Microanalysis data depe nd strongly on the electron beam energy used in the S E M and the effects are more pronou nced with bulk samples than with thin sections. The situation can be used to advantage but there are also significant problems. The data and any quantitative corrections can be quite different at different beam energies. Th e spectra look different and this can be both helpful and confusing, with low ene rgy x-ra y lines generally more prominent at lower voltages. S p ec tra at several different voltages are particularly informative, both for quantitative analysis and to deco nvo lute three dimensional geometries. There may be no single perfect voltage for an analysis.

Although high resolution (2nm), low voltage (lkV), SEM of bio-organic materials can now be performed more or less routinely using instruments fitted with a field emission source, virtually no low voltage x-ray microanalysis has been carried out on this type of specimen. Boyes and Nockolds showed that quantitative microanalytical information could be obtained from polished inorganic samples at a spatial resolution of l00nm at 5kV and Johnson et al obtained similar type of data at a spatial resolution of 150nm at 3kV. High spatial resolution (l0nm) microanalysis can be achieved in frozen dried or chemically compromised sections of biological material examined at high voltage in the TEM but frozen hydrated chemically unfixed sections are damaged. The other approach is to use the SEM with frozen hydrated, chemically uncompromised samples, usually at about 10-15kV, in order to obtain sufficient signal from the elements of interest which typically lie in the range Na (Z=l 1) to Ca (Z=20).

The contraction of muscle depends on the ability of intracellular organelles to rapidly release and take up large amounts of activator calcium. In cardiac muscle the intracellular sodium concentration is known to also modulate this Ca2+ release: Na+-Ca2+ exchange creates narrow cytosolic microdomains just internal to the sarcolemma (approximately 20 nm wide) with locally elevated concentrations of Ca2+ and Na+. The existence of such “functional” compartments with transient high ionic concentrations has been postulated for explaining the nigh efficiency of signal transmission during excitation-contraction coupling. We present results showing that electron probe microanalysis (EPMA) at the high spatial resolution of scanning transmission electron microscopy (STEM) can detect changes in sodium and calcium concentration of membrane-limited organelles and functional microdomains provided the cardiac myocytes are rapidly frozen at defined times during activation of contraction.

The development of energy dispersive x-ray spectrometry (EDS) has had a profound impact on the methodology of quantitative x-ray microanalysis of thick specimens (i.e., thickness≫ electron range) as performed in electron beam instruments. By equipping the scanning electron microscope (SEM) with EDS, quantitative x-ray microanalysis has become commonly available to a wide range of users, at least some of whom have only a modest background in analytical science. An important aspect of the development of quantitative analysis by EDS has been the extensive analytical experience gained during the development of the electron probe microanalyzer (EPMA) equipped with wavelength dispersive x-ray spectrometers (WDS). The critical measurement step for quantitative WDS analysis was recognized to be the determination of the “k-value”:

k = Iunk / Istd (1)

where I is the measured characteristic intensity of a specific x-ray peak, corrected for background and peak overlaps, for both the unknown and the standard.

We report measurements of chemical shifts in the Fe-L x-ray lines of different Fe compounds from xray emission spectra acquired using a microcalorimeter energy dispersive spectrometer (EDS). The observed changes in peak position and relative intensity of the Fe-L x-ray lines are in agreement with measurements obtained using a wavelength dispersive spectrometer (WDS), demonstrating the usefulness of microcalorimeter EDS for high-energy-resolution x-ray microanalysis.

Chemical shifts result from changes in electron binding energies with the chemical environment of atoms. In x-ray spectra, chemical shifts lead to changes in x-ray peak positions, relative peak intensities, and peak shapes. These chemical bonding effects can be significant (with x-ray peak shifts on the order of 1 eV), particularly for x-ray lines resulting from transitions involving valence electrons of light elements such as B and C.

High performance Si(Li) detectors for EDS applications must be cooled in order to eliminate excess leakage current and to reduce noise as a result of thermal excitation. The most common cooling method is the use of liquid nitrogen (LN), which has been commonly used during the last four decades. The LN method is relatively simple, however, it has some disadvantages such as: requiring a large dewar for storing the LN, the necessity of refilling it with LN, as well as some hazardous handling issues.

Several years ago, Kevex introduced a new no-LN x-ray detector, the SuperDry, offering the customer a solution to the previously mentioned cooling problems. The SuperDry uses a thermoelectric device, a Peltier solid-state refrigerator, without any moving parts. The SuperDry covers all the disadvantages of the LN method, however, the temperature achieved has not reached the same low level as the LN products.

Function dependent changes in the intracellular (i.c.) concentrations of calcium and sodium play a crucial role in the regulation of biological processes such as contraction of heart muscle. However, accurate measurements of both elements are difficult since in i.e. compartments they are present in low concentrations, hence the Ca and Na peaks show small count rates and small peak to continuum ratios. The accurate quantification of Ca and Na is rendered even more difficult by the partially overlap of the NaK with CuL peak and CaKα with KKβ peak. In some cases electrophysiologist substitute in the experiments K+ with Cs+ or the pathologist is interested in the storage of mercury. For analysis of such elements (Cs, Hg) with higher atomic number the stopping power of the EDS crystal play an important role for system sensitivity. Quantitative analysis of thin biological cryosections with the continuum method must be corrected for influence of X-rays emit from holder, supporting film and grid.

