A deceptive staining artifact in ultramicrotomed polymer sections.
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Artifacts↗
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Cross-sectional transmission electron microscopy of X-ray multilayer thin film structures.
A simple method for preparing cross-sectional transmission electron microscopy specimens and discussions of possible artifacts from specimen preparation and observation of x-ray multilayer thin film structures are presented. The specimen preparation method employs mechanical grinding and polishing to approximately 20 microns, followed by ion milling, without dimpling. Artifacts such as preferential ion milling and crystallization under the electron beam, as well as effects of Fresnel fringes at interfaces, are important factors in interpretation of the images. Care in identifying them is required to avoid erroneous results in studies of morphology and microstructures within the layers and at their interfaces. Example high-resolution TEM results of cross-sectional W/C, Ru/C, and Mo/Si multilayers are presented.
Artifacts↗
Ultrathin sectioning of different areas of the same semithin section.
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Microtomy↗
Orientation of human spermatozoa for electron microscopy: a fast, simple method.
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Humans↗
Ultrastructural morphology and allergen detection in birch pollen after aqueous, anhydrous-liquid, and vapor fixation techniques.
In order to find a good compromise between preservation of ultrastructural morphology and retention of antigenicity, birch pollen grains were chemically fixed in aqueous p-formaldehyde or glutaraldehyde, in p-formaldehyde or glutaraldehyde dissolved in anhydrous glycerol, and in p-formaldehyde or glutaraldehyde vapor. Representative cytoplasmic areas were inspected for the preservation of ultrastructural morphology and for their capacity to bind a monoclonal antibody against Bet v I, the major birch pollen allergen. The experiments showed that cytoplasmic morphology was best preserved after vapor fixation in p-formaldehyde. This fixation also led to the highest degree of specific antibody binding.
Allergens↗
A method for rapidly collecting serial sections for light microscopy.
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Immunologic Techniques↗
Controlled-depth and cross-section preparation techniques for transmission electron microscopy subsurface studies in metals.
Thin film specimen preparation from bulk material at a controlled depth below the surface and cross-section thin film preparation for transmission electron microscope investigations of electrically conducting materials are described. Both techniques are illustrated by austenitic stainless steel, where they have been used complementary to each other for microstructural studies of subsurface ion irradiation damage. The advantages and limitations of both techniques are discussed.
Metals↗
Selective etching of AlAs for preparation of III-V semiconductor thin foils.
A new method of thin section preparation of III-V semiconductors and multilayers for transmission electron microscopy (TEM) is presented that exhibits considerable advantages over conventional methods such as ion beam milling and jet thinning. GaAs thin films and multilayers of GaAs/In chi Ga1-chi As/GaAs are grown over an etch release layer of AlAs on GaAs substrates by molecular beam epitaxy (MBE). Planar TEM sections prepared by selective etching from these samples show improved ability to image film morphology and dislocation arrangements, and the resulting large thin electron transparent areas facilitate dislocation density measurements and detection of spatial variations. Avoidance of radiation effects and wedge shaping, both common to ion milled samples, allows this method to be used to prepare uniform thickness standards of single layer GaAs films for EDS analysis or lattice imaging.
Aluminum↗
A novel method for the cross-sectional TEM preparation of thin films deposited onto water-soluble substrates.
A preparational method was developed solving the problem of cross-sectional TEM preparation of thin films and layer systems deposited onto water-soluble substrates. The technique is based on the replacement of the sample onto steady substrate, followed by mechanical and ion beam thinning. Cross-sectional TEM micrographs of Ag and Ag/Ag2Se layers are shown presenting the efficiency of this novel technique.
Histological Techniques↗
A step section method for full histopathological assessment of carcinogen-affected target tissue during respiratory carcinogenesis.
Studies of carcinogenesis that are not limited to overt neoplasms but also involve evaluations of preneoplastic stages require histopathological assessment of the entire carcinogen-affected tissue so that the true nature and sequence of the progressive process can be determined. The customary serial sectioning approach achieves this goal, but at an inordinate logistic cost. In studies of hamster bronchial carcinogenesis, a step section method was compared to a quasi-random approach and to the customary serial section method. The step section method achieved the same diagnostic completeness as serial sectioning, but at a two orders of magnitude reduction in costs.
