Automated electron tomography with scanning transmission electron microscopy

We report the successful implementation of a fully automated tomographic data collection system in scanning transmission electron microscopy (STEM) mode. Autotracking is carried out by combining mechanical and electronic corrections for specimen movement. Autofocusing is based on contrast difference of a focus series of a small sample area. The focus gradient that exists in normal images due to specimen tilt is effectively removed by using dynamic focusing. An advantage of STEM tomography with dynamic focusing over TEM tomography is its ability to reconstruct large objects with a potentially higher resolution.     

TEM-, SEM- and STEM-studies of sputter-coated cytoskeletons

A technique for sputter coating of cytoskeletons from detergent extracted cells is described. The method allows their study in high resolution TEM, SEM and STEM. The use of cytoskeletons is also a valuable model system for the evaluation of metal coating techniques, allowing studies of deposited metal on fine filamentous structures of varying thicknesses. Pt and W were sputtered at 1 to 5 nm, and comparisons were made with reference to contrast and granularity. Both metais gave good topographical contrast, but Pt showed a coarse structure and a greater decorating tendency than W. The method provides a simple system for studies of the three-dimensional cytoskeletal organization, without the use of cumbersome replica-techniques.     

Observation of dislocations and microplasma sites in semiconductors by direct correlations of STEBIC,STEM and ELS

A high voltage electron microscope, equipped with scanning transmission (STEM) attachment, electron beam induced conductivity (EBIC) facilities, and electron energy loss spectrometer (ELS), has been used to investigate semiconductor devices. The capability of STEM to produce, simultaneously or sequentially, conductive and transmission images of the same specimen region, which can also be ELS analysed, is exploited in order to establish direct and unambiguous correlations between EBIC and STEM images of defective regions (dislocations and microplasma sites) in silicon devices. The results obtained are discussed in terms of correlations, resolution, contrast, and radiation damage; in addition, a comparison is made between this method and the other correlation methods based on EBIC/SEM (scanning electron microscope) and TEM (transmission electron microscope).     

Secondary electron detection in the scanning transmission electron microscope

In a dedicated scanning transmission electron microscope (STEM) secondary electron images with high spatial resolution and good contrast can be obtained. Two types of detector are described. These take into account the secondary electrons which depend on the post-specimen field strength of the objective lens. Due to the thinness of the samples and the collection geometry the images differ from those obtained in a convectional scanning microscope. Examples are given where secondary electron images augment the information obtained by the more commonly used imaging modes.     

Structure analysis of sputter-coated and ion-beam sputter-coated films: a comparative study

Gold, platinum and tungsten films were deposited by low energy input (7 mA, 450 V), or high deposition rate (80 mA, 1500 V), diode sputter coating and by ion beam sputter coating. Film structures on Formvar coated grids and on the surface of coated erythrocytes, resin embedded, sectioned, and recorded at high magnification in a TEM were compared using computer-assisted measurements and analysis of film thickness and grain size. The average grain size of the thinnest gold and platinum films was relatively independent of the mode or rate of deposition but as the film thickness increased, significant differences in grain size and film structure were observed. Thick platinum or gold films deposited by low energy input sputter coating contained large grain size and electron transparent cracks; however, more even films with narrower cracks but larger grain size were produced at high deposition rates. Ion beam sputter coated gold had relatively large grain size in 10 nm thick films, but beyond this thickness the grains coalesced to form a continuous film. Platinum films deposited by ion beam sputter coating were even and free of electron transparent cracks and had a very small grain size (1–2 nm), which was relatively independent of the film thickness. Tungsten deposition either by low energy input or ion beam sputter coating resulted in fine grained even films which were free of electron transparent cracks. Such films remained granular in substructure and had a grain size of about 1 nm which was relatively independent of film thickness. Tungsten films produced at high deposition rates were of poorer quality. We conclude that thick diode sputter coated platinum and gold films are best deposited at high deposition rates provided the specimens are not heat sensitive, the improvement in film structure being more significant than the slight increase in grain size. Thick diode or ion beam sputter coated gold films should be suitable for low resolution SEM, and thin discontinuous gold films for medium resolution SEM. Diode sputter coated platinum should be suitable for medium resolution SEM and ion beam sputter coated platinum for medium and some high resolution SEM. 1–5 nm thick tungsten films, deposited by low energy input or ion beam sputter coating should be suitable for high resolution SEM, particularly where contrast is of less importance than resolution.     

