Revealing local variations in nanoparticle size distributions in supported catalysts: a generic TEM specimen preparation method

The specimen preparation method is crucial for how much information can be gained from transmission electron microscopy (TEM) studies of supported nanoparticle catalysts. The aim of this work is to develop a method that allows for observation of size and location of nanoparticles deposited on a porous oxide support material. A bimetallic Pt‐Pd/Al₂O₃ catalyst in powder form was embedded in acrylic resin and lift‐out specimens were extracted using combined focused ion beam/scanning electron microscopy (FIB/SEM). These specimens allow for a cross‐section view across individual oxide support particles, including the unaltered near surface region of these particles. A site‐dependent size distribution of Pt‐Pd nanoparticles was revealed along the radial direction of the support particles by scanning transmission electron microscopy (STEM) imaging. The developed specimen preparation method enables obtaining information about the spatial distribution of nanoparticles in complex support structures which commonly is a challenge in heterogeneous catalysis.     

A controlled atmosphere specimen holder for transmission electron microscopy

A controlled atmosphere specimen holder (CASH) has been developed for transmission electron microscopy (TEM) experiments. It is designed for studying the specimen's microstructure before and after treatments in various gases (H₂, O₂, N₂, Ar, etc.) at temperatures up to 600°C. The experiments are carried out without exposing the specimen to the ambient atmosphere. No modification of the electron microscope itself is needed. The same area of the specimen can be easily located after each gas treatment, thus the changes in the microstructure can be studied directly. Preliminary results on the cyclic oxidation and reduction of a model cobalt catalyst are presented.     

Embedding in Spurr's resin is a good choice for immunolabelling after freeze drying as shown with chemically unfixed dendritic cells

Immunocytochemical reactions on biological specimens depend on many factors, the most crucial one being the maintenance of antigenicity. Antigens are vulnerable at each stage during preparation for electron microscopy. One of the least traumatic methods of preparing biological tissues for post‐embedding immunolabelling includes the following steps: (1) physical stabilization of the native biological material by rapid freezing (cryofixation) and keeping the immobilized biological sample at low temperature, thereby avoiding any movements of water, ions and macromolecules; (2) dehydrating the frozen biological material by freeze‐drying at low temperature; (3) embedding of the dehydrated specimen. Here we show that embedding of chemically unfixed dendritic cells in Spurr's resin after cryofixation and freeze‐drying enables the conservation of fine ultrastructure without cell distortion or shrinkage. Furthermore, we demonstrate the feasibility of protein localization in ultrathin sections by immunolabelling of the major histocompatibility class II molecules.     

Standards for the application of X-ray microanalysis to biological specimens

A review on the subject of compounds used as standards for biological X-ray microanalysis is presented. The general approach used for standardization has been to use standards which resemble the specimen closely in composition. Thus, standards based on proteins have been used for analysis of quench-frozen cryosectioned specimens, whereas standards based on embedding resins have been used for resin-embedded material. The properties of, and problems associated with, each type of standard are recognized and have been well documented. The choice and analysis of standard should not be a drawback to fully quantitative analysis of biological material. Attention is drawn to the fact that the problems associated with any quantification procedure need to be kept in mind when analysis of standards is undertaken.     

Effect of counterface material for abrasive wear of Cercidiphyllum japonicum wood on three-body abrasion

A three-body abrasion test with a loose abrasive grain scattered on a variety of plastic counterface materials is conducted for Cercidiphyllum japonicum wood (katsura wood). The effect of the counterface material in rubbing with the katsura wood is investigated. The results show that a peak wear coefficient exists for the axial, tangential and radial sections of the katsura wood specimen when rubbed with a counterface material. The peak in the wear coefficient is also recognized in the plastic specimen experiments. The peaks in three-body abrasion experiments for both the katsura wood and plastic specimens are closely related to the variable of material yield stress. The peak on katsura wood specimen occurs when the yield stress of the counterface material is approximately twice as large as that of katsura wood, and the peak on the plastic specimen occurs when the yield stress of the counterface material is approximately the same as that of the specimen. The difference in the results between the katsura wood and plastic material could appear to be due to the change in embedding balance of the loose abrasive grain, which is likely affected by the porous wood structure.     

Low‐temperature glycol methacrylate resin embedding method: A protocol suitable for bone marrow immunohistochemistry,PCR, and fish analysis

Molecular analyses such as fluorescence in situ hybridization (FISH) and polymerase chain reaction (PCR) are demanded to improve diagnostic accuracy in addition to immunohistopathology of bone marrow (BM) trephine specimens. Conventional BM embedding method needs decalcification, and its procedure may impair tissue morphology and DNA quality. Here, we report an undecalcified method by which glycol methacrylate resin is polymerized at low temperature (4°C). Using this method, BM enzyme activity and antigenic determinants are well preserved, and moreover, DNA extracted from plastic embedding sections is suitable for PCR amplification and sequencing, FISH analysis can be well done because of the DNA integrity of BM sections. If working with BM trephine specimen, our protocol offers the possibility to combine superior morphology with modern molecular analysis. Microsc. Res. Tech. 73:1067–1071, 2010. © 2010 Wiley‐Liss, Inc.     

