The preparation of cross-section specimens for transmission electron microscopy

The structure and chemistry of thin solid films are best studied by transmission electron microscopy (TEM) when they are viewed in cross-section—that is, when the surface normal of the film is made perpendicular to the electron beam. In this orientation, the substrate, the thin film layers, and the interfaces between them can be imaged either simultaneously or individually. Further, information from each of these regions remains distinct from that obtained from the others, eliminating the problems of superimposition that are a consequence of viewing a layered structure in the conventional manner (i.e., parallel to the surface normal). A technique for fabricating TEM specimens that can be viewed in cross-section is described here. Although the majority of our work is with silicon-based materials, the technique can be readily adapted to the study of other systems.     

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.     

Chemical polishing of palladium for transmission electron microscopy

Chemical polishing methods for thinning palladium for TEM are reviewed. The quality of the foils produced by the different solutions is compared and the possible influence of hydrogen absorption on the microstructure is discussed.     

A balanced technique for preparation of specimens from pathogenicity studies for scanning electron microscopy

This paper reports our experiences with preparing delicate biological specimens for scanning electron microscopy. Three different washing methods were evaluated: One method allowed the analysis of the location of the bacterium Mycoplasma mobile on piscine gill epithelium and the optimal evaluation of histopathologic changes caused by this microbe. These results were achieved when specimens were washed three times in a cacodylic acid buffer after completion of the in vitro infection experiment in gill explant cultures. We also found that of three different concentrations of glutaraldehyde, a fixation with a 1.5% solution was sufficient to achieve excellent structural preservation, even without using post fixation in osmium tetroxide. Furthermore, this study showed that the use of acetone-carbon dioxide in the critical point drying procedure resulted in well-preserved piscine gill epithelium and mycoplasmas. Finally, long-term storage of tissue specimens in 0.1 M cacodylic acid buffer is possible if the buffer is changed on a monthly basis to avoid growth of unwanted microorganisms, such as fungi.     

The preparation of cross-sectional transmission electron microscopy specimens of Nb/Al multilayer thin films on sapphire substrates

We have developed a technique for preparation of cross-sectional transmission electron microscopy samples of reacted and unreacted Nb/al multilayer thin films on sapphire substrates. The choice of substrate was found to be extremely important. Sapphire sputters more slowly than Nb and Nb-compounds and therefore makes it possible to obtain the electron transparent regions in the thin films rather than in the substrate. However, the brittle nature of the sapphire restricts the types of thinning techniques that can be used, requiring extensive ion thinning as a final stage.     

Preparation of high-quality ultrathin transmission electron microscopy specimens of a nanocrystalline metallic powder

This article explores the achievable transmission electron microscopy specimen thickness and quality by using three different preparation methods in the case of a high-strength nanocrystalline Cu-Nb powder alloy. Low specimen thickness is essential for spatially resolved analyses of the grains in nanocrystalline materials. We have found that single-sided as well as double-sided low-angle Ar ion milling of the Cu-Nb powders embedded into epoxy resin produced wedge-shaped particles of very low thickness (<10 nm) near the edge. By means of a modified focused ion beam lift-out technique generating holes in the lamella interior large micrometer-sized electron-transparent regions were obtained. However, this lamella displayed a higher thickness at the rim of ≥30 nm. Limiting factors for the observed thicknesses are discussed including ion damage depths, backscattering, and surface roughness, which depend on ion type, energy, current density, and specimen motion. Finally, sections cut by ultramicrotomy at low stroke rate and low set thickness offered vast, several tens of square micrometers uniformly thin regions of ～10-nm minimum thickness. As major drawbacks, we have detected a thin coating on the sections consisting of epoxy deployed as the embedding material and considerable nanoscale thickness variations.     

Preparation of lithium specimens for transmission electron microscopy

A method of preparing undeformed thin lithium specimens for TEM is described. A solution of dehydrated methanol and toluene is used both for initial dishing of the foil by chemical polishing and also for final thinning. Under electron beam irradiation in the TEM, new pure Li crystals can grow out of the existing Li specimen. These in situ crystals can be used for the study of the microstructure and electronic structure of Li using TEM and electron energy loss spectrometry.     

An improved preparation method for cross‐sectional TEM specimens of films deposited on metallic substrates

Cross‐sectional TEM analysis is one of the most important techniques to characterize microstructures of films. However, the complex process, low efficiency, and low success rate of specimen preparation limit its application. This paper analyzed the main causes of low success rate and proposed an improved method for specimen preparation of films deposited on metallic substrates. This method consisting of twin‐jet electropolishing and one‐sided rocking ion milling is high in efficiency and success rate. Microsc. Res. Tech. 79:276–279, 2016. © 2016 Wiley Periodicals, Inc.     

Calcium alginate encapsulation of small specimens for transmission electron microscopy

A technique of encapsulating small objects in calcium alginate for further processing for transmission electron microscopy is described. Five methods are outlined which enable a variety of specimens including single cells (in suspension and on agar plates), small organisms and monolayers of tissue culture cells to be processed. A method for immunolabelling alginate-entrapped material is also outlined.     

