Three-dimensional imaging in double aberration-corrected scanning confocal electron microscopy, part I: elastic scattering

A transmission electron microscope fitted with both pre-specimen and post-specimen spherical aberration correctors enables the possibility of aberration-corrected scanning confocal electron microscopy. Imaging modes available in this configuration can make use of either elastically or inelastically scattered electrons. In this paper we consider image contrast for elastically scattered electrons. It is shown that there is no linear phase contrast in the confocal condition, leading to very low contrast for a single atom. Multislice simulations of a thicker crystalline sample show that sample vertical location and thickness can be accurately determined. However, buried impurity layers do not give strong, nor readily interpretable contrast. The accompanying paper examines the detection of inelastically scattered electrons in the confocal geometry.     

In situ transmission electron microscope study of single asperity sliding contacts

Direct observation of events taking place at the contacting interfaces is important to understand many tribological phenomena. Transmission electron microscope (TEM) has the ability to look through materials at very high magnifications. Most of the TEM observations are done long after the deforming loads and stresses have been relaxed and the material state is further disturbed during the specimen preparation. We have developed a specimen holder in which two electron transparent surfaces can be brought in contact and moved relative to each other in JEOL 2000FX microscope. This holder enables visualization of not only the contacting surfaces at nanoscale but also the subsurface deformation resulting from the contact interaction. Sliding experiments have been carried out mimicking a single asperity sliding contact. A sharp tungsten probe is moved laterally against a tip mounted on a cantilever. Magnitude of the contact instability, when the contact is broken is found to be dependent on the local geometry of the contact.     

When is parallel illumination best for alchemi?

The role of the tangential component of parallelism of an electron beam in a conventional transmission electron microscope, where the specimen is immersed in a magnetic field, in the performance of ALCHEMI experiments is investigated. It has been shown that the best results are not always obtained for the case of a probe convergent on the specimen surface, even when the convergence angle is less than the operating Bragg diffraction angle. A simple experiment to determine when this tangential component would be critical is described and some preliminary experimental results on “parallelism” are provided.     

Quantitative phase-sensitive imaging in a transmission electron microscope

This paper presents a new technique for forming quantitative phase and amplitude electron images applicable to a conventional transmission electron microscope. With magnetised cobalt microstructures used as a test object, we use electron holography to obtain an independent measurement of the phase shift. After a suitable calibration of the microscope, we obtain quantitative agreement of the phase shift imposed on the 200 keV electrons passing through the sample.     

Computer simulation and analysis of high-resolution electron microscope images and diffraction patterns with partial coherence,hollow cone illumination,and virtual apertures

A general method for computing high-resolution conventional transmission electron microscope images and diffraction patterns, when there are different types of partially coherent illumination conditions, is described. Examples of convergent beam, hollow cone, and virtual aperture illumination conditions are given in the context of interpreting image features. A comparison of real and computed diffraction patterns shows that, in practice, many innovative imaging modes are possible, which can be verified prior to real microscope experiments.     

Determination of the mean inner potential in III-V semiconductors by electron holography

The mean inner potential of GaAs(14.18V), InAs(14.50V), GaP(14.35V) and InP(14.50V) has been measured by transmission electron holography using the phase shift of the (000)-beam of the first hologram sideband. To provide a defined specimen geometry we used 90 degrees wedges obtained by the cleavage technique. The exact excitation condition as well as the acceleration voltage of the electrons were determined from convergent beam electron diffraction images. The magnification is extracted from two-beam lattice fringe images and dynamical effects are taken into account by Bloch-wave calculations.     

Transmission electron microscopic X-ray quantitative analysis of human dentin at 200 kV accelerating voltage

Qualitative and quantitative x-ray energy dispersive spectroscopy is now used successfully to analyze many features and processes in inorganic samples. When applied to inorganic samples, however, the results are often less satisfactory due to problems of preparation of organic samples, difficulty of measuring x-rays from organic samples, damage of the sample by the electron beam, and other practical problems. In the present study we used a high voltage transmission electron microscope equipped with an energy dispersive x-ray spectrometer to examine accurate quantitative standardless analysis of thin sections of an organic sample, human dentin. Based on our experiments we found the important parameters for quantitative analysis were sample thickness and appropriate choice of model sample. Further, we show that the method of Cliff and Lorimer can be used with biological samples at 200 kV, and we show that quantitative analysis of human dentin can be carried out at 200 kV. Finally, we show that areas of human dentin can be differentiated by their morphological characteristics and x-ray analyses obtained in the transmission electron microscope.     

