Quantitative EPMA (electron probe microanalysis) intensity measurements require an accurate correction for the X-ray continuum (or background) created by the Bremsstrahlung effect from the primary electron beam. This X-ray continuum, as measured on a wavelength-dispersive spectrometer at any particular wavelength, is primarily a function of the mean atomic number of the material being analyzed. One can calibrate the dependence of the continuum on mean atomic number by measuring and curve fitting the X-ray intensities at the analytical peak in pure elements, oxides, and binary compound standards that do not contain any of the analyte or any interfering elements and use that calibration to calculate the X-ray background correction. For unknown samples, the mean atomic number is determined from the elemental concentrations calculated by the ZAF or φ(ρz) matrix correction, and the fit regression coefficients are used iteratively to calculate the actual background correction. Over a large range of mean atomic number we find that the dependence of the continuum intensity on mean atomic number is well described by a second-order polynomial fit. In the case of low-energy X-ray lines (<1 to 2 keV), this fit is significantly improved by correcting the X-ray continuum intensities for absorption. For major and most minor element analyses, the improved mean atomic number background correction procedure presented in this paper is accurate and robust for a wide variety of samples. Empirical mean atomic number background data are presented for a typical 10-element silicate and a 15-element sulfide analytical set up that demonstrate the validity of the technique as well as some potential limitations.

Procedures were developed to sample, store, ship, and process precipitated and metamorphosed snow crystals, collectively known as “snowflakes,” from remote sites to a laboratory where they could be observed and photographed using low temperature scanning electron microscopy (LTSEM). Snow samples were collected during 1994–96 from West Virginia, Colorado, and Alaska and sent to Beltsville, Maryland for observation. The samples consisted of freshly precipitated snowflakes as well as snow that was collected from pits that were excavated in winter snowfields measuring up to 1.5m in depth. The snow crystals were mounted onto copper plates, plunged into lN2 and then transferred to a storage dewar that was shipped to the laboratory. Observations, which could be easily recorded in stereo format (three-dimension), revealed detailed surface features on the precipitated crystals consisting of rime, graupel, and skeletal features. Samples from snowpacks preserved the metamorphosed crystals, which had unique structural features and bonding patterns resulting from temperature and vapor pressure gradients. In late spring, the surface of a snowpack in an alpine region exhibited a reddish hue. Undisturbed surfaces from these snowpacks could be sampled to observe the snow crystals as well as the organisms responsible for the coloration. Etching the surface of samples from these sites exposed the presence of numerous cells believed to be algae. The results of this study indicate that LTSEM can be used to provide detailed information about the surface features of precipitated and metamorphosed snow crystals sampled at remote locations. The technique can also be used to increase our understanding about the ecology of snow. The results have application to research activities that attempt to forecast the quantity of water in the winter snowpack and the amount that will ultimately reach reservoirs and be available for agriculture and hydroelectric power.

The methods of immunolabeling make visible the presence of specific antigens, proteins, genetic sequences, or functions of a cell. In this paper we present examples of imaging immunolabels in a scanning transmission x-ray microscope using the novel method of dark-field contrast. Colloidal gold, or silver-enhanced colloidal gold, is used as a label, which strongly scatters x-rays. This leads to a high-contrast dark-field image of the label and reduced radiation dose to the specimen. The x-ray images are compared with electron micrographs of the same labeled, unsectioned, whole cell. It is verified that the dark-field x-ray signal is primarily due to the label and the bright-field x-ray signal, showing absorption due to carbon, is largely unaffected by the label. The label can be well visualized even when it is embedded in or laying behind dense material, such as the cell nucleus. The resolution of the images is measured to be 60 nm, without the need for computer processing. This figure includes the x-ray microscope resolution and the accuracy of the label positioning. The technique should be particularly useful for the study of relatively thick (up to 10 μm), wet, or frozen hydrated specimens.

