contrast, both topographical and compositional, can be achieved using both conventional environmental scanning electron microscopy (ESEM) and a transmission detector giving an environmental scanning transmission electron microscope (ESTEM) configuration. Controllable charging effects present in the ESEM were observed, giving rise to a novel voltage contrast. This shows the potential of such contrast to provide excellent images of phase structure and charge distributions.     

Human chromatid ultrastructure: new observations with scanning and transmission electron microscopy

Two new observations have been made on human chromatid/chromosome ultrastructure using both scanning and transmission electron microscopy (SEM, TEM). A bipartite, apparently half-chromatid-like structure was observed in whole human chromosomes studied with SEM and in longitudinally sectioned chromosomes analyzed with TEM. In addition, we also observed a zipper-like configuration as the parallel sister chromatids separated likely due to the supercoiled structure of the chromosome and chromatid. It is possible that either or both of these new observations resulted from our (improved) method of preparing the chromosomes for SEM and TEM.     

Energy filtering transmission electron microscopy using the new JEM-2010FEF

The new JEM-2010FEF electron microscope provides useful techniques based on energy filtering as an omega-type energy filter is integrated into a thermal field-emission 200 kV transmission electron microscope. For example, the zero-loss imaging improves the contrast of high resolution lattice images as well as images of precipitates or lattice defects in alloys. The acquisition time for elemental mapping with core-loss electrons is one order in magnitude shorter than with energy-dispersive X-ray spectroscopy. The removal of inelastically scattered electrons enables us to observe weak lines in convergent-beam electron diffraction patterns from a thicker specimen with a probe size 1–2 nm in diameter. A combination of the field emission gun and sensitive recording media such as an imaging plate and a slow-scan CCD camera makes the energy filtering more powerful.     

A procedure to prepare cultured cells in suspension for electron probe X-ray microanalysis: application to scanning and transmission electron microscopy

We describe a simple procedure to prepare cultured cells in suspension to analyse elemental content at the cellular level by electron probe X-ray microanalysis. Cells cultured in suspension were deposited onto polycarbonate tissue, culture plate well inserts, centrifuged at low g , washed to remove the extracellular medium, cryofixed and freeze-dried, and analysed in the scanning mode of a scanning electron microscope. We tested the effect of different washing solutions (150 m m ammonium acetate, 300 m m sucrose, and distilled water) on the elemental content of cultured cells in suspension. The results demonstrated that distilled water was the best washing solution to prepare cultured cells. In addition, the low Na content, high K content and high K/Na ratio of the cells indicated that this procedure, based on the centrifugation at low g followed by cryopreparation, constitutes a satisfactory method to prepare cultured cells in suspension. We also investigated the effects of different accelerating voltages on X-ray signal collection. The results showed that moderate accelerating voltages, i.e. 10–11 kV, should be used to analyse whole cells in the scanning mode of the scanning electron microscope. We show that this method of preparation makes it possible to prepare cryosections of the cultured cells, thus permitting analysis of the elemental content at the subcellular level, i.e. nucleus, cytoplasm and mitochondria, using a scanning transmission electron microscope.     

Investigating the optical properties of dislocations by scanning transmission electron microscopy

The scanning transmission electron microscope (STEM) allows collection of a number of simultaneous signals, such as cathodoluminescence (CL), transmitted electron intensity and spectroscopic information from individual localized defects. This review traces the development of CL and atomic resolution imaging from their early inception through to the possibilities that exist today for achieving a true atomic-scale understanding of the optical properties of individual dislocations cores. This review is dedicated to Professor David Holt, a pioneer in this field.     

What atomic resolution annular dark field imaging can tell us about gold nanoparticles on (1 1 0)

Annular dark field scanning transmission electron microscopy imaging was recently applied to a catalyst consisting of gold nanoparticles on TiO₂ (1 1 0), showing directly that the gold atoms in small nanoparticles preferentially attach to specific sites on the TiO₂ (1 1 0) surface. Here, through simulation, a parameter exploration of the imaging conditions which maximise the visibility of such nanoparticles is presented. Aberration correction, finite source size and profile imaging are all considered while trying to extracting the maximum amount of information from a given sample. Comment is made on the role of the thermal vibration of the atoms in the nanoparticle, the magnitude of which is generally not known a priori but which affects the visibility of the nanoparticles in this imaging mode.     