Complementary TEM characterization of a squeeze-cast metal-matrix composite by dissolution of the sample in butanol

Transmission electron microscopy observations of ion-beam thinned samples and samples extracted using a butanol dissolution technique gave information regarding the interface microstructure which could not be obtained from the ion-beam thinned samples, and that therefore both these sample preparation routes should be considered when observing aluminium metal-matrix composites in the transmission electron microscope.     

Construction and testing of a nanomachining instrument

This paper presents a nanomachining instrument that was developed for conducting nanocutting, nanoscratching, and nanoindentation experiments. A piezoelectric tube scanner (PZT) is employed to generate three-dimensional machining motions. The sample is moved by the PZT, and the tool is kept stationary during machining. The machining forces are measured by force sensors with a resolution of sub-milliNewtons. The instrument is compact and can be used inside optical microscopes and scanning electron microscopes. In this paper, depth-sensing indentation experiments were performed to test the basic performance of the instrument. The indentation displacement was measured by a capacitance probe situated inside the PZT tube. An experimental system was constructed to locate and image indentations. The system consists of a high magnification microscope to measure coordinates of the indentation relative to a reference corner point on the sample, and an AFM equipped with an on-axis optical imaging system for locating the indentation. A technique was also employed to establish the tool-sample contact to nanometer accuracy. Indentation experiments were carried out on three kinds of materials with different hardness. Experimental results demonstrated the instrument has the ability of performing depth-sensing indentations. The frame compliance was also evaluated from the indentation results.     

Off-axis electron holography of electrostatic potentials in unbiased and reverse biased focused ion beam milled semiconductor devices

Off‐axis electron holography in the transmission electron microscope (TEM) is used to measure two‐dimensional electrostatic potentials in both unbiased and reverse biased silicon specimens that each contain a single p–n junction. All the specimens are prepared for examination in the TEM using focused ion beam (FIB) milling. The in situ electrical biasing experiments make use of a novel specimen geometry, which is based on a combination of cleaving and FIB milling. The design and construction of an electrical biasing holder are described, and the effects of TEM specimen preparation on the electrostatic potential in the specimen, as well as on fringing fields beyond the specimen surface, are assessed.     

Focused ion beam sample preparation of continuous fibre-reinforced ceramic composite specimens for transmission electron microscopy

The microanalysis of interfaces in fibre-reinforced composite materials is dependent on the successful preparation of specimens suitable for transmission electron microscope (TEM) inspection. Ideal samples should possess large amounts of structurally intact and uniform thin area in the fibre/matrix interface regions of the samples. Because fibre/matrix interfaces in this class of materials are often designed to fail under mechanical stress, conventionally prepared samples are prone to interfacial failure and differential thinning, both of which preclude detailed TEM microanalysis. These effects were seen in a conventionally dimpled and ion-beam-thinned specimen prepared from a continuous fibre reinforced ceramic composite composed of CaWO₄-coated Nextel 610^TM fibres in an alumina matrix. The dimpled specimen showed large amounts of interfacial failure, with only thick regions of the specimen left intact. To overcome these limitations, a focused ion beam (FIB) technique was applied to this same material. The superiority of the FIB-produced sample is evident in both the morphology and scanning transmission electron microscopy analyses of the sample.     