The use of a thin annular detector in a scanning transmission electron microscope is shown, theoretically and experimentally, to allow several imaging modes that may be of value for the study of thin specimens. The diffraction pattern on the detector plane may be expanded or contracted by means of post-specimen lenses to vary the collection angle of the thin annular detector to form dark- or bright-field images. Dark-field images obtained from annuli of various radii in the diffraction pattern can selectively reveal different components of the sample, as illustrated in the case of a sample containing platinum crystallites, amorphous carbon, and carbon nanotubes. Amorphous materials of different composition can be distinguished by selecting the main maxima in their diffraction patterns. If the central beam is enlarged so that it just fills the inner aperture of the detector, the “marginal” imaging mode so achieved gives an image contrast that is proportional to the square of the differential of the projected potential distribution. Any deflection of the incident beam spot due to a change of the projected potential gives bright contrast in the image.

Alternating bright/dark anomalous subunitcell contrast in HREM images along or near the close-packed direction of 6H-SiC, Ti5Si3, α-Ti, and 4H-SiC, all of which are hexagonal, was examined using computer-generated crystal models, HREM image simulations, and digital diffractograms from the corresponding experimental images. The primary variables were crystal tilt and thickness. Crystal model projections showed that the scattering potential was smeared anisotropically within the unit cells by small crystal tilts, which reproduced the experimentally observed anomalous subunit-cell contrast modulations in the corresponding simulations. The effect increased with thickness, but it did not occur in exact zone axis simulations for any crystal thickness. Structural considerations indicated that the contrast resulted from tilt-induced violations of Gjonnes-Moodie dynamical extinctions and excitation of kinematically forbidden reflections in the imaging zone. Digital diffractograms from experimental HREM images confirmed their presence in the imaging zone diffraction patterns. These effects were absent in HREM images from cubic crystals in this material system because the structurally induced requisite kinematically forbidden reflections do not occur in the imaging zone.

Dense, polycrystalline MgO was infiltrated with monticellite (CaMgSiO4) liquid to study the penetration of liquid along the grain boundaries of MgO. Grain growth was found to be restricted with increasing amounts of liquid. The inter-granular regions were generally found to be comprised of a two-phase mixture: crystalline monticellite and a glassy phase rich in the impurities present in the starting MgO material. MgO grains act as seeding agents for the crystallization of monticellite. The location and composition of the glassy phase with respect to the MgO grains emphasizes the role of intergranular liquid during the devitrification process in “snowplowing” impurities present in the matrix.

Serious difficulties are encountered when SIMS analysis is applied to plant cells because of the cells' basic organization. In most plant cells, the cytoplasm is present as a thin layer that surrounds a large central vacuole, and is surrounded externally by a porous semi-rigid cell wall. Due to the high internal hydrostatic pressure typical of plant cells, large-scale solute redistribution may occur when tissues are excised. Relatively small solute decompartmentation is sufficient to collapse the native solute gradients between the cytoplasm and the adjacent compartments, due to the small volume of the former. For these reasons, most of the SIMS analyses in plant cells have been performed on elements bound to non-diffusible structures such as proteins, cell wall polymers, or in dry seeds. Sample preparation remains a limiting factor when imaging the distribution of soluble compounds. Cryotechniques have generated considerable interest to circumvent these problems. Cryofixation followed by cryosectioning would a priori be the best procedure, but encouraging results indicate that cryofixation followed by cryosubstitution is an interesting alternative.

Backscattered electron imaging (BEI) and transmission electron microscopy (TEM) were used to examine the effects of treatment with intermittent slow-release sodium fluoride (SRNaF) and continuous calcium citrate on bone architecture and crystallinity. Examination was performed in nondecalcified biopsies obtained from patients following up to four years of therapy (placebo or SRNaF) and compared to pretreatment biopsies from each patient, as well as to bone from young, normal subjects. BEI images disclosed increased areas of recent bone formation following fluoride administration. There was no evidence of a mineralization defect in any biopsy and both cortical and trabecular architecture remained normal. TEM analysis demonstrated intrafibrillar platelike crystals and extrafibrillar needlelike crystals for both the pre- and post-treatment biopsies as well as for the bone from young normal subjects. There was no evidence of increased crystal size or of an increase in extrafibrillar mineral deposition. These observations suggest that intermittent SRNaF and continuous calcium therapy exerts an anabolic action on the skeleton not accompanied by a mineralization defect or an alteration of bone mineral deposition. The use of BEI and TEM holds promise for the study of the pathophysiology and treatment of metabolic bone diseases.

