The range of electrons for a given beam energy and atomic number is one of the most valuable piece of information a microscopist must know before carrying out qualitative and quantitative analysis of heterogeneous samples in a scanning electron microscope (SEM). The frequently used parametrization of Kanaya & Okayama is only « valid » at high energy (EO > 10 keV). However, with the advent of Field Emission Gun SEM (FEGSEM) most of the effort has been toward low energy analysis where no parametrization is available yet. In this paper, the parametrization of the range of electrons at low energy as a function of atomic number and beam energy will be presented for both the backscattered and the internal electrons.

The distribution of the maximum depth reached by 250 k electrons generated by the CASINO Monte Carlo program2 was used to compute the range for 10 elements at 20 energies.

Determination of the variation of absolute and relative electron-excited x-ray production rates as a function of electron beam energy and sample atomic number is necessary for calculation of the "stopping power" atomic number correction and the relative amount of characteristic fluorescence and for development of “standardless” and Monte Carlo algorithms for quantitative x-ray analysis. Critical to the calculation of x-ray production rates is an accurate expression for the inner shell electron ionization cross section. A large number of expressions have been proposed for the relative x-ray production rates (used in the fluorescence correction)1 and for the ionization cross section used in the atomic number correction, and these yield quite different results. In order to evaluate which expressions gave the most accurate results when applied to quantitative x-ray emission measurements, we performed a series of high precision measurements of x-ray intensities as a function of electron beam accelerating potential for a series of pure element and simple oxide, phosphide, sulfide, and chloride standards for 65 elements ranging in Z from C to U

There has been much interest in the last few years in the technique for focusing x-rays into high intensity spots using tapered glass capillaries or other forms of grazing incident x-ray reflectors. The resulting microbeams have been used in applications that include microfluorescence, microdiffraction, tomography and lithography. Instead of focusing x-rays to a spot, a collimating optic can be used to capture x-rays from a point source and turn them into a collimated parallel beam at the exit aperture to the optic. Kirkland et. al. have pointed out that the use of such an optic could provide enhanced detection sensitivity in wavelength dispersive spectroscopy.

We have developed a grazing incidence collimating x-ray optic that can be coupled to a simple wavelength dispersive spectrometer (WDS). This combined instrument was designed to enhance the intensity of x-rays from a sample by an order of magnitude or more in the energy range of 0 to 1 keV compared to a conventional WDS.

Changes in shape and position of long-wavelength X-ray lines measured by wavelength dispersive spectrometry (WDS) reflect changes in the density of bonding states of the emitting atom. However, some features of soft X-ray spectra are not due to bonding. For example, small peaks on the shorter-wavelength side of a characteristic peak probably arise from double ionization, absorption effects must be considered and there may be artefacts from the diffracting crystal itself. Nonetheless, when there are dramatic shape changes, spectrometry of soft X-rays can usefully augment microanalysis particularly in the study of tiny particulates which may be difficult to prepare for transmission microscopy.5 Two examples of spectrometry of O Kα follow.

Oxidation of very fine TiB2 particulates is a problem in the ceramics industry. The backscattered electron (BSE) micrograph in fig. 1 shows a particle section with a central core of TiB2 and a darker, oxidised rim analysing roughly like a borate, interspersed with brighter regions, probably TiO2.

Microanalytical techniques for the analysis of beryllium using the electron probe have been evaluated for Be metal, Be-rich silicate minerals, and three Be-rich synthetic silicate glasses. Despite difficulties typical of ultra-light element analysis in geologic materials, quantitative data for Be may be obtainable through careful analytical measurements, determination of optimal operating conditions, and consideration of parameters such as sample orientation and beam-induced migration.

Analyses for Be were obtained using a layered synthetic microstructure “crystal” (Mo/B4C) on the JEOL 8900 electron microprobe at the USGS, Denver, Colorado. The optimum accelerating voltage for Be detection was determined to be 8kV (Fig.l). Beryllium analyses in glass and mineral phases were successful using a beam current of 30nA and a 50 μm beam diameter. Due to low count rates, peak counting times of 10 minutes (beryl) and 30 minutes (glass) were required in order to achieve reasonable counting statistics and detection limits.