Electron diffraction from nanovolumes of amorphous material using coherent convergent illumination

Using a conventional transmission electron microscope that incorporates a field emission gun it is possible to focus an electron beam to form a small probe (<1nm full-width at half-maximum). Such a probe can then be used to perform high spatial resolution diffraction experiments. The high spatial resolution allows technologically interesting amorphous volumes, such as those found in glassy intergranular phases or in semiconductor implantations, to be investigated directly. In order to achieve the probe characteristics necessary to investigate nanovolumes of material the probe must be highly convergent which results in it being highly coherent. In this paper we examine the effect of coherent convergent illumination on electron diffraction data taken from nanovolumes of amorphous material. It is shown that, for amorphous volumes as small as 1.2nm in diameter, the additional interference effects induced in the diffraction data by the use of coherent convergent illumination are largely suppressed by the lack of order in amorphous materials. This allows the use of deconvolution techniques, developed for the correction of broadening of the diffraction pattern in the case of incoherent illumination, and the subsequent application of reduced density function (G(r)) analysis, to also be used for coherent illumination.     

Microwave-assisted rapid plant sample preparation for transmission electron microscopy

The preparation of plant leaf material for transmission electron microscopical investigations can be a very time- and labour-consuming task as the reagents infiltrate the samples quite slowly and as usually most steps have to be performed manually. Fixation, buffer washes, dehydration, resin infiltration and polymerization of the resin-infiltrated leaf samples can take several days before the specimen can be cut ultrathin and used for ultrastructural investigations. In this study, we present a microwave-assisted automated sample preparation procedure that reduces preparation time from at least 3 days to about 5 h – with only a few steps that have to be performed manually – until the plant sample can be ultrathin sectioned and observed with the transmission electron microscope. For studying the efficiency of this method we have compared the ultrastructure of different leaf material ( Arabidopsis thaliana , Nicotiana tabacum and Picea abies ) which was prepared with a conventional, well-established chemical fixation and embedding protocol and a commercially available automated microwave tissue processor. Despite the massive reduction in sample preparation time no negative effects on cutting properties of the blocks, stability of the sections in the electron beam, contrast and ultrastructure of the cells were observed under the transmission electron microscope when samples were prepared with the microwave-assisted protocol. Additionally, no negative effects were detected on the dimensions of fine structures of grana stacks (including membranes, inter- and intrathylakoidal spaces), the nuclear envelope and the plasma membrane as the diameter of these structural components did not differ between leaf samples (of the same species) that were processed with the automated microwave tissue processor or by conventional fixation and embedding at room temperature.     

Cross-section analysis of organic light-emitting diodes

The 'lift-out' technique using a focused ion beam microscope was applied to prepare cross-sectional specimens of organic light-emitting diodes for use in transmission electron microscopy. The focused ion beam equally thins the organic/inorganic hybrid devices despite the difference in material hardness of the compounds. This allowed to overcome preparation difficulties of conventional techniques such as ion thinning or ultra-microtomy. Two different samples were prepared and studied by both conventional transmission electron microscopy and analytical electron microscopy to display some of the investigation possibilities which become available with this sample preparation method.     

Preparation of transmission electron microscopy cross-section specimens of crack tips using focused ion beam milling

The preparation of transmission electron microscope (TEM) thin foil specimens from metal alloys containing cracks is usually thwarted by the difficulty in preventing preferential erosion of material along the flanks and at the tips of cracks. Recent developments in focused ion beam (FIB) micromachining methods have the potential to overcome this inherent problem. In this article we describe the development of new procedures, one using FIB alone and the other using a combination of FIB with more conventional ion milling to generate TEM specimens that largely retain the microstructural information at stress corrosion cracks in austentic alloys. Examples of corrosion product phase identification and interfacial segregation are included to verify that detailed information is not destroyed by ion bombardment during specimen preparation.     

Extracting physically interpretable data from electron energy-loss spectra

Principal component analysis is routinely applied to analyze data sets in electron energy-loss spectroscopy (EELS). We show how physically meaningful spectra can be obtained from the principal components using a knowledge of the scattering of the probe electron and the geometry of the experiment. This approach is illustrated by application to EELS data for the carbon K edge in graphite obtained using a conventional transmission electron microscope. The effect of scattering of the probe electron is accounted for, yielding spectra which are equivalent to experiments using linearly polarized X-rays. The approach is general and can also be applied to EELS in the context of scanning transmission electron microscopy.     