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.     

First experimental test of a new monochromated and aberration-corrected 200 kV field-emission scanning transmission electron microscope

The first 200 kV scanning transmission electron microscope (STEM) with an imaging energy filter, a monochromator and a corrector for the spherical aberration (Cs-corrector) of the illumination system has been built and tested. The STEM/TEM concept with Koehler illumination allows to switch easily between STEM mode for analytical and TEM mode for high-resolution or in situ studies. The Cs-corrector allows the use of large illumination angles for retaining a sufficiently high beam current despite the intensity loss in the monochromator. With the monochromator on and a 3 microm slit in the dispersion plane that gives 0.26 eV full-width at half-maximum (FWHM) energy resolution we have obtained so far an electron beam smaller than 0.20 nm in diameter (FWHM as measured by scanning the spot quickly over the CCD) which contains 7 pA current and, according to simulations, should be around 0.12 nm in true size. A high-angle annular dark field (ADF) image with isotropic resolution better than 0.28 nm has been recorded with the monochromator in the above configuration and the Cs-corrector on. The beam current is still somewhat low for electron energy-loss spectroscopy (EELS) but is expected to increase substantially by optimising the condenser set-up and using a somewhat larger condenser aperture.     

Quantitative analysis of image contrast in phase contrast STEM for low dose imaging

This work quantitatively evaluates the contrast in phase contrast images of thin vermiculite crystals recorded by TEM and aberration-corrected bright-field STEM. Specimen movement induced by electron irradiation remains a major problem limiting the phase contrast in TEM images of radiation-sensitive specimens. While spot scanning improves the contrast, it does not eliminate the problem. One possibility is to utilise aberration-corrected scanning transmission electron microscopy (STEM) with an Ångstrom-sized probe to illuminate the sample, and thus further reduce irradiation-induced specimen movement. Vermiculite is relatively radiation insensitive in TEM to electron fluences below 100,000 e⁻/Ų and this is likely to be similar for STEM although different damage mechanisms could occur. We compare the performance of a TEM with a thermally assisted field emission electron gun (FEG) and charge coupled device (CCD) image capture to the performance of STEMs with spherical aberration correction, cold field emission electron sources and photomultiplier tube image capture at a range of electron fluences and similar illumination areas. We show that the absolute contrast of the phase contrast images obtained by aberration-corrected STEM is better than that obtained by TEM. Although the STEM contrast is higher, the efficiency of collection of electrons in bright field STEM is still much less than that in bright field TEM (where for thin samples virtually all the electrons contribute to the image), and the SNR of equivalent STEM images is three times lower. This is better than expected, probably due to the absence of a frequency dependent modulation transfer function in the STEM detection system. With optimisation of the STEM bright field collection angles, the efficiency may approach that of bright field TEM, and if reductions in beam-induced specimen movement are found, STEM could surpass the overall performance of TEM.     

The formation and interpretation of defect images from crystalline materials in a scanning transmission electron microscope.

The technique of scanning transmission electron microscopy (STEM) has been employed usefully in studies of amorphous materials, and the theory of image formation and interpretation in this case has been well developed. Less attention has been given to the practical and theoretical problems associated with the use of STEM for the examination of crystalline materials. In this case the contrast mechanisms are dominated by Bragg diffraction and so they are quite different from those occurring in amorphous substances. In this paper practical techniques for the observation and interpretation of contrast from defects in crystalline materials are discussed. It is shown that whilst images of defects are obtained readily under all typical STEM operating conditions, the form of the image and the information it contains varies with the angle subtended at the specimen by the detector. If this angle is too large significant image modifications relative to the "conventional" transmission electron microscope case may occur and the resolution of the image may degrade. If this angle is too small, then signal to noise considerations make an interpretation of the image difficult. In this paper we indicate how the detector angle may be chosen correctly, and also present techniques for setting up a STEM instrument for imaging a crystalline material containing lattice defects.     