Developments of new Lowicryl® resins for embedding biological specimens at even lower temperatures

Two new Lowicryl resins have been developed for embedding biological materials at temperatures down to 210K (hydrophilic K11M) and to 190K (hydrophobic HM23). They have similar properties to Lowicryl K4M and HM20. The new resins were first tested for low temperature applications by the ‘progressive lowering of temperature’ procedure and this shows that the low viscosity of K11M and HM23 is favourable for the infiltration of biological specimens. Hardening is achieved through photo-polymerization at these lower temperatures. These properties make K11M and HM23 suitable for cryosubstitution of rapidly frozen material and it is speculated that the preservation of antigenicity may be further improved.     

Applicability of ultrasonic technique for evaluation of elastic plastic fracture toughness of high manganese steel at low temperatures

A study on the evaluation of elastic-plastic fracture toughness, J_IC, by ultrasonic technique is described for a newly developed high manganese steel at low temperature. In order to see the applicability of the ultrasonic technique based on pulse echo method at low temperature, special attention was paid to detect change point of ultrasonic echo due to the onset of stable crack growth. The J_IC values are evaluated by the ultrasonic method (which needs single specimen) and by stretched zone method (which needs several specimens) for compact tension (CT) specimens and three-point bend (3PB) specimens. The temperature dependence of these J_IC values of theCT specimens by the ultrasonic method show almost the same values to 3PB specimens over the temperature range tested. The J_IC values of 3PB specimens by the stretched zone method show slightly higher values than those of theCT specimens at low temperature. The J_IC values evaluated by the ultrasonic method give more conservative values than those evaluated by the stretched zone method for bothCT and 3PB specimens.     

Quantitative electron holographic tomography for the 3D characterisation of semiconductor device structures

Electron tomography and electron holography experiments have been combined to investigate the 3D electrostatic potential distribution in semiconductor devices. The experimental procedure for the acquisition and data reconstruction of holographic tilt series of silicon p-n junction specimens is described. A quantitative analysis of the experimental results from specimens of two different thicknesses is presented, revealing the 3D electrostatic potential variations arising from the presence of surfaces and damage generated by focused ion beam (FIB) sample preparation. Close to bulk-like properties are measured in the centre of the tomographic reconstruction of the specimen, revealing higher electrically active dopant concentrations compared to the measurements obtained at the specimen surfaces. A comparison of the experimental results from the different thickness specimens has revealed a 'critical' thickness for this specimen preparation method of 350nm that is required for this device structure to retain 'bulk'-like properties in the centre of the membrane.     

Cryo-SEM specimen preparation under controlled temperature and concentration conditions

Cryogenic temperature scanning electron microscopy (cryo-SEM) is an excellent technique for imaging liquid and semi-liquid materials of high vapour pressure, which are highly viscous or contain large (>0.5 μm) aggregates, in which nanometric details are to be studied. However, so far there have been no adequate tools for controlled cryo-specimen preparation. The specimen preparation stage is critical, because most of those samples are very sensitive to concentration and temperature changes, leading to nanostructural artefacts in the specimens. We designed and built a system for easy and reliable cryo-SEM specimen preparation under controlled conditions of fixed temperature and humidity. We describe this new methodology, and demonstrate its applicability, by showing imaging data of three liquid material systems. We have studied carbon nanotubes (CNTs) dispersions in superacid. We also characterized a number of systems made of water/isooctane/nonionic and cationic surfactant that showed different microemulsion phases as function of the system composition and temperature. In all of the examples given, we demonstrate artefact- and contamination-free specimens, which have preserved their native nanostructure. Our new system paves the way for a new methodology for the newly emerging field of cryo-SEM.     

The isector: a simple and direct method for generating isotropic,uniform random sections from small specimens

The very simple and strong principle of vertical sections devised by Baddeley et al. has been a major advance in stereology when any kind of anisotropy is present in the specimen under study. On the other hand, some important stereological estimators still require isotropic, uniform random sections. This paper deals with a simple technique for embedding specimens in rubber moulds with spherical cavities. After the embedding, any handling of the resulting sphere independent of the specimen will induce isotropy of the final histological sections.     

