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.     

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.     

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.     

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.     

Comparative study of a block copolymer morphology by transmission electron microscopy and scanning force microscopy

Using transmission electron microscopy (TEM) and scanning force microscopy (SFM) together, it was possible to verify important structural features of a nanostructured bulk material such as the kp‐morphology in an ABC triblock copolymer. By applying suitable imaging techniques during the SFM measurements it was possible to determine the morphology without additional manipulation steps in between. In comparison, TEM investigations on this type of material usually require selective staining procedures prior to the measurement. Also electron beam damage is often encountered during TEM measurements especially if components such as poly(methacrylates) are present. In contrast, SFM measurements can be assumed not to significantly change the phase dimensions of the components.     

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.     

A new experimental procedure to quantify annular dark field images in scanning transmission electron microscopy

A new procedure to quantify the contrast in annular dark field images recorded without lattice resolution in a scanning transmission electron microscope is proposed. The method relies on the use of an in‐column energy filter prior to the annular dark field detector and the acquisition of a series of energy‐filtered images as a function of the inner detection angle. When the image contrast of an interface between two materials in such energy‐filtered annular dark field images is plotted vs. camera length and extrapolated to zero (i.e. infinite scattering angle), the contrast is shown to behave exactly as predicted by Rutherford's scattering formula (i.e. intensity scales ∝Z²). This can then be used to determine the local chemistry at and the effective chemical widths of interfaces or thin films without any additional spectroscopy method for calibration, provided the global chemical composition is known. As examples, the systems SiGe/Si and InGaAs/Ge are considered in detail.     

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.     

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.     

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.     

Imaging an optical disc by the combined use of scanning tunnelling microscopy and scanning electron microscopy

We present the data obtained by scanning tunnelling microscopy combined with scanning electron microscopy of the digitally encoded structure on a stamper used to fabricate optical discs. The combination allows us to focus the STM tip on a preselected spot with a precision of ?0·3 μm. The data show the superiority of STM for a more detailed characterization of shape, width, length, height and fine structure appearing on the sample. We also show the influence of tip shape on STM resolution. Simultaneous use of both microscopes is possible but high electron doses produce an insulating layer of contaminants thick enough to make STM operation impossible.     

Use of sputter coating to prepare whole mounts of cytoskeletons for transmission and high-resolution scanning and scanning transmission electron microscopy

This paper describes the use of sputter coating to prepare detergent-extracted cytoskeletons for observation by scanning (SEM), scanning transmission (STEM), inverted contrast STEM, and transmission (TEM) electron microscopy. Sputtered coats of 1–2 nm of platinum or tungsten provide both an adequate secondary electron signal for SEM and good contrast for STEM and TEM. At the same time, the grain size of the coating is sufficiently fine to be just at (platinum) or below (tungsten) the limit of resolution for SEM and STEM. In TEM, the granular structure of platinum coats is resolved, and platinum decoration artifacts are observed on the surface of structures. The platinum is deposited as small islands with a periodic distribution that may reveal information about the underlying molecular structure. This method produces samples that are similar in appearance to replicas prepared by low-angle rotary shadowing with platinum and carbon. However, the sputter-coating method is easier to use; more widely available to investigators; and compatible with SEM, STEM, and TEM. It may also be combined with immunogold and other labeling methods. While TEM provides the highest resolution images of sputter-coated cytoskeletons, it also damages the specimens owing to heating in the beam. In SEM and STEM cytoskeletons are stable and the resolution is adequate to resolve individual microfilaments. The best single method for visualizing cytoskeletons is inverted contrast STEM, which images both the metal-coated cytoskeletal structures and electron-dense material within the nucleus and cytoplasm as white against a dark background. STEM and TEM were both suitable for visualizing colloidal gold particles in immunolabeled samples.     