Scanning image detection (SID) system for conventional transmission electron microscope (CTEM) images

A new image detection system has been developed to display transmission electron microscope (TEM) images on a CRT without a video camera system. Deflection coils placed in both the upper space of an objective lens and in the lower space of the first intermediate lens scan a small electron probe simultaneously. The electrical signal acquired through an improved scintillator and a photomultiplier is synchronized with the scanning signal and displayed in a similar fashion to a conventional scanning TEM (STEM) instrument. A preliminary system using a 100 kV conventional TEM (CTEM) equipped with a hairpin-type electron gun, produced an image with a spatial resolution of 1 nm.     

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.     

Simulation of scanning transmission electron microscope images on desktop computers

Two independent strategies are presented for reducing the computation time of multislice simulations of scanning transmission electron microscope (STEM) images: (1) optimal probe sampling, and (2) the use of desktop graphics processing units. The first strategy is applicable to STEM images generated by elastic and/or inelastic scattering, and requires minimal effort for its implementation. Used together, these two strategies can reduce typical computation times from days to hours, allowing practical simulation of STEM images of general atomic structures on a desktop computer.     

Reflection electron microscopy methods for the study of surface structure

The techniques of reflection electron microscopy (REM) using TEM instruments and scanning reflection electron microscopy (SREM) using STEM instruments have been explored as means for the observation of surface structure with high spatial resolution, better than 1 nm in each case. Under the ordinary environment of a commercial TEM instrument, we have studied the contrast in REM images of atomic steps and made comparison with the calculated results from the multi-slice dynamical diffraction theory. Comparison has also been made between the REM images of defects and the calculated images based on the column approximation. The influence of surface resonances on the contrast has been investigated. By SREM performed in a modified HB5 STEM with attached high vacuum preparation chamber, we have observed the formation of periodically distributed Pd particles on the surface of cleaved MgO.     

A new cryo‐STEM method for imaging food colloids in the scanning electron microscope

A new cryo‐scanning transmission electron microscopy (cryo‐STEM) technique for imaging casein micelles in a field emission scanning electron microscope is presented. Thin films of micellar casein suspensions on lacey carbon grids were prepared using a modified sample holder developed by Gatan UK. Bright and dark field images were obtained at ?135°C showing casein micelles in their frozen hydrated state and in the size range 30–500 nm. Results were compared favorably with published images of casein micelles obtained with conventional cryo‐transmission electron microscopy, suggesting that cryo‐STEM is a useful alternative technique for visualizing food colloids close to their native state. SCANNING 32: 150–154, 2010. © 2010 Wiley Periodicals, Inc.     

Preservation and visualization of molecular structure in detergent-extracted whole mounts of cultured cells.

