Charles E. Fiori

Advanced Scanning Electron Microscopy and X-Ray Microanalysis

1. Modeling Electron Beam-Specimen Interactions.- 2. SEM Microcharacterization of Semiconductors.- 3. Electron Channeling Contrast in the SEM.- 4. Magnetic Contrast in the SEM.- 5. Computer-Aided Imaging and Interpretation.- 6. Alternative Microanalytical Techniques.- 7. Specimen Coating.- 8. Advances in Specimen Preparation for Biological SEM.- 9. Cryomicroscopy.- References.

SEM Microcharacterization of Semiconductors

The electronics industry is now one of the principal users of the scanning electron microscope, with as many as half of all new SEMs being bought, directly or indirectly, for semiconductor applications. There are several reasons for this popularity. First, the SEM provides a variety of contrast modes which are of great value in qualitatively and quantitatively assessing the properties of semiconductor materials. Second, the SEM offers modes which allow the operation of devices such as switches, transistors, and even complete integrated circuits, to be observed under conditions which approximate those of normal use. As the size of devices has been reduced to the micrometer scale, and as devices themselves become more complex, the fact that the SEM can combine imaging and chemical microanalysis with such facilities as the ability to identify electrically active defects, or measure voltages, makes it in many cases the most versatile tool for characterization, diagnosis, and failure analysis. The major techniques in current use for semiconductor studies are discused below, but other modes of operation including electron channeling and x-ray microanalysis are also of value and the chapters dealing with these topics should be consulted as well.

Mass thickness determination by electron energy loss for quantitative X-ray microanalysis in biology

As is well known, electron energy loss spectroscopy can be used to determine the relative sample thickness in the electron microscope. This paper considers how such measurements can be applied to biological samples in order to obtain the mass thickness for quantitative X‐ray microanalysis. The important quantity in estimating the mass thickness from an unknown sample is the total inelastic cross section per unit mass. Models for the cross section suggest that this quantity is constant to within ±20% for most biological compounds. This is comparable with the approximation made in the continuum method for measuring mass thickness. The linearity of the energy loss technique is established by some measurements on evaporated films and quantitation is demonstrated by measurements on thin calcium standards. A significant advantage of the method is that the energy loss spectrum can be recorded at very low dose, so that mass thickness determination can be made before even the most sensitive samples suffer damage resulting in mass loss. The energy loss measurements avoid the necessity to correct the continuum measurement for stray radiation produced in the vicinity of the sample holder. Unlike the continuum method the energy loss technique requires uniform mass thickness across the probe area, but this is not usually a problem when small probes (<100 nm diameter) are used.

Quantitative X-ray mapping biological cryosections

The potential for applying X-ray mapping to the elemental microanalysis of biological cryosections is discussed. Methods are described for acquiring and processing data, including use of the top-hat digital filter to remove the average effects of the background contribution. Practical considerations for X-ray mapping are discussed in terms of typical counts per pixel and minimum detectability which depends on the number of pixels chosen to integrate the signal. These aspects are illustrated with elemental maps (Na, P, K, Ca and Fe) from freeze-dried cryosections of mouse cerebellar cortex. A calcium sensitivity in the range 0.5 to 2.5 mmol/kg wet weight of tissue is demonstrated. The correction for overlap of potassium K beta and calcium K alpha is demonstrated with X-ray maps from cryosectioned synaptosomes of squid optic lobe. Quantitative results obtained using internal standards to determine wet weight concentrations are in reasonable agreement with expected values. Alternate schemes applicable to X-ray maps for determining the dry mass concentration, such as the peak/continuum (Hall method), are also discussed.

Combined elemental and stem imaging under computer control

Abstract A computer-controlled analytical electron microscope has been used to combine digitally acquired elemental maps and STEM images. Real time processing of the electron energy loss and energy-dispersive X-ray signals allows an accurate subtraction of the spectral background at each pixel. The resulting images reflect the true elemental distribution rather than mass thickness variations in the sample. Acquisition of several signals one pixel at a time allows elemental distributions and morphology to be correlated.

Relative transition probabilities for the x‐ray lines from the K level

The ratio of emission of the electron‐excited Kβ lines to the sum of Kα and Kβ lines was measured for several elements with a lithium‐