Imaging of extraradicular biofilm using combined scanning electron microscopy and stereomicroscopy

Microorganisms are able to survive and induce persistent infection in extraradicular areas. The objective of this study was to evaluate the relationship between extraradicular biofilm and persistent periapical periodontitis. Thirty‐five apical samples with different stages of pulp and periapical pathology (vital pulp, pulp necrosis without radiographically visible periapical lesions, chronic periapical periodontitis that had not received root canal therapy and persistent periapical periodontitis) were initially evaluated using scanning electron microscopy. The same samples were then processed using Brown and Brenn‐modified Gram staining. We detected extraradicular biofilm in all samples with persistent periapical periodontitis and in three samples with chronic periapical periodontitis. The extraradicular bacteria predominantly had rod and filament morphology and were surrounded by varying amounts of amorphous extracellular material. The surfaces outside the root of the apical samples with vital pulp and pulp necrosis were covered by fibers, and no extraradicular microorganisms were present, which suggests that extraradicular biofilm is closely associated with failed endodontic treatments, thus resulting in persistent infection. Microsc. Res. Tech. 76:979–983, 2013. © 2013 Wiley Periodicals, Inc.     

A rapid and simple technique for correlating light microscopy,transmission and scanning electron microscopy of fixed tissues in Epon blocks

A simplified and standardized technique for close correlation between light microscopy (LM), transmission electron microscopy (TEM) and scanning electron microscopy (SEM) is described. Perfusion and immersion fixed tissue specimens were embedded in Epon 812 and cut for conventional LM and TEM. The Epon blocks with remaining tissue were thereafter treated with epoxy solvent (ethanol-NaOH solution) for partial epoxy resin removal only (dissolving rate approx 33μm/h). The blocks with partially blotted tissue specimens were then critically point dried and gold coated for SEM. This method, in an easy way, allows repeated observations with LM, TEM and SEM with preserved fine structure and exact correlation. Since the technique is so simple and there is no need for special equipment the method can easily be adopted in all laboratories with basic SEM standards.     

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.     

A scanning and transmission electron microscopy study of the parabronchial unit in quail (Coturnix coturnix) and town pigeons (Columba livia).

A combined scanning electron (SEM) and transmission electron microscopy (TEM) investigation was undertaken to gain insight into the complex structural pattern of the atrial compartment and the gas exchange tissue of parabronchial units in quail and town pigeons. The aim was also to depict the changes taking place in the parabronchial unit in the late prehatching and early posthatching periods in quail. The standard SEM and TEM investigation was carried out in 13 mature quail and 8 town pigeons. The developmental study involved embryonic quail (Days 15, 16, 17), newly hatched quail, quail 24 h after hatching, and quail aged 2, 10, 19, and 25 days (3 individuals per group). The luminal relief of the parabronchus is formed by anastomosing interatrial septa delineating the atrial pits, which are thinner and shallower in pigeons. The atrial bottom opens in mature individuals into 3-6 infundibula. The extracellular material represented by trilaminar substance, which does not appear until hatching, veils the surface relief of the parabronchial epithelium, which is consequently hardly accessible to three-dimensional visualization. Only in town pigeons with fewer discontinuous layers of extracellular material was it possible to visualize the surface of the atrial epithelium, that is, of the granular and squamous atrial cells. The SEM analysis has convincingly shown the intricate spatial organization of atria, infundibula, and air and blood capillaries of the gas exchange tissue. The retinacula, that is, parallelly arranged processes of squamous respiratory cells bridging the air-capillary lumina, were evidenced by SEM and TEM. The complex structure of the avian parabronchus has been successfully demonstrated in the present SEM and TEM study.     

Elimination of scanning electron microscopy image periodic distortions with digital signal-processing methods

Electromagnetic interference is one of the main distortion sources in scanning electron microscopy. Electromagnetic interference‐generated scanning electron microscopy image distortions are usually visible as edge blur (at low scan rates) or vibration (at high scan rates). Hardware solutions to this problem, e.g. electrostatic and magnetic shielding, are expensive and, in some cases, difficult to implement. The current investigations led to a significant decrease in the periodic distortions by a novel adaptation of software‐based digital signal processing to scanning electron microscopy problems, without any hardware modification.