Today's electron microscopes have a resolution sufficient to resolve supramolecular structures. However, the methods used to prepare biological samples for electron microscopy often limit our ability to achieve the resolution that is theoretically possible. We use whole mounts of detergent-extracted cells grown on Formvar-coated gold grids as a model system to evaluate various steps in the preparation of biological samples for high resolution scanning electron microscopy (SEM). Factors that are important in determining the structure and composition of detergent-extracted cells include the nature of the detergent and the composition of the extraction vehicle. Chelation of calcium is extremely important to stabilize and preserve the cytoskeletal filaments. We have also demonstrated both morphologically and by gel electrophoresis that treatment of cells with bifunctional protein crosslinkers before or during extraction with detergent can significantly enhance the preservation of both proteins and supramolecular structures. The methods used to dry samples are a major determinant of the quality of structural preservation. For cytoskeletons freeze-drying (FD) is superior to critical point-drying (CPD), one reason being that CPD samples have to be dehydrated, thereby causing more shrinkage as compared to FD samples. The high pressures to which samples are exposed during CPD may also cause increased shrinkage, and water contamination during CPD causes severe structural damage. We have obtained the best structural preservation of detergent-extracted and fixed cells by manually plunging them into liquid propane and drying over night in a freeze-dryer. The factor that most limits achievement of high resolution in SEM is the metal coat, which has to be very thin, uniform, and free of grain in order not to hide structures or to create artifactual ones. We have found that sputter-coating with 1-3 nm of tungsten (W) or niobium (Nb) gives extremely fine-grained films as well as satisfactory emission of secondary electrons. These samples can also be examined at high resolution by transmission electron microscopy (TEM) and scanning transmission electron microscopy (STEM). The best preservation and visualization of supramolecular structures have been obtained using cryosputtering, in which the samples are freeze-dried and then sputter-coated within the freeze-dryer while still frozen.     

Nanometric crystal defects in transmission electron microscopy

Transmission electron microscopy (TEM) is revisited in order to define methods for the identification of nanometric defects. Nanometric crystal defects play an important role as they influence, generally in a detrimental way, physical properties. For instance, radiation-induced damage in metals strongly degrades mechanical properties, rendering the material stronger but brittle. The difficulty in using TEM to identify the nature and size of such defects resides in their small size. TEM image simulations are deployed to explore limits and possible ways to improve on spatial resolution and contrast. The contrast of dislocation loops, cavities, and a stacking fault tetrahedra (SFT) are simulated in weak beam, interfering reflections (HRTEM), and scanned condensed electron probe (STEM) mode. Results indicate that STEM is a possible way to image small defects. In addition, a new objective aperture is proposed to improve resolution in diffraction contrast. It is investigated by simulations of the weak beam imaging of SFT and successfully applied in experimental observations.     

Preparation of cultured mammalian cells for transmission and scanning electron microscopy using aclar film

Common methods for the preparation of cultured cells for concurrent light microscopy (LM), scanning electron microscopy (SEM), and transmission electron microscopy (TEM) are not completely satisfactory. This article describes how we grow mammalian cells on plastic disks made from Aclar film. Aclar is a transparent fluorinated-chlorinated thermoplastic that contains no volatile components and is, for all practical purposes, chemically inert. Cells adhere to it readily and remain attached after fixation, dehydration, and critical-point drying or embedding. The film also accepts heavy metal coating by ionic bombardment and is extremely stable in the vacuum of the SEM. LM observations are unhindered by Aclar, since the film is as transparent as glass. Fluorescence microscopy is possible with this film, since it exhibits no detectable autofluorescence. During SEM observation, the film has great dimensional stability, and the cells and heavy metal coating remain attached to the Aclar even under high-resolution operating conditions. TEM processing of specimens grown on Aclar is simplified by the fact that Aclar does not stick to the epoxy resins used in EM. Furthermore, Aclar is easily sectioned and does not damage knives used in ultramicrotomy. The use of Aclar film considerably simplifies the preparation of cultured cells for all types of microscopy. This method is particularly useful in correlating surface features between SEM and TEM observations.     

Polyethylene glycol (PEG) embedding and subsequent de-embedding as a method for the correlation of light microscopy,scanning electron microscopy,and transmission electron microscopy

The polyethylene glycol (PEG) embedding and subsequent deembedding method was applied to the observation of general tissues in scanning electron microscopy (SEM). Resulting SEM images were of high quality. It was demonstrated that intermicroscopic correlation of images between light microscopy (LM), scanning electron microscopy (SEM), and transmission electron microscopy (TEM) is easily and reliably done by means of the PEG method. In particular, the exact correlation of immuno-LM with SEM is shown to be of potential value.