Animals↗
Planing frozen hydrated plant specimens for SEM observation and EDX microanalysis.
A procedure is described for forming a flat face on a frozen piece of plant tissue, which may then be observed fully-hydrated or lightly etched, and coated or uncoated with a metal film, in scanning electron microscopy (SEM). The frozen sample was planed with a glass knife at -80 degrees C in a cryo-ultramicrotome. The sections were discarded, and the planed block face placed on the cold stage in the microscope column, either for observation uncoated at low kV, or for light etching (-90 degrees C) to reveal the cell outlines. If a higher accelerating voltage was needed, the face was given an evaporative coating of Al in the cryo-preparation chamber and returned to the column. The advantages of the planed face over the usual fracture face are illustrated: imaging at a chosen rather than a chance position; clearer cellular and subcellular detail; preservation of hydrated gels like mucilage and swollen cell walls; the possibility of making serial parallel sections through the same piece of tissue; opportunities for accurate morphometric analyses on the planed face; capacity to produce longitudinal sections; preservation of very delicate structures that are destroyed by fixation and drying. A major advantage of the Al-coated planed face is the increased accuracy of energy-dispersive X-ray (EDX) microanalyses on a smooth rather than a rough surface. Tests are included which show that neither the light etching employed, nor successive planing, interferes with the analyses of elements in the frozen face.
Cryopreservation↗
A negative contrast stain for ultra-thin frozen sections.
Ultra-thin frozen sections are ideal substrates for immunolabelling in high resolution electron microscopy. However, visualization of subcellular structures is inferior to that obtained with corresponding plastic sections. Although negative staining is generally effective and even superior to positive staining, the accumulated stain is often too heavy, obscuring morphology and markers used for immunocytochemical localization of antigens. This paper describes the development of a modified negative contrast staining technique in which a high concentration of uranyl acetate is mixed with methyl cellulose at a low pH. Application of this stain to cryosections of cells and tissue resulted in improved visualization of morphological structures characterized by negative images of membranes and cell organelles. Use of this stain is advantageous for morphological and immunocytochemical studies involving ultra-thin frozen sections.
Adrenodoxin↗
Fine structure of Tritrichomonas foetus as seen using cryotechniques.
Tritrichomonas foetus was studied using different physical and chemical fixation methods such as fast-freezing (by high pressure, "slam-freezing," and jet-propane), freeze-substitution, conventional freeze-fracture and deep-etching, cryoultramicrotomy, and routine preparation for transmission electron microscopy. The use of fast-freezing fixation (FFF) proved to be superior in terms of structural preservation due to the rapidity of this fixation compared to that obtained using conventional chemical fixation. The low temperature techniques used here were useful to confirm data already obtained by conventional freeze-fracture using chemical fixation and cryoprotection, such as the presence of flagellar rosettes and costa structure. Cryoultramicrotomy and slam-freezing also demonstrated the presence of hair-like structures projecting out from the protozoan surface. New aspects of organelles of T. foetus were demonstrated.
Animals↗
Preparation of oxide superconductor specimens for TEM examination.
We have investigated a wide variety of oxide superconductors and report here on a number of techniques that can be effectively used to prepare transmission electron microscopy (TEM) specimens from these materials. Crushing, cleaving, ion milling, ultramicrotomy, and jet polishing all were successfully utilized, and details of each technique, as well as equipment used, are described. Selection among these methods depends both on the starting form of the material and the information required. Ion milling and crushing generally give the best results and have the widest applicability in our particular work, while crushing and cleaving involve the least equipment cost. In some cases, particularly with ion milling and jet polishing, small variations in the details of preparation have a dramatic effect on the success rate. We have found it to be a great advantage that the same techniques can be applied in a similar manner to a whole range of oxide materials, even (with some refinements and special precautions) to those that are extremely oxygen or moisture sensitive.