Deconvolution of electron diffraction patterns of amorphous materials formed with convergent beam

To perform reduced density function (G(r)) analysis on electron diffraction patterns of amorphous materials formed with convergent beams, the effects of convergence must be removed from the diffraction data. Assuming electrons incident upon the sample in different directions are incoherent, this can be done using deconvolution (Ultramicroscopy 76 (1999) 115). In this letter we show that the combination of an energy filtering transmission electron microscope with an image plate, increases the accuracy with which diffraction data can be measured and, subsequently, the accuracy of the deconvolution.     

Effects of the sample preparation temperature on the nanostructure of compression moulded ultrahigh molecular weight polyethylene

In this study, the effects of the sample sectioning temperature on the surface nanostructure and mechanical response of compression moulded ultrahigh molecular weight polyethylene (UHMWPE) at a nanometer scale (nanomechanical properties) have been characterized. The primary focus of this work was to determine if the sample sectioning temperature significantly changed the nanostructure of UHMWPE, while the secondary focus was to characterize the effect on the mechanical response due to the changes in the sectioned surface nanostructure. The goals of this study were: (a) to investigate the potential possibility of creating surface artefacts by the sample preparation technique by sectioning at different temperatures relative to the published range of glass transition temperatures, Tg, for PE (-12, -80 and -25 degrees C); (b) to determine the possibility of molecular orientation induced by plastic deformation of the UHMWPE sample during the process of sample preparation; (c) to measure the relative difference in nanomechanical properties owing to evolution of different nanostructures as a function of sample sectioning temperature. Field emission scanning electron microscopy (FESEM), atomic force microscopy (AFM) and nanoindentation were used to demonstrate that the sectioning temperature caused a change in nanostructure of the compression moulded UHMWPE sectioned surface, explaining the change in mechanical response to indentation at a nanoscale. In this study, it was demonstrated that significant plastic deformation occurs when a shear stress is applied between the glass or diamond blade and the UHMWPE during sample preparation under ambient conditions at a temperature of 22 degrees C. These results also suggest that an optimum sample sectioning temperature should definitely be below the measured Tg of the polymer.     

Avoiding artefacts during electron microscopy of silver nanomaterials exposed to biological environments

Electron microscopy has been applied widely to study the interaction of nanomaterials with proteins, cells and tissues at nanometre scale. Biological material is most commonly embedded in thermoset resins to make it compatible with the high vacuum in the electron microscope. Room temperature sample preparation protocols developed over decades provide contrast by staining cell organelles, and aim to preserve the native cell structure. However, the effect of these complex protocols on the nanomaterials in the system is seldom considered. Any artefacts generated during sample preparation may ultimately interfere with the accurate prediction of the stability and reactivity of the nanomaterials. As a case study, we review steps in the room temperature preparation of cells exposed to silver nanomaterials (AgNMs) for transmission electron microscopy imaging and analysis. In particular, embedding and staining protocols, which can alter the physicochemical properties of AgNMs and introduce artefacts thereby leading to a misinterpretation of silver bioreactivity, are scrutinized. Recommendations are given for the application of cryogenic sample preparation protocols, which simultaneously fix both particles and diffusible ions. By being aware of the advantages and limitations of different sample preparation methods, compromises or selection of different correlative techniques can be made to draw more accurate conclusions about the data.     

Sensitivity optimization of the scanning microdeformation microscope and application to mechanical characterization of soft materials

In this article we present the study of the sensitivity optimization of our system of micromechanical characterization called the scanning microdeformation microscope. The flexural contact modes of vibration of the cantilever have been modeled. We discuss the matching between the cantilever stiffness and the contact stiffness which depends on the sample material. In order to obtain the best sensitivity, the stiffnesses must be the closest one to each other. Because the length of the cantilever directly affects its stiffness, the cantilever geometry can be optimized for different materials. We have validated this study with measurements on a soft material the polydimethylsiloxane with a cantilever optimized for materials of Young's moduli of some megapascals. Experimental results obtained with two different samples have shown the high sensitivity of the method for the measurement of low Young's moduli and have been compared with nanoindentation and dynamic mechanical analysis results.