Multivariate statistical analysis (MSA) is a powerful tool for the analysis of series of spectra. This paper explores an application of MSA to a series of energy dispersive X-ray (EDX) spectra acquired in the scanning electron microscope (SEM) from a series of particles. The raw data were series of spectra previously acquired to test analytical procedures for trace element detection. This paper explores the possibility of performing the trace element detection with MSA components that have been extracted from the raw data without any a priori assumptions about the information content of the particle spectra. Particles were prepared from two analytical glasses, dispersed onto carbon substrates and coated with carbon. The compositions of the two glasses are substantially similar, except that one glass (K-3106) contains 0.7 wt.% Fe, whereas the other glass (K-3069) does not contain Fe at a detectable level.

The introduction of Multivariate Statistical Analysis techniques such as Principal Component Analysis (PCA) to the study of EDS and EELS spectra has opened up new possibilities for processing spectral data. In conventional EDS analysis each spectrum provides information about the elemental composition of the sample at a specific probe position. Where the signal arises from a single phase this elemental composition corresponds to the chemical composition of the phase. However, where the probe is incident on a multiphase region of the sample it is impossible to make a direct identification of the different chemical phases contributing to the overall spectrum.

With PCA it is no longer necessary to consider the spectra as isolated pieces of information. It is now possible to analyze a series of spectra as a single entity, looking for correlations between the variations in the elemental signals present in the individual spectra.

This paper present new results about x-ray emission from porous materials obtained from Monte Carlo simulations. This is an update from the results which have been presented for the first time last year. In particular, the absorption correction is now simulated in the same way as the electron scattering. In the results presented last year, x-ray absorption was calculated with the integration of the φ(ρz) curves via the usual equation :

where I is the intensity emitted from the porous specimen, I0 is the intensity emitted from a thin foil with no porosity, x is the mass absorption coefficient times the cosine of the take off angle, z is the depth in the specimen and ρ is the density of the porous materials. Since the density term does not include the effect of the size of the pores, integration of this equation will give wrong results when the size of the pores are big relative to the electron range.

The overall precision of electron microprobe measurements has not been studied extensively. It is often only described in terms of the precision of individual x-ray measurements or worse yet by relying on some rule of thumb based on historical experience. This is unfortunate because the analyst is expected to determine whether his or her measurements can distinguish between two samples or if a particular component can be detected above background or in the presence of other elements. Thus precision is at the heart of establishing composition/structure/property relationships and clearly deserves more attention.

There are many sources of variability encountered in determining composition whether using energy dispersive (EDS) or wavelength dispersive (WDS) spectrometery. These include Instrumental Variables, i.e., beam voltage and current, specimen position and orientation, detector position, and measurement electronics; Specimen Preparation, i.e., how representative the sample is of the problem to be solved, specimen preparation and contamination, and quality of standards; Data Processing, i.e.,

There are no “absolutes” in terms of performance and wherever possible, performance measures need to reflect conditions similar to those in the intended application. Unfortunately, measurements are frequently constrained by practical and economic considerations. For example, the most convenient radiation source for testing EDX systems is the Fe55 radioactive source which produces Mn Kα radiation at 5.9keV and can be used with an air path between source and detector. However, the fwhm resolution at 5.9keV is a rather poor measure of what the resolution will be for x-ray energies below 0.5 keV. Furthermore, “P/B”, the ratio of Mn Kα peak height to background level at IkeV, is only a crude indication of incomplete charge collection ICC, and does not reveal problems that may give rise to shift and broadening of the C Kα peak for example.

In high-resolution biological energy-dispersive x-ray (EDX) microanalysis it is often necessary to measure very low elemental concentrations. As an example, calcium, a physiologically important element, typically occurs in subcellular compartments at concentrations of 10-100 atomic parts per million (corresponding to 1-10 millimole/kg dry weight of sample) and it is ultimately desirable to measure the concentration of this element with a standard error of ±1 atomic ppm (or ± 0.1 millimole/kg). Detection of calcium in biological specimens is further complicated by the presence of relatively high levels of potassium (around 0.5 atomic % or 500 millimole/kg), which gives rise to overlap of the K Kβ and Ca Kα peaks in the EDX spectrum. Counting statistics are frequently the limiting factor for detectability, but this is not necessarily the case because in the analytical electron microscope it is possible to collect spectra for long periods using a high probe current.

Monitoring the performance and capabilities of energy dispersive X-ray spectrometers (EDS) and related x-ray analysis electronics and software is important for maintaining and improving the reliability, sensitivity, and accuracy of the x-ray analysis system. There is growing demand for quality systems through laboratory accreditation, ISO 9000, ISO Guide 25 and related programs that require set quality control procedures for analytical instrumentation. In such cases it is frequently more useful to have one national/international standard. This approach is not only more efficient than having each analyst devise their own system, but the use of the same standard procedures among labs would allow direct intercomparison of results. This intercomparison can help labs and manufacturers determine what are normal versus abnormal results and lead to higher quality instruments and analyses.