Electric Conductivity↗
Design and implementation of a database and program for 3D reconstruction from serial sections: a data-driven approach.
This paper describes a structural approach for a standard setup of a computer program for 3D reconstruction from serial sections. Three-dimensional reconstruction as a technique increases in importance as, along with modern immunohistochemical techniques, it is a tool in the understanding of three-dimensional development patterns. In order to apply 3D reconstruction technique in a standard laboratory setup, an attempt was made to streamline the input and the manipulation of the data such that results are obtained easily. One will find a combination of two approaches in this paper: the first is a strict ordering of the complex data, and the second is an ordering of the processes that one wishes to apply on the data (together, these two approaches constitute an information analysis); because it was observed that developmental biologists tend to work from simple lines to describe their observations, the contour model was chosen as the vehicle to build a reconstruction model from. Consequently, the data is ordered in a database that has to be manipulated to get the data out in the desired format. The most important output format is a display of the reconstructed contour stack on a graphical computer screen. Together with the other data manipulation processes, such as the input, the inspection, the revision (correction), and the reconstruction, all processes are described using the reconstruction of an 11 embryonic days (ED) rat embryo as an example. Finally, the merits of the program are illustrated with an example from the development of the human embryonic heart.
Animals↗
Confocal microscopy of thick sections from acrylamide gel embedded embryos.
The preparation of optically clear, thick sections of fragile embryonic tissues greatly aids the power of confocal scanning laser microscopy in imaging three-dimensional structures. We report here conditions for embedding, sectioning, and staining embryos in polyacrylamide gels for a variety of confocal imaging techniques. Infiltration of tissues in standard mixtures of 10-15% acrylamide monomer yields, upon polymerization, blocks that cut easily by vibratome between 50 and 1,000 microns. These conditions worked well for tissues previously stained or for staining gel sections with low molecular weight water-soluble fluorochromes (MW < 5 kD [e.g., propidium iodide, phalloidin]). For immunostaining of tissue after embedding and sectioning, the acrylamide concentration was reduced to 2-3% acrylamide to allow access of immunoglobulins to antigenic sites; such gels were supplemented with 1% agarose to facilitate sectioning and handling. Either method yielded abundant, optically clear, and easily handled sections for mounting and examination in water-miscible media.
Acrylic Resins↗
Uranium-contaminated soils: ultramicrotomy and electron beam analysis.
Uranium-contaminated soils from the U.S. Department of Energy (DOE) Fernald Site, Ohio, have been examined by a combination of backscattered electron imaging (BSE) and analytical electron microscopy with electron diffraction (AEM). The inhomogeneous distribution of particulate uranium phases in the soil required the development of a method for using ultramicrotomy to prepare transmission electron microscopy (TEM) thin sections from the SEM mounts. A water-miscible resin was selected that allowed comparison between SEM and TEM images, permitting representative sampling of the soil. Uranium was found in iron oxides, silicates (soddyite), phosphates (autunites), and uraninite (UO2 + x). No uranium was detected in association with phyllosilicates in the soil.
Electron Probe Microanalysis↗
Ultramicrotomy of nanocrystalline materials.
The methodology and one of the first attempts to produce transmission electron microscopic (TEM) specimens of nanocrystalline metals, alloys and ceramics by ultramicrotomy are presented. Samples of the pure elements Co, Pd; alloys of Y-12 at. % Fe, Al-7 at. % Ag and W-30 at. % Ga; and ZnO ceramic, were found to section successfully to varying degrees. Advantages of sections prepared through ultramicrotomy over ion beam methods include extensive electron-transparent regions of uniform thickness and absence of ion beam damage. Typical artefacts were observed (knife marks, tearing, pull-out, shear lamellae, section curling, and anodic dissolution) but did not impede TEM analysis significantly. A potentially important effect observed was that of a texture development upon sectioning of the Co and Pd samples. It is thought that this unusual phenomenon results from the extremely fine scale of the microstructure and the purity of the Co and Pd samples, and may be enhanced by frictional heating effects and the state of the knife edge.
Alloys↗