We examine the problem of building quantitative elemental maps from X-ray absorption images (radiography). As we suggested in a previous publication in Microbeam Analysis, factorial analysis of correspondence is shown to be an optimal method, in the least squares sense, for solving the multilinear equation system given by Beer's law: it relates to an efficient description of the problem in the concentration phase space. We explain how factorial analysis is related to singular value decomposition and we give a complete description of the algorithm. The method can be applied to any multilinear analytical technique as well. Programs are written in C and Mathematica® languages. Academic users may obtain the relevant software (source and code) as freeware directly from the authors.

Depth profiling has been performed by using Auger electron spectrometry and X-ray photoelectron spectrometry in combination with Ar-ion sputtering. The data obtained by both surface-analytical methods have been evaluated by means of factor analysis and partly by applying artificial neural network in order to determine the compositional layering of different thin-films such as TiNx on Ti, Cr2O3/CrN sandwich layer, and copper oxide on Cu. Both multivariate statistical methods applied to the same data sets lead to results that agree well within statistical deviations provided that the structure of the artificial neural network is constructed appropriate to the actual problem.

We determined the effect of non-uniform specimen thickness underlying an electron probe on specimen thickness measurements by EELS and discussed its effect on quantitation of trace element concentrations. Our results are applicable especially, though not solely, to biological specimens, and show that if the probed area is not uniform, the specimen thickness measured by either the conventional or the fitting method could deviate from the true mean thickness by more than 30%. However, errors associated with these two methods are in the opposite directions and, consequently, the average of the two methods yields the best empirical estimate of the true mean thickness. The error due to non-uniformity of the specimen in the trace element concentration measured in typical cryosections can reach 30%, and its direction (overestimate or underestimate) depends on the methods used for calculating the concentration.

We describe a new electron-optical configuration for transmission electron microscopy (TEM). In this novel mode, the objective mini-lens is strongly excited so that the back focal plane of the objective lens is imaged onto the plane of the selected-area aperture with a magnification of 3.2. Thus, the selected-area aperture can function as an objective aperture either in place of or in addition to the conventional objective aperture. This new configuration, which has been implemented on a JEOL 4000EX high voltage electron microscope, provides improved resolution and contrast in images of thick biological specimens and also facilitates the use of beam tilt for stereo image acquisition.

Several examples are presented that show the successful application of uranyl acetate and ammonium molybdate negative staining in the presence of trehalose for TEM studies of filamentous and tubular structures. The principal benefit to be gained from the inclusion of trehalose stems from the considerably reduced flattening of the large tubular structures and the greater orientational freedom of single molecules due to an increased depth of the negative stain in the presence of trehalose. Trehalose is likely to provide considerable protection to protein molecules and their assemblies during the drying of negatively stained specimens. Some reduction in the excessive density imparted by uranyl acetate around large assemblies is also achieved. Nevertheless, in the presence of 1% (w/v) trehalose, it is desirable to increase the concentration of negative stain to 5% (w/v) for ammonium molybdate and to 4% for uranyl acetate to produce satisfactory image contrast. In general, the ammonium molybdate-trehalose negative stain is more satisfactory than the uranyl acetate-trehalose combination, because of the greater electron beam sensitivity of the uranyl negative stain. Reassembled taxol-stabilized pig brain microtubules, together with collagen fibrils, sperm tails, helical filaments, and reassociated hemocyanin (KLH2), all from the giant keyhole limpet Megathura crenulata, have been studied by negative staining in the presence of trehalose. In all cases satisfactory TEM imaging conditions were readily obtained on the specimens, as long as regions of excessively deep stain were avoided.