Improvements in instrumentation for energy-dispersive X-ray microanalysis (EDX) and electron energy-loss spectroscopy (EELS) have underlined the need for suitable standards for measuring performance. We report the results from several laboratories that were supplied with a test specimen consisting of a thin film of nickel oxide supported on a molybdenum grid. The Ni-Kα/Mo-Kα count ratio was used as an indication of number of stray electrons and/or X-rays in the TEM column; the Ni-Kα peak/background ratio provided a measure of the total background in the EDX spectrum, including bremsstrahlung contributions and the effect of detector electronics. By providing values typical of current instrumentation, the results illustrate how the test specimen can be used to evaluate TEM/EDX systems prior to purchase, during installation, and (periodically) during operation. The NiO films were also used to test EELS acquisition and quantification procedures: measured Ni/O elemental ratios were all within 10% of stoichiometry.

Phosphoenolpyruvate carboxykinase (PEPCK) is the rate-limiting gluconeogenic enzyme and in liver occurs in a lobular gradient from periportal to pericentral regions. The subcellular distribution of cytoplasmic PEPCK molecules within hepatocytes and its relationship to organelles have not been determined previously. In this study, we have used immunogold electron microscopy to evaluate the subcellar distribution of the enzyme, in addition to brightfield and epipolarized light microscopy. Cryosections (10 μm) of perfusion-fixed rat liver were collected on silanated slides and immunostained using goat anti-rat PEPCK followed by 5-nm gold-labeled secondary and tertiary antibodies. Additionally, free-floating vibratome sections (25, 50, and 100 μm) of perfusion-immersion-fixed rat liver were immunogold stained using goat anti-rat PEPCK and 5-nm gold-labeled secondary antibody, with and without silver enhancement. The immunogold labeled sections from both procedures were embedded in epoxy resin for the preparation of thin sections for electron microscopy. The results showed that the gold-labeled antibodies penetrated the entire thickness of cryosections, resulting in a high signal for PEPCK, but membranes in general, the smooth endoplasmic reticulum in particular, were not identifiable as electron dense unit membranes. On the other hand, the vibratome sections of well-fixed tissue allowed good visualization of the ultrastructure of cellular organelles, with the smooth endoplasmic reticulum appearing as vesicles and tubules with electron dense unit membranes; however, the penetration of the gold-labeled antibody was limited to cells at the surface of the vibratome sections. In both procedures, PEPCK, as indicated by gold particles, is predominantly in the glycogen areas of the cytosome and not in mitochondria, nuclei, Golgi apparatus, or other cell organelles. Hepatocytes in periportal regions have a compact subcellular distribution of PEPCK shown by gold particles; hepatocytes in pericentral regions have a diffuse subcellular distribution of PEPCK and thus more scattered gold particles. When normal serum replaced the first antibody in the immunogold staining procedures, the background was very low.

The atomic force microscopy is increasingly being used in analytical laboratories to study material surface phenomena. Whereas its use is not free of artifacts itself, the AFM, because of the ways it produces topography images, can shed some light on problems associated with other analytical techniques. This article describes the use of Atomic Force Microscopy (AFM) in visualizing and evaluating the extent of some well-known artifacts produced by three techniques widely used in analytical laboratories. The three different types of artifacts demonstrated here are caused, respectively, (1) by a stylus profilometer used for topography characterization of the pole tip area of magnetic heads, (2) by an accumulation of an organic contamination caused by a stationary electron beam positioned on an analyzed surface during SEM/EDX analysis, (3) by an enhancement of aluminum grain structure produced by a rastered monodirectional sputtering ion beam during Auger depth profile analysis. The analytical consequences of each of the presented artifacts are discussed. The images were collected on the TOPOMETRIX TX 2000 “Discoverer” AFM, using standard 4-μm pyramidal tips and forces within a few nanometers.

An efficient method of merging images from electron crystallographic data has been developed that is several orders of magnitude faster than the method of phase shift origin search. The results of this new cross-correlation method are compared with the phase residual in terms of speed, consistency with the phase residual method, and the ease of incorporating symmetry constraints along with the image-to-image merging. The program is further accelerated by a more efficient sorting procedure.

Changes in microstructure of Zr, Ti, and Ti-6%Al-%V resulting from ion-beam sputtering used to prepare samples for transmission electron microscopy have been correlated with hydrogen absorption. Zr was particularly sensitive to this phenomenon, resulting in extensive hydride formation in thin foil samples. Hydrogen enrichment extending to several micrometers in depth could also be produced in bulk samples in a few hours of sputtering. The performance of various sputtering units in different configurations has been examined. It is concluded that hydride formation appears to be caused primarily by the presence of hydrocarbons, for example, from the backstreaming of diffusion pump oil, in the residual vacuum background of the sputtering chamber.