Improved handling of structural fragile cell-biological specimens during electron microscopic preparation by the exchange method

An exact method of preparation of soft biological specimens for electron microscopic analysis of surface fine structures is described. It allows routine preparations of fragile specimens for SEM and TEM imaging modes. With this procedure physical preparation parameters such as mechanical loads on the specimen surface or changes of temperature are controlled. The wet specimens are premounted in cheap disposable BEEM-containers or glass boats and are constantly kept under liquid in a closed system. The exchange of preparation media is performed continuously and, if necessary, over gradients. For comparative investigations with different EM-modes, at each step of the procedure parts of the specimens may be removed for individual processing. Conventionally prepared critical-point dried specimens are compared to those processed by the exchange technique and preservation of surface fine structures is demonstrated. Shadow-casted clathrin cages and stereo-replicas of virus infected cell cultures are shown in TEM preparations. For SEM, coverslip cell cultures and isolated glomerulus basement membranes are prepared and an additional flat embedding for TEM ultrathin sections is demonstrated.     

The use of pattern recognition for classification of stochastic specïmens by a set of standard specimens

This paper describes an empirical method for the experimental assigning of specimens to a set of standard specimens. The procedure was developed especially for stochastic scenes. Typical applications of such a procedure are found in material sciences in assigning specimens to a standard series. The assignment is based on feature vectors obtained from analyses of the specimen texture by means of erosion or opening. In cases where the arrangement of particles is characteristic of the specimen, dilation or closing are used to obtain a feature vector. To increase the sensitivity of the method described the use of more than one structuring element is recommended in the analysis. Besides the classification of an unknown specimen the procedure provides information on the separability of the standard specimen and the significance of assignment.     

A versatile multi-specimen holder for processing and critical point drying of materials for examination in the scanning electron microscope (SEM)

A specimen holder has been designed specifically for use in the Polaron E.3000 critical point drier (CPD) and is capable of drying up to twenty different specimens within a size range of 4.5 mm to 30 μm by utilization of a variable grid system. The principle, however, could be employed in designs for most other critical point dryers. A large number of designs have been produced for handling small specimens during preparation and CPD procedures prior to observation in the scanning electron microscope (SEM). In a great number of cases these have been specific to one particular organism or preparation method. Marchant (1973) suggested culturing organisms on Millipore filters which could then be used in conjunction with a plain filter as a sieve mechanism to contain the specimens, the complete unit being housed in a BEEM embedding capsule. Scott et al. (1973) produced another design, again utilizing the BEEM capsule together with 100 grade copper mesh as the specimen retaining section. Both these designs were flexible in that different grade filters and meshes could be employed. The disadvantage of using BEEM capsules was noted by Taylor (1975) in that the chemical plasticizers present in the capsule material could leak out in the presence of substitution solvents. To overcome this he produced an all metal design for a container employing a similar type of grid system as in this multispecimen holder. His design was for a single chamber specimen holder with a maximum specimen thickness of 1 mm and was such that the complete chamber could be placed directly into the SEM. The need for a multispecimen holder arose when large numbers of specimens, each specific in its preparation required critical point drying without tedious repetition of critical point drying runs. It was necessary to consider the inflexible features of the design. These were, the external dimensions of the container, which had to fit within the existing aluminium specimen-holder of the Polaron E.3000 CPD, and the dimensions of the transmission electron microscope (TEM) grids used as the specimen retainers. Transmission electron microscope grids are available in two sizes, 3.01 mm and 2.3 mm, with a large range in patterns and materials to choose from to suit most types of specimen preparation. From these fixed dimensions a design was drawn up which allowed specimens of up to 4.5 mm across to be prepared and yet still gave the large number of individual chambers required. The construction of the holder can be seen, from the drawing (Fig. 1) and the photographs (Fig. 2) to be comprised of large dimension chambers with a TEM grid housed at each end to contain the specimens and yet still allow the free flow of dehydrating and substitution agents. The complex arrangement of screws was necessary to facilitate the assembly and use of a container which has a separate lid section that can be dismantled to allow different grids to be inserted depending on the dimensions of the material under preparation. The specimens are supported on the lower grid system which can also be varied and for the ease of removal of larger specimens the chamber section divides leaving the specimens readily accessible in the lower half. Where the specimens are very small, the chamber section can be completely removed by carefully removing all screws and then screwing a stud down the centre thread and extracting the chamber section leaving all the specimens supported on their grids on the base plate. These can easily be transferred directly onto the SEM stub and secured either by double-sided sellotape or careful application of an adhesive such as Durafix. It has even been found that discs punched out of Visking tubing can be used in place of the TEM grids to provide a finer sieve mechanism. It was noted, however, that the tubing was hardened by substitution solvents but this still did not seem to impair the results as satisfactory preparations of Penicillium expansum sporangiophores and pollen of Dactylis glomeratus have been achieved (Medi-Cine, 1976). The container has been made out of high quality brass because of its good machining properties necessary when such fine work has to be carried out. The metric design utilizes standard milling cutters with the inclusion of the ?th cutter to produce satisfactory grid housings allowing free movement to ensure that they always settle on the base plate. The versatility of the design can be further increased with the production of two accessory structures (see Fig. 1). The first, simply produced by cutting brass tubing, is for the preparation of small numbers of specimens per chamber, and ensures that the specimens are deposited onto the grid. The holes drilled through the tube wall allow easy removal with fine forceps. The second structure simply partitions the large chambers increasing the capacity to eighty specimens where such a large specific separation is required. The production of artifacts resulting from specimen handling is reduced considerably with this specimen holder. Once the specimens have been loaded, all the processing stages can be carried out on all the material at once and the specimens are ready for mounting prior to observation.     

