We evaluated factors affected the accuracy and precision of quantitating trace concentration of Ca with electron energy loss spectroscopy (EELS). These factors include internal reflection in the spectrometer, precision of correlation between standards and experimental spectra, and radiation damage-induced spectral changes. We present methods of correcting for these effects and improving the reliability of trace Ca quantitation. A two-step fitting procedure is described that improves the retrieval of small Ca signals from the large background common to biological specimens. After optimizing the experimental conditions and data processing procedures, our current system can detect about 2.2 mmol/kg Ca in a 730-Å thick specimen at a total dose of about 410 nA . sec at 95% confidence level by fitting the first difference spectra. Because of the 0.1% residual gain variation after gain normalization, the first difference spectrum fitting is still the preferred method for trace Ca quantitation. Our study also demonstrates the clear advantage of using a 200 keV system, instead of 120 keV or lower accelerating voltages, for EELS analysis of relatively thick biological cryosections.

We have used a two-step casting procedure to create replicas of fibrillar collagen. A negative replica or mold is first created using either Mercox casting resin or a prepolymerized methacrylate resin that was originally described by Murakami. In the second step, a positive replica is made from the first using a solution of polystyrene or polyurethane to coat the methacrylate cast. The resulting replica is removed from the methacrylate cast. Results indicate the highest level of spatial resolution is obtained using a modified Murakami resin for the negative cast and polystyrene for the positive replica. The 29-nm-wide repeat banding structure present on collagen fibrils could be clearly identified in both the methacrylate cast and in the polystyrene positive replica of the methacrylate cast. Thus high-resolution replicas can be obtained. Such replicas may be useful to imprint inplantable devices with biologically relevant textures to improve tissue integration or for the development of textured surfaces for the in vitro expansion of cultured cells.

Electron-excited X-ray maps recorded with the scanning electron microscope (SEM)/electron probe X-ray microanalyzer (EPMA) are a major method of presenting compositional information. Digitally recorded maps are processed in a variety of ways to improve the visibility of features. Scaling of the recorded signal to match the 8-bit gray-scale intensity range of a typical computer display system is almost always necessary. Inherent limitations of gray-scale displays have led to other intensity-encoding methods for X-ray maps, including clipping, histogram normalization, and pseudocolor scales. While feature visibility is improved by applying these scales, comparisons among image sets are difficult. Quantitative comparisons must be based on standardized intensities corrected for background to produce intensity ratio (k-value) maps. We have developed a new logarithmic, multiband color-encoding method to view these k-value maps more effectively. Three color bands are defined, starting with a dark primary color and grading to a bright pastel: blue = trace (0.001 to 0.01); green = minor (0.01 to 0.1); and red = major (0.1 to 1.0). Within each band, the color is assigned according to a logarithmic scale that depends on intensity ratio or compositional measurements. Logarithmic multiband color encoding permits direct comparisons of maps, such as maps of different elements in the same field of view or maps of the same element in different areas, because the color scale is identical for all maps.

Abstract: Uncommon, micro-sized particles with different morphologies were found in sintered aluminous electrical porcelains. The presence of these particles seems to be relatively insensitive to changes in composition of the porcelain system as well as in firing temperatures. X-ray energy-dispersive spectrometry (EDS) microanalysis showed that some particles, those with very well–defined geometries, were composed of K, Si, Al, and O with empirical formula KAlSi2O6—that is, leucite particles. Other particles showed less well–defined geometries, including some composed of Na, Si, Al, and O, corresponding to a sodium aluminum silicate structure, and others of K, Si, Na, Al, and O, corresponding to the leucite composition, where some K has been substituted for Na. The well–defined geometry particles were detected most often, and diffraction analysis with backscattering electrons showed that their unit cell was cubic, with unit cell parameter a = 1.343 nm. The formation of these particles may be the result of the relatively high concentration of K2O and/or Na2O in some very localized zones of the glassy phase due to weak coupling (e.g., alkali-enriched or -depleted melts) during diffusion at a slow cooling rate in the presence of catalytic agents such as TiO2.

The measurement of absolute crystal polarity is crucial to understanding the structural properties of many planar defects in compound semiconductors. Grain boundaries, including twin boundaries, in the sphalerite lattice are uniquely characterized by the crystallographic misorientation of individual grains and the direction of the crystal polarity in domains adjoining the grain boundary. To evaluate crystal polarity in gallium phosphide (GaP), asymmetrical interference contrast in convergent-beam electron-diffraction (CBED) patterns was used to ascertain the nature and direction of polar bonds. The direction of the asymmetry in the electron diffraction reflections was correlated with the crystal polarity of a sample with known polarity. The CBED technique was applied to determine the polar orientation of grains adjoining Σ = 3 coherent and lateral twin boundaries in polycrystalline GaP.

