Abstract: A method is described to prepare a high-resolution electron micrograph for quantitative comparison with a simulated high-resolution image. The experimental data are converted from the darkening of film used to acquire the image to units of electrons per incident electron, the same units used in the simulation. Also, distortions in the image arising from distortions in the image-forming lenses of the electron microscope are removed to improve the quality of the data. Finally, an alignment procedure is described which gives precise, pixel-by-pixel alignment of the experimental image with the simulated image. Examples of the procedure are shown to illustrate how actual data are prepared for quantitative analysis.

Abstract: It has long been debated whether the 30-nm fiber of chromatin is packed in an orderly array. The fiber may be condensed by supercoiling, producing structures of varying diameter. Alternatively, technical problems may have prevented the detection of higher-order structures. We developed a strategy to distinguish between these two possibilities. One potential obstacle to studying the order of packing was the effect of fixatives, dehydrating agents, heat, and embedding polymers on the native structure of chromatin prepared for viewing by electron microscopy. The known tendency of proteins to be degraded by osmium tetroxide and subsequently to be extracted in the conventional protocols for embedding might be particularly damaging. To avoid such denaturants and ensure the retention of proteins in chromatin, the embedding resin HACH was employed. Drosophila mimica polytene chromosomes were thin sectioned, stained with uranyl acetate, and viewed in the transmission electron microscope. Images were digitized and subjected to computerized image processing. Raw data files, containing boundary coordinates of all closed figures in the image, were edited to retain only those regions of interest (ROIs) that exhibited dimensions similar to those of 30-nm fibers in projection views. Euclidean distances between the centroids of such structures were calculated to obtain linear intercepts between recognizable 30-nm fibers. According to stereology theory, the dimensions of a lamellar structure can be determined from the volume distribution function of such intercepts. Therefore, intercept values were pooled for final data files from five processed images of chromatin. The resulting frequency histogram, showing the number of observations at different intercept values, had a sigmoidal inflection that was diagnostic of a major, new spacing at 95 nm. The 95-nm minimum was sandwiched between maxima in the 85 to 90 nm interval and throughout the range 105 to 120 nm. The results suggest that established stereological theory will be a useful tool for investigating the intractable problem of higher-order chromatin structure.

Abstract: The purpose of this study was to determine whether a polymer could be formed from relatively innocuous monomeric ingredients and, if so, if it might serve as a suitable embedding medium for maximizing antigen retention. Such a polymer, HACH, was made up from a mixture of 2-hydroxyhexanedial and carbohydrazide. It polymerized spontaneously at room temperature within 24 hr. Preservation of protein antigenicity and subsequent immunocytochemical localization were demonstrated by three methods. To determine whether protein antigens were retained up to the polymerization stage, we studied hemoagglutination of red blood cells using antibodies directed against their protein antigens. In these trials, HACH-treated cells exhibited the same agglutination responses as control, untreated cells. Second, a guinea pig antibody was used to immunodecorate insulin in β cells of the islets of Langerhans. The number of gold particles, indicating sites where the antibody was bound, was several-fold greater in HACH- than in Lowicryl K4M-embedded pancreatic β cells. To assess the limit of detection of protein antigens in thin sections, an example of a protein present in mitochondria, lipoamide dehydrogenase, was also studied. An indirect procedure for immunodecoration, employing rabbit immunoglobulin G followed by gold-tagged secondary antibody, indicated that the enzyme was present at several sites within cross-sectioned mitochondria. The results suggest that the HACH polymer will be useful for the localization of antigens that are present in relatively few copies.

Atomic Structure and Chemistry of Boundaries/Interfaces

Abstract: Atomistic simulations were performed for the {222}MgO/Cu interface by local density functional theory (LDFT) methods, within the plane-wave-pseudopotential representation, and by (classical) molecular dynamics and statics. The electronic spectra obtained with LDFT calculations showed a localized interface state within the bulk MgO gap, approximately 1 eV above the MgO valence band edge. LDFT adhesive energy calculations, as a function of interface spacing and translations parallel to the interface, were employed to devise an interatomic potential suitable for large-scale atomistic simulation. The interface structure, which was obtained with molecular dynamics (and statics) calculations based on the resultant potential, exhibited a misfit dislocation network with trigonal symmetry, and no standoff dislocations.

Abstract: The atomic-scale structure of tilt grain boundaries has been studied by high-resolution electron microscopy (HREM) in several ceramics and metals such as NiO, yttria stabilized zirconia, Au, and Al. It is found that when grain boundaries are formed between two crystals a considerable variety of modes of relaxations, i.e., the rearrangements of atoms in and near the grain boundary core are possible, depending on grain boundary geometry and the interatomic interactions. The atomic relaxations within and near the grain boundary core often include relaxations that involve the formation of stacking faults in low-stacking-fault energy fcc materials. Grain boundary dissociations are also observed for several grain boundary geometries. Computer simulations of grain boundary structures in metals have in general been able to reproduce the relaxation features at least on a qualitative level. In contrast, ceramic grain boundaries typically are more complex and not always tractable by simple computer relaxation procedures, since the formation of point defects and partial occupancy of atomic columns may be involved in the relaxation processes. It is found that when a boundary is not allowed to relax to its lowest energy state due to geometrical constraints, a multitude of grain boundary core structures can exist. Such conditions are expected to be characteristic for nanocrystalline materials. Because of its close connection to grain boundary properties, quantification of the volume expansion by HREM techniques is an important goal of grain boundary research. Image simulations of grain boundaries suggest that for most materials of interest the 3-fold astigmatism must be corrected to better than 100 nm to achieve the desired accuracies.

Abstract: The compositional profile of a narrow layer of InAsxPl−x in InP has been determined using energy-filtered Fresnel contrast analysis, high-resolution electron microscopy (HREM), and high-angle annular dark-field (HAADF) imaging. The consistency of the results obtained using the three techniques is discussed, and conclusions are drawn both about the validity of interpreting the magnitude of Fresnel contrast data quantitatively and about the degree to which high-angle annular dark-field images of such materials are affected by inelastic scattering and strain.

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