We are designing a standard 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.

Few quantitative analysis techniques attempt as large an extrapolation between the compositions of standards and samples than is attempted in electron microbeam x-ray emission analysis. In-situ x-ray microanalysis can be performed for essentially all elements in the periodic table in complex matricies that may contain, in extreme cases, thirty or more detectable elements. Analyses are attempted for the same elements, using the same standards, in various matrices whose average atomic numbers might range from 4 to 94. Unlike most analytical techniques, where suites of standards are synthesized having similar bulk compositions as the samples and bracketing the concentrations of the elements of interest, the standards employed in microbeam analysis are most commonly pure elements, simple oxides, or other binary element compounds. This is true even though matrix effects on electron retardation and scattering, x-ray absorption, and secondary x-ray fluorescence can cause major variations in the differences between relative intensity and relative concentration.

Interest in electron beam x-ray microanalysis with low incident beam energies, defined arbitrarily as 5 keV and below, has been greatly stimulated in recent years by the development of the high performance field emission gun scanning electron microscope (FEG-SEM), which can produce a nanometer-scale probe with sufficient current to operate with both energy dispersive (EDS) and wavelength dispersive (WDS) spectrometers. Microanalysis in this regime requires the analyst to confront new spectrometry problems that are not typically encountered, or that can be safely ignored, when operating with conventional beam energies, 10 keV or greater. With low energy operation, the choice of atomic shells that can be accessed is restricted, forcing the analyst to make use of shells that have low fluorescence yields for intermediate and high atomic number elements, and possibly strong chemical effects, which are evident with high resolution x-ray spectrometry.

Wavelength-dispersive analysis of the ultra-light elements boron, carbon, nitrogen, and oxygen requires much more complicated experimental procedures than the analysis of medium-to-high Z (Z >11) elements. For a large part this is the result of a number of fundamental physical problems which cannot be circumvented. The most difficult problems of this nature are those related to the differences in the x-ray emission profiles from one ultra-light element compound to another. These differences are caused by the fact that in exciting ultra-light element x-rays electronic transitions of the bonding electrons are involved. As a consequence one has to deal with large peak shifts and large peak shape alterations in WDS analysis of ultra-light elements. While peak shifts can easily be accounted for by simply retuning the spectrometer when moving from standard to specimen the effects of peak shape alterations are much more difficult to deal with and in general it is imperative to perform the intensity measurements in an integral fashion.

A number of problematic analytical situations are known to exist in electron probe microanalysis (EPMA) where characteristic x-ray spectral overlaps are not only severe, but are also of the “self-interfering” or “cascade” variety. The “self-interfering” variety is exemplified by the innocuous Ba Lα ↔ Ti Kα; to the fearsome Pb Lα↔As Kα binaries, while “cascade” interferences are often seen among the transition metal series as in Ti Kβ → V Kα - V Kα Cr Kα or as seen with a secondary fluorescence interference effect as in Ni K ⇒ Fe Kα - Fe Kβ → Co Kα. Unlike simple interferences of the type Mn Kβ → Fe Kα, both of these types of spectral interferences are often quite troublesome for the analyst to correct for, especially for Si(Li) and Ge energy dispersive spectrometers (EDS) where the analytical peaks are often so overlapped as to prevent graphical deconvolution, and even for the higher resolution wavelength dispersive spectrometers (WDS) along with the yet to come bolometric energy dispersive detectors (based on projected resolution), the task can still be formidable.

Quantitative analysis of Mg and Fe in olivines, pyroxenes, and garnets is important for geochemical studies. Natural olivines have compositions close to the forsterite (Mg2SiO4) and fayalite (Fe2SiO4) binary system, with relatively minor substitution of other components. Pyroxenes and garnets are problematic in that they exhibit more diverse solid solution, and may contain both Fe2+ and Fe3+, the relative concentrations of which cannot be directly measured with the microprobe. The forsterite-fayalite olivine binary is a convenient system for studying the accuracy of Mg and Fe analysis without other complicating factors.

Bence and Albee utilized the a-factor approach for quantitative analysis of silicates, based on the empirical observation:

where CA,AB is the weight fraction of the oxide A in a binary oxide system AB, and KA,AB is the measured x-ray intensity of A relative to that of the pure oxide A.

Among the more important semiconductor materials, with commercially important applications in microelectronics, photonics, and nanodevice development are alloys in the system GaP-GaAs-AlAs- InAs (e.g., 1). These components have extensive regions of solid solution. With the wide variety of phases that can be grown in this system, it is possible to “tailor” heterojunctions that have desired band offsets or quantum wells that have a given shape of confinement potential, which is the basis of “bandgap engineering” (e.g., 2-4). In order to characterize the properties of these phases and maintain quality control in the manufacturing of devices utilizing these phases, it is critical to be able to perform accurate determinations of their compositions. This can be especially difficult, since many of the applications utilizing these phases involve micro- or nano-devices or thin multilayer coatings. In terms of the spatial analysis requirements, electron microbeam x-ray microanalysis is ideally suited for characterization of such specimens.