Preparation of thin ceramic monofilaments for characterization by TEM

A method for preparing transmission electron microscopy specimens from ceramic fibers has been developed that is particularly useful when only a small amount of the material is available for characterization. Fiber segments are lined up and sandwiched between two glass slides using high-temperature epoxy. The resulting specimen is then polished flat from both sides using tripod polisher to remove the glass and produce thin (< 2 microm) longitudinal section of the fibers. The specimen is then ion-milled for a short time to produce electron-transparent areas. The method is also suitable for preparing very flat specimens for site-specific optical and SEM analysis.     

Formation of tangential pre-stress as a residual stress and its influence on the tribological performance of nodular cast irons

Since any state of residual stress influences the service behavior of a material, it is of particular interest for engineers and designers to know its benefits to machine parts, and where it can be utilized successfully. From this point of view, mechanical tangential pre-stress as circumferential compressive residual stress has been investigated for wear performance. For this purpose, a thick walled cylinder specimen model was established as a tribo-element, and the force, which will form the desired tangential pre-stress representing residual stress, has been calculated by the rules of elasticity theory. In order to compare the effect of residual stress on the wear performance under dry and lubricated conditions, 0.8 R_e (R_e: unidirectional yield strength) were applied to the nodular cast iron (DDK-40, DDK-60) specimens. Wear test results have been evaluated in terms of two different stress levels, 0 R_e and 0.8 R_e, formed on the specimen.     

The crack healing behavior of ZrO2/SiC composite ceramics with TiO2 additive

This paper was carried out to assess the crack-healing behavior of ZrO₂ composite ceramics. The ZrO₂ composite ceramics were made by adding 10 wt.% SiC and TiO₂. The TiO₂ additives were changed to 1, 3 and 5 wt.%. The characteristics of crack-healing behavior were studied as a function of annealing temperature and time. Specimens were mirror polished. The cracks of about 100 ??m were introduced on the specimen surface by Vickers indenter and specimen were heat-treated. The strength of crack-healing was conducted by a three-point bending, and the each specimens was measured by XRD. The results obtained in this study are as follows: The ZST composite ceramics contained crack-healing properties, and the strength of ZST smooth specimens have been shown to be better than ZS specimens. The optimum crack-healing condition of the ZTS3 specimen was 1073 K, 5 hours in the air. Specimens with heat treatment had micro-cracks and large cracks on the surface, but the micro-cracks on the surface did not affect the bending strength. It was found that specimens display phase transformation from tetragonal crystal to monoclinic crystal after heat treatment.     

Preparation of ceramic fibre TEM cross-sections using ultramicrotomy and ion beam thinning methods

The preparation of specimens for detailed TEM microanalysis of micrometre-diameter, ceramic fibre cross-sections is described. The starter material is ceramic fibre in powder form and both ultramicrotomy-based and ion beam thinning-based methods are described. Requirements for specimens of uniform and adequate thinness, for easy selection of representative fibre cross-sections within the same specimen and for a reliable and time-efficient preparation method, resulted in choice of the ultramicrotomy-based method and the associated development of a novel extrusion and sedimentation technique of embedding the fibres to provide necessary pre-alignment and packing.     

Estimating the length of a bounded curve in three dimensions using total vertical projections

A new method is proposed to estimate the total length of a bounded, isolated linear feature in three dimensions from ‘total vertical projections’, obtained by rotating the curve about a fixed axis (arbitrarily called ‘vertical’) and projecting it onto a fixed vertical plane. No sections are required. Properly stained and embedded neuron dendrites, mycelial trees, fluorescent cytoskeletal filaments within a cell, etc., are candidate specimens for the method, especially in combination with the new devices for non-invasive three-dimensional microscopy. It is necessary that the specimen curve is rigid (i.e. of constant shape), that its length density is not too high (so that overlapping effects are not important) and that the embedding medium is fairly transparent. Given these requirements, the method can be very accurate and convenient to use, as is exemplified here.