A variation of transmission electron microscope sample preparation for VLSI analysis

Sample preparation for VLSI analysis is often slow due to long ion milling time and because the location of the thin area of the sample is difficult to control. By modifying the standard techniques used with a VCR Group (and perhaps other) mechanical dimpler, the ion milling time can be reduced to less than 30 min. and the location on the thinned area reasonably controlled. These modifications involve the use of a radiused edge on the dimpling tool, a rubber O-ring on the polishing tool, and not rotating the sample platen during polishing. The modifications to the dimpling and polishing tools allow more control of the geometry of the dimple, while not rotating the sample platen allows a thinner sample to be produced and permits the use of the sample translation micrometers to shift the location of the thinned area during polishing. The quality of samples produced using this modified procedure is equivalent to that obtained with the more standard methods.     

A method for preparing powdered specimens for transmission electron microscopy

A new method of preparing TEM samples from powders is described. The method consists of compacting epoxy-powder composite mixtures in special molds by centrifuging prior to polymerization. The compact is then ion milled by standard methods. Samples prepared in this way are currently being used to study dislocation structures developed during processing of ceramic powders.     

Specimen preparation technique for high resolution transmission electron microscopy studies on model supported metal catalysts

A new technique is described which can be used for preparing transmission electron microscopy (TEM) specimens suitable for high resolution studies on supported metal catalysts. By conventional silicon processing techniques 200 × 200 μm² Si₃N₄ membranes on Si wafers are produced. These membranes are extremely flat and have a uniform thickness of 13 nm. They can be used as a support in various kinds of thin film deposition. A TiO₂ film, optimally structured with respect to the requirements for high resolution TEM work in TiO₂–metal cluster systems, is deposited on the Si₃N₄ layer. It consists of one monolayer of 10–25 nm TiO₂ crystallites. TiO₂ lattice images show that a line resolution down to 0.19 nm is possible. Examples of TiO₂–Pd and TiO₂–Rh are given using respectively photodeposition and impregnation reduction to produce l.5–4 nm metal clusters.     

Redundancy of washing in the preparation of biological specimens for transmission electron microscopy

A marine unicellular organism, human trophoblast tissue and cultured trophoblast cells of human origin have been satisfactorily preserved for electron microscopy without resort to washing either before dehydration or between different stages of fixation. The time required to fix and dehydrate a specimen using this method is 55 min.     

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.     

Preparation of cross-sectional transmission electron microscopy samples by electron beam lithography and reactive ion etching

A cross-sectional sample preparation technique is described that relies on lithographic and dry-etching processing, thus avoiding metallographic polishing and ion milling. The method is capable of producing cross-sectional transmission electron microscopy samples with a large amount of transparent area (1 μm × 2.5 mm) which allows the examination of many patterned test sites on the same sample from the same chip of a silicon wafer. An example of the application of the technique is given for localized oxidation through a mask.     

A simple method for orienting silk and other flexible fibres in transmission electron microscopy specimens

When microstructures are characterized by transmission electron microscopy (TEM), the interpretation of results is facilitated if the material can be sectioned in defined orientations. In the case of fibres, it is especially useful if transverse and longitudinal sections can be obtained reliably. Here we describe a procedure for orienting spider silk and other flexible fibres for TEM investigation. Prior to embedding in epoxy resin, the silk is wound around a notched support made from polyester film. No glue is required. After the silk and its supporting film have been embedded and the resin has been cured the film can be peeled away to reveal nearly perfectly orientated silk threads. Both transverse and longitudinal sections can then be cut with a microtome. The method can be extended to obtain sections at any intermediate orientation.     

Interfacial energies and surface-tension forces involved in the preparation of thin,flat crystals of biological macromolecules for high-resolution electron microscopy

It is generally agreed that surface-tension forces and the direct interaction between the specimen and either the air-water interface or the water-substrate interface can influence significantly the preparation of biological materials for electron microscopy. Even so, there is relatively little systematic information available that would make it possible to control surface-tension forces and interfacial energies in a quantitative fashion. The main objective in undertaking the present work has been to understand somewhat better the factors that influence the degree of specimen flatness of large, monolayer crystals of biological macromolecules. However, the data obtained in our work should be useful in understanding the preparation of specimens of biological macromolecules in general. Data collection by electron diffraction and electron microscopy at high resolution and high tilt angles requires thin crystals of biological macromolecules that are flat to at least 1°, and perhaps less than 0·2°, over areas as large as 1 μm² or more. In addition to determining empirically by electron diffraction experiments whether sufficiently flat specimens can be prepared on various types of modified or unmodified carbon support films, we have begun to use other techniques to characterize both the surfaces involved and the interaction of our specimen with these surfaces. In the specific case of large, monolayer crystals of bacteriorhodopsin prepared as glucose-embedded specimens on hydrophobic carbon films, it was concluded that the initial interfacial interaction involves adsorption of the specimen to the air-water interface rather than adsorption of the specimen to the substrate. Surface-tension forces at the air-water interface and an apparently repulsive interaction between the specimen and the hydrophobic carbon seem to be major factors influencing the specimen flatness in this case. In the more general case it seems likely that interfacial interactions with either the substrate or the air-water interface can be variously manipulated in the search to find desirable conditions of specimen preparation.     

High resolution electron microscope observations of microstructures in A15 type Nb3X superconductors

Microstructures of superconducting Nb₃X(A15) compounds are studied by means of high resolution electron microscopy. Structure images are obtained with the 400kV high resolution electron microscope for both the annealed and ion-irradiated crystals. The images obtained from the annealed crystals show bright contrast patterns similar to the projections of Nb and X atoms comprising the A15 structure. Defects observed in the annealed crystal irradiated with 40 kV Nb⁺ ions show characteristic features in contrast. The dependence of image contrast on the element X and the atomic structures of the defects are discussed by comparison with the calculated images.