Electron Probe Micro Analyzer (EPMA) uses the characteristic X-ray for analysis. Therefore the depth of analysis area is spread to a few micrometer. It is difficult to make the quantitative or state analysis of surface thin-film. However, it is possible to measure the thickness of thin-film by using the X-ray intensity ratio between the film and bulk standard specimen under an assumed X-ray depth distribution function. In this paper, a known composition thin-film was measured by EPMA and the result was applied to thin-film area analysis.

Film thickness was measured as mass thickness (ρz) by the intensity ratio of the characteristic X-ray intensities between the thin-film and bulk standard. As the X-ray distribution function ϕ(ρz) the model due to Packwood and Brown1) was used, which is written as.

(1)

An error is thought to exist in the thickness calculation because the backscattering yield near the interface is different when the difference in the atomic numbers is large between the thin-film and the substrate.

Yttria-doped zirconia plasma-sprayed thermal barrier coatings (TBC’s) have been used successfully to improve the performance and extend the life of industrial equipment such as jet engines. For this reason, there is considerable interest in understanding and improving the plasma spray process and resulting TBC’s. A collaborative research program exists between Sandia National Laboratories and the National Institute of Science and Technology (NIST) to study the materials and processes used in preparing the coatings as well as the relationship between the physical properties of the coating and its microstructure.

The purpose of this work was to determine the elemental compositions on a micrometer scale of an yttria-doped zirconia coating. The cubic and tetragonal crystal forms of the yttria-doped zirconia have the most favorable thermal expansion coefficient, therefore providing the most effective thermal barrier. The Y concentration in these crystal phases is known to be 6-7 wt%, while the less desirable monoclinic phase, having a less favorable thermal expansion coefficient, contains less that 3 wt% Y.

In the last 10 years the development of new polymer type detector window materials has dramatically increased the opportunities for light element analysis with Energy Dispersive Spectrometers (EDS). With the introduction of light-element analysis the need also arises to accurately quantify X-ray spectra. Traditional quantification techniques, using a probe current measuring device and pure elemental standards, have been introduced into the field of EDS, but these techniques prevented much of the conveniences of the EDS techniques with respect to speed and ease of use. Many users are therefore willing to sacrifice part of the maximum achievable accuracy in return for a method that is more convenient: standardless analysis.

With EDS analysis the widely published ZAF and φ(ρz) models can be used to convert relative intensities into weight percentages, but for standardless analysis the inaccuracy of the result is mainly caused by the reference intensities.

One of the main goals of automated scanning electron microscopy analysis (ASEM) of particles is to provide macroscopic phase and compositional information on particle populations based on the energy dispersive x-ray spectrometry, EDS, analysis of several hundred to several thousand individual particles from the sample. The selection and identification of the various elemental groupings is often accomplished by applying techniques for grouping data, such as multivariate or cluster analysis methods, to characteristic x-ray intensities or normalized elemental concentrations. Particle groupings are often based on major and minor elements with concentrations greater than about 1-2 wt. percent. At these concentration levels, the peak-to-background ratios for the characteristic x-ray peaks are sufficiently large that in the absence of severe peak overlaps the elements can be easily identified even in spectra with poor counting statistics. Additional refinement in particle groupings may be possible if reliable information can be obtained on the trace elements present in particles at less than about 1-2 wt.%.

Monitoring the performance capabilities of energy dispersive X-ray spectrometers (EDS) and related x-ray analysis electronics and software is important for determining and improving the reliability, sensitivity, and accuracy of the x-ray analysis system. In addition, there is a growing popularity of quality systems through laboratory accreditation and ISO 9000 related programs that require set quality control procedures for analytical instrumentation. Having similar standard procedures amongst labs would allow direct intercomparison of results. This intercomparison would help labs and manufacturers determine what are normal versus abnormal results and lead to higher quality instruments and analyses. We have been developing a standard operating procedure for the characterization of EDS x-ray analysis systems on electron beam instruments.

We are designing the procedure to maximize the efficiency of each quality control (QC) measurement so that we spend as little time monitoring the analysis system as is possible. We first chose useful QC specimens and then designed data collection methods.

The Advanced Photon Source (APS) at Argonne National Laboratory is a third-generation synchrotron x-ray source, optimized for producing x-rays from undulators. Such undulator sources provide extremely bright, quasi-monochromatic radiation which is ideal for an x-ray microprobe. Such microprobes can be used for trace element quantification with x-ray fluorescence, or for chemical state determination with x-ray absorption spectroscopy. The GeoSoilEnviroCARS (GSECARS) sector at the APS is building an x-ray microprobe for research in earth, planetary, soil and environmental sciences.