Error rates in buccal-dental microwear quantification using scanning electron microscopy

Dental microwear, usually analyzed using scanning electron microscopy (SEM) techniques, is a good indicator of the abrasive potential of past human population diets. Scanning electron microscopy secondary electrons provide excellent images of dental enamel relief for characterizing striation density, average length, and orientation. However, methodological standardization is required for interobserver comparisons since semiautomatic counting procedures are still used for micrograph characterization. The analysis of normally distributed variables allows the characterization of small interpopulation differences. However, the interobserver error rates associated with SEM experience and the degree of expertise in measuring striations are critical to population dietary interpretation. The interobserver comparisons made here clearly indicate that the precision of SEM buccal microwear measurements depends heavily on variable definition and the researcher's expertise. Moreover, error rates are not the only concern for dental microwear research. Low error rates do not guarantee that all researchers are measuring the same magnitudes of the variables considered. The results obtained show that researchers tend to maintain high intrapopulation homogeneity and low measurement error rates, whereas significant interobserver differences appear. Such differences are due to a differential interpretation of SEM microwear features and variable definitions that require detailed and precise agreement among researchers. The substitution of semiautomatic with fully automated procedures will completely avoid interobserver error rate differences.     

Estimation of mass thickness response of embedded aggregated silica nanospheres from high angle annular dark‐field scanning transmission electron micrographs

In this study, we investigate the functional behaviour of the intensity in high‐angle annular dark field scanning transmission electron micrograph images. The model material is a silica particle (20 nm) gel at 5 wt%. By assuming that the intensity response is monotonically increasing with increasing mass thickness of silica, an estimate of the functional form is calculated using a maximum likelihood approach. We conclude that a linear functional form of the intensity provides a fair estimate but that a power function is significantly better for estimating the amount of silica in the z‐direction. The work adds to the development of quantifying material properties from electron micrographs, especially in the field of tomography methods and three‐dimensional quantitative structural characterization from a scanning transmission electron micrograph. It also provides means for direct three‐dimensional quantitative structural characterization from a scanning transmission electron micrograph.     

Imaging of high-angle annular dark-field scanning transmission electron microscopy and observations of GaN-based violet laser diodes

The first part of this paper is devoted to physics, to explain high‐angle annular dark‐field scanning transmission electron microscopy (HAADF‐STEM) imaging and to interpret why HAADF‐STEM imaging is incoherent, instructing a strict definition of interference and coherence of electron waves. Next, we present our recent investigations of InGaN/GaN multiple quantum wells and AlGaN/GaN strained‐layer superlattice claddings in GaN‐based violet laser diodes, which have been performed by HAADF‐STEM and high‐resolution field‐emission gun scanning electron microscopy.     

Investigation of cholesterol,bilirubin, and protein distribution in human gallstones by color cathodoluminescence scanning electron microscopy and transmission electron microscopy

The application of color cathodoluminescent scanning electron microscopy (CCL-SEM) for qualitative luminescence analysis of cholesterol, bilirubin, and protein in human gallstones was demonstrated. Images of these deposits (cholesterol, bilirubin, and protein) were formed in real colors (blue—cholesterol, red, orange—bilirubin, yellow, green—protein) in accordance with the cathodoluminescent spectrum for each control material. The other method described for transmission electron microscopy (TEM) of ultra-thin sections provides more detailed characterization of the ultrastructure of cholesterol-containing regions and their spatial interrelations with bilirubin-containing regions. Using CCL-SEM combined with TEM permits the receipt of more complete information about the chemical composition and ultrastructure of gallstones and may lead to more effective understanding of the pathogenesis of cholesterol cholelithiasis.