One of the first samples analyzed by Castaing in his electron probe microanalyzer (EPMA) some 50 years ago was an iron meteorite. The Widmanstatten pattern microstructure of iron meteorites can be observed at very low magnifications ( Fig. 1). These meteorites are ideal samples for microanalysis because of the Ni gradient which extends over 10 to 1000 microns in the parent taenite phase of these Fe-Ni samples (Fig. 3). The Ni gradient is the result of very slow cooling of the iron meteorite, in terms of millions of years, within a parent'asteroid.

The scanning electron microscope (SEM) has been used to characterize the microstructure of meteorites, as well as samples from the moon and mars. For example, the microstructure of the dark etching taenite areas (T in Fig. 1) of the Carleton iron meteorite is shown in Fig 2. In this example, precipitates are observed along original martensite laths which form during the cooling of the iron meteorite at low temperatures.

Abstract: The SIMS channelplate used for recording image signals is characterized by local sensitivity differences caused by increasing aging of the central channelplate regions, which are subject to much more intense ion bombardment than the outer regions. These sensitivity differences lead to inhomogenous illumination of the sample, i.e., to images with disturbed intensity distributions. We present here a novel method to correct these illumination discrepancies by using the intensity changes due to sample position shifts to find the interfering background function that can be used to correct the original image. The proposed method has low presuppositions about the regarded images, and is stable with regard to noise and easy to apply. It is suitable not only for correction of channelplate local sensitivity differences but for all sorts of microscopic images that suffer from illumination inhomogeneity.

Abstract: Quantitative X-ray mapping in the analytical electron microscope (AEM) could improve the statistics of grain-boundary segregation measurements if high spatial resolution can be maintained at lower magnifications (<500 kX). Typically, only about 10 boundaries are analyzed because of the difficulty of conventional AEM measurements; however, a low-magnification quantitative X-ray map could contain twice this number of boundaries in a single field of view. Microscope conditions and mapping parameters have been explored for operation at ∼250 kX, under a variety of conditions to illustrate the trade-offs between various characteristics, such as analytical resolution, counting statistics, magnification, and acquisition time. From these data, it is possible to extrapolate to maps generated under different conditions and estimate their limitations with respect to these characteristics. A simple model has been developed to describe the behavior of inclined grain boundaries that can be used to estimate the detectability of segregant as a function of boundary tilt. Using quantitative X-ray maps, grain boundary Cu coverage has been measured from 55 boundaries in Al–4 wt.% Cu with minimal user effort. For fine-grained thin films, mapping is substantially more efficient than other methods of data acquisition and may be used to measure segregation at large numbers of boundaries.

This study provides a quantitative validation of qualitative automated three-dimensional (3-D) analysis methods reported earlier. It demonstrates the applicability and quantitative accuracy of our method to detect, characterize, and count Feulgen stained cell nuclei in two tissues (hippocampus and testes). A laser-scanned confocal light microscope was used to record 3-D images i which our algorithms automatically identified individual nuclei from the optical sections given an estimate of minimum nuclear size. The hippocampal data sets were also manually counted independently by five trained observers using the STERECON 3-D image reconstruction system. The automated and manual counts were compared. A nucleus-by-nucleus comparison of the manual and automated counts verified that the automated analysis was accurate and reproducible, and permitted additional quantitative analyses not available from manual methods. The algorithms also identified subpopulations of nuclei within the hippocampal samples, and haploid and diploid nuclei in the testes. Our methods were shown to be repeatable, accurate, and more consistent than manual counting.

We have fabricated amorphous thin-film specimens containing light elements (B, C, N, O, F, and Mg) in addition to silicon, and have measured their composition using electron energy-loss spectroscopy (EELS). The films appear to be stable during storage in air and during irradiation by an electron beam, for doses below 105 C/cm2. Used with a transmission electron microscope fitted with an energy-dispersive X-ray (EDX) detector, they provide a convenient means of determining light-element k-factors for X-ray microanalysis. For a TEM equipped with an electron energy-loss spectrometer, the specimens can also be used to check the EELS instrumentation and elemental-quantification procedures.

The accuracy of Na measurements in biological cryosections has been improved through replacement of the 8 μm Be window of the Si(Li) detector with a Super Atmospheric Thin Window (SuperATW) window mounted on a germanium electron microscope (GEM) detector. Greater accuracy of Ca measurements was also attained when a GEM detector instead of an Si(Li) detector was used for analysis of total Ca concentrations in cardiac muscle. Although the advantage of improved sensitivity for Na and Ca is lost in spot mode analysis because of the overriding contribution of biological variability, this advantage becomes important in X-ray mapping. We compared the two systems by analyzing cryosections of identical biological material under comparable conditions. We found that Si contamination in thin biological cryosections is unpredictable; it may be widespread and differ in degree in various cell compartments of the same cell. Hence, a correction for Si contamination should be included in the calculations of elemental concentrations. We suggest here a procedure for measuring and subtracting Si contamination from elemental spectra.

Phagocytosis of particulate wear debris from arthroplasties by macrophages induces an inflammatory response that has been linked to implant loosening and premature failure of artificial joints. Inflammatory mediators released by phagocytic macrophages such as tumor necrosis factor-a (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), and prostaglandin E2 (PGE2) are believed to play a central role in the pathogenesis of aseptic loosening. The objective of this study was to characterize titanium alloy particulates that closely match wear debris found around joint arthroplasties and to study their effects on the biosynthesis of inflammatory mediators by cultured monocytes. Peripheral blood monocytes were isolated from healthy human volunteers. Monocytes were cultured in 96-well plates for 24 h, washed, and exposed to three concentrations of titanium particulates and controls from 18Ð24 h. Supernatants were assayed for TNF-α, IL-1β, IL-6, and PGE2 activity. Energy dispersive X-ray spectroscopy (EDX) verified the titanium alloy to be Ti6A14V. Scanning electron microscopy (SEM) analysis showed significant titanium particulate heterogeneity with approximately 95% of the particles <1 micrometer in diameter. SEM and EDX technology was useful in the characterization of the titanium particulates utilized for in vitro models of titanium-induced cytokine release by monocytes. Incubation of titanium particulates (in concentrations similar to those found around loosened prosthetic joints) with cultured monocytes significantly increased their production of TNF-α, IL-1β, and PGE2.

Abstract: In this work, we show how field emission–scanning electron microscopy (FE-SEM) can be a useful tool for the study of self-assembled monolayer systems. We have carried out a comparative study using FE-SEM and atomic force microscopy (AFM) to assess the morphology and coverage of self-assembled monolayers (SAM) on different substrates. The results show that FE-SEM images present the same qualitative information obtained by AFM images when the SAM is deposited on a smooth substrate (e.g., mica). Further experiments with rough substrates (e.g., Al grains on glass) show that FE-SEM is capable of unambiguously identifying SAMs on any type of substrate, whereas AFM has significant difficulties in identifying SAMs on rough surfaces.

The steady evolution of computer controlled electron microscopes is dramatically changing the way we teach microscopy. For today’s microscopy student, an electron microscope may be just another program on the desktop of whatever computer platform he or she uses. This is reflected in the use of the term Desktop Microscopy. The SEM in particular has become a mouse and keyboard controlled machine, and running the microscope is not very different from using a drawing program or a word processor. Transmission electron microscopes are headed in the same direction.

While one can debate whether or not it is wise to treat an SEM or a TEM as just another black-box computer program, it is a fact that these machines are here to stay, which means that microscopy educators must adapt their traditional didactic tools and methods. One way to bring electron microscopes into the classroom is through the use of remote control software packages, such as Timbuktu Pro or PC-Anywhere. The remote user essentially opens a window containing the desktop of the microscope control computer and has all functions available. On microscopes with specialized graphics boards, integration of the image and control display for remote operation may not be straightforward, and often requires the purchase of additional graphics boards for the remote machine.

An in-lens Schottky field emission scanning electron microscope (SEM) combined with a transmission electron microscope (TEM)-type cold-stage and a chromium (Cr) sputter-coating system was developed to rapidly prepare and cryo-image biological specimens to attain accurate nanometer-level structural information. High-resolution topographic images at high primary magnification ([eg ]200,000 times) were digitally recorded with very short dwell times and without beam damage. Plunge freezing in ethane, followed by fracturing, Cr coating, and in-lens cryo-high-resolution scanning electron microscope (HRSEM) imaging directly revealed macromolecular features of yeast cells, platelets, and cell-free elastin analogues. The 'vitreous' nature of bulk water in its solid state appeared featureless in cryo-HRSEM images, suggesting that if ice crystals were present they would be [el ]2–3 nm (the approximate instrument resolution on cryo-specimens). Compared to technically difficult and indirect freeze-fracture TEM replicas, cryo-HRSEM samples are fully hydrated, unfixed, noncryoprotected specimens immersed in featureless ice. The time necessary to cryo-immobilize the specimen and record the image is <3 hr. The hexagonal arrays of intramembrane particles on the protoplasmic face of yeast cells and differences in surface morphology between thrombin-stimulated and quiescent platelets were assessed. A clear interface line between collapsed elastin fibril lacework and vitreous lakes was commonly observed. These experiments demonstrate the feasibility of this technique to rapidly evaluate macromolecular features in cryofixed cells and cell-free systems.

Light and electron microscopy are exceedingly versatile disciplines having many applications in pharmaceutical research. Many corporate and university facilities are organized with a centralized microscopy laboratory that serves the needs of many different investigators. This is also true at the Novartis Pharmaceuticals Corporation in East Hanover, New Jersey. A centralized microscopy laboratory is not only more efficient, but also most cost effective. The microscopy lab at Novartis is designed to meet the needs for a wide range of applications serving all of Research and Development. The lab contains seven functional areas for microscopy applications. A large preparation and processing area, necropsy, microtomy, TEM, SEM, wet and dry dark rooms, and an image analysis suite including telepathology, complete the lab.

Microscopy service is widely used in Pathology, Toxicology, Drug Discovery, Technical Research and Development, Production and Quality Assurance, and Compliance. Microscopy services have also been involved in marketing (packaging labels and commercials) for Novartis products.