The GSECARS undulator source is a standard APS Undulator “A” which is a 3.3 cm period device with 72 periods. The energies of the undulator peaks can be varied by adjusting the gap, and hence the magnetic field of the undulator. The energy of the first harmonic can be varied in this way from approximately 3.1 keV to 14 keV. A measured undulator spectrum is shown in Figure 1.

X-ray microscopy with soft X-rays is well suited for investigations of aqueous samples of some microns thickness when the resolution is required to be better than in visible light microscopy an not better than 30 nm. Sample preparation is as simple as for light microscopy, i.e. no fixation or metal coating is needed.

In Aarhus, an X-ray microscope is used for investigations in fields as biology, medicine and soil sciences. A ray diagram of the Aarhus X-ray microscope is shown in Fig.l. Synchrotron radiation at a wavelength of 2.4 nm from the Aarhus Storage Ring is focused by a condenser zone plate onto an object. Another zone plate as an objective behind the object forms the image on a CCD camera. Objects are located under atmospheric pressure. For dry samples, almost any kind of holder can be mounted in the microscope. Wet samples are placed between thin silicon foils in a sealed chamber. With typical liquid layer thickness of 5-15 µm, samples can be kept in the chamber for many hours without drying out.

Phase segregation is important in determining the physical and chemical properties of many complex polymers, including polyurethanes. Achieving a better understanding of the connections between formulation chemistry, the chemical nature of segregated phases, and the physical properties of the resulting polymer, has the potential to greatly advance the development of improved polyurethane materials. However, the sub-micron size of segregated features precludes their chemical analysis by most existing methods. Near edge X-ray absorption spectroscopy carried out with sub micron spatial resolution provides one of the few suitable means for quantitative chemical analysis (speciation) of individual segregated phases. We have used the NSLS and ALS scanning transmission x-ray microscopes (STXM) to record images and spectra of both model and real polyurethane polymers. Relative to energy loss spectroscopy in a transmission electron microscope, STXM has remarkable advantages with regard to a much lower radiation damage rates and much higher spectral resolution (∼0.1 eV at the C ls edge), with a spatial resolution (∼0.1 μm) adequate for many real world problems in polymer analysis.

Soft x-rays provide a high signal-to-background ratio and an energy resolution of 0.1 - 0.5 eV for quantitative microanalysis of thin organic specimens. For specimens with non-uniform thickness, it it necessary to take spatially resolved spectra for quantitative analysis. In the Stony Brook scanning transmission x-ray microscope, spectra can be acquired from ∼ 0.2 micron spots, and images at selected energies can be acquired at < 55 nm Rayleigh resolution. This spectromicroscopy capability has been used for biological applications including the mapping of protein and DNA so as to rule out certain models of chromosome packing in sperm, the mapping of calcium in bone and tendon tissue, and for polymer science applications.

We have investigated x-ray absorption near edge structure (XANES) of amino acids and peptides at the carbon K-edge. We find strong evidence that spectra of small proteins can be predicted from amino acid spectra

Ever since the recognition of calcium as a major intracellular messenger of signal transduction, its subcellular localization and intracellular movements have been intensively sought through electron and light optical methods. Electron probe microanalysis (EPMA), X-ray mapping, electron energy-loss spectroscopy (EELS) and energy-filtered imaging still provide the highest spatial resolution for measuring total calcium, whereas with light optical methods (fluorescent, luminescent and absorbance dyes) free [Ca2+]i can be measured with high sensitivity and time resolution. This presentation will summarize the relationship, whether collision or convergence, between the results of electron and light optical methods, with particular reference to mitochondrial Ca, and consider the potential for further improvements in detection sensitivity and spatial resolution.

Sarcoplasmic and endoplasmic reticulum: Early attempts to quantitate Ca in cellular organelles with EPMA were directed at the sarcoplasmic reticulum (SR) of skeletal muscle, where EPMA could also address questions not amenable to studies of isolated SR.

Many cell functions, including such critical processes as secretion, neuronal communication, muscle contraction, and even gene expression, are regulated by spatial and temporal changes in the intracellular concentration of Ca2+ ions. The high spatial resolution and analytical sensitivity of energy dispersive x-ray (EDX) microanalysis are ideally suited to characterizing biologically relevant changes in total concentration of cellular Ca. However, EDX results have not yet had a wide impact in the field of Ca regulation, with most laboratories preferring to focus on optically measured changes in free Ca concentrations as the important determinant of biological activity. This paper advocates a more positive perspective for the role of EDX microanalysis in studies of Ca regulation.

It is well known that the large majority of cellular Ca is in the bound form, with typical bound/free ratios on the order of l03-104 5. However, the most dramatic changes during Ca2+ -dependent cell activation occur as transients in the free Ca2+ pool.

During myocardial ischemia, several factors have been identified which contribute to the development of cardiac arrhythmias. These include membrane depolarization, increased cytosolic Na+ and decreased intracellular pH. This increase in intracellular Na+ has been shown to be associated with the development of ventricular arrhythmias. Changes in intracellular Cl- may also be involved in development of cardiac arrhythmias since decreasing extracellular Cl- concentration in the perfusion medium can prevent development of arrhythmias during myocardial ischemia in vitro. Extracellular ATP is known to increase in the heart during myocardial ischemia. ATP activation of p2 purinergic receptors on cardiac myocytes has been proposed to contribute to the initiation of the arrhythmias and ventricular fibrillations which characterize myocardial ischemia. Activation of p2 purinergic receptors results in membrane depolarization and decreased intracellular pH, thus reproducing some of the changes that occur during ischemia. We recently showed that activation of p2 purinergic receptors in both quiescent and electrically stimulated ventricular myocytes triggers spontaneous oscillatory contractions and Ca2+ transients.

The major storage site for calcium in cardiac muscle is the sarcoplasmic reticulum (SR). It has been shown using indirect methods that the amount of calcium stored in the SR can be altered by various agonists and anesthetics. The only technique to date which directly quantifies the amount of calcium in the SR is Electron Probe Microanalysis (EPMA). Using EPMA, an accurate measurement of the size of the SR calcium store can be made following treatments with known agonists.

Isoproterenol (ISO) causes an increased inotropic response in cardiac muscle via the (3-adrenergic pathway. When ISO binds to the (β-receptor on the plasma membrane, it causes the activation of Protein Kinase A (PKA) through a cascade of events. PKA phosphorylates the sarcolemmal calcium channels causing an increase in the rate of calcium influx. PKA also phosphorylates Tnl, which sensitizes the myofilaments to calcium, thereby increasing the rate of calcium release from the myofilaments.

Myocardial ischemia is defined as a cessation of blood flow to the heart, and reperfusion as a return to normal flow conditions following ischemia. It has been shown that contractility decreases immediately during ischemia and diastolic dysfunctiuon may persist during reperfusion. Changes in contractility can be directly attributed to intracellular Ca2+ handling, either by altering the myofilament sensitivity to calcium or by affecting Ca2+ cycling by the sarcoplasmic reticulum (SR). Previous studies have demonstrated that there are changes in intracellular Ca2+ cycling following ischemia and during reperfusion. The goals of the present study were: 1) to assess the effects of varying periods of ischemia on the size of the releasable (diastolic) SR Ca2+ store; 2) to determine whether changes in the size of the SR Ca2+ store are related to ischemia or reperfusion; 3) to correlate changes in the size of the SR Ca2+ store following ischemia with functional recovery of the heart.

Mg is present in all living cells as free and bound Mg. Previous observations have shown a positive correlation between total Mg content and proliferative rate in several different cell types (1). To investigate the association between Mg and cell proliferation, we utilized a human promyelocytic leukemia cell line, HL60, that can be induced to differentiate in vitro to neutrophil-like cells, by retinoic acid (RA) and other compounds.

To further investigate this question, we analyzed elemental compartmentalization in HL60 cells before and after differentiation using electron probe microanalysis (EPMA). Differentiation to neutrophil-like cells was induced by culturing the HL60 cells for 96 hours in the presence of 1 μM RA. Cells were washed, pelleted, then rapidly frozen in liquid ethane, cooled to -185°C with liquid N2. Ultrathin cryosections were cut from the surface of the frozen cells, freeze-dried overnight and carbon coated. X ray spectra were collected at -100°C and at 80 keV accelerating voltage in a Philips CM 12 STEM equipped with LaB6 gun and an EDAX 30-mm2 Si(Li) energy dispersive x-ray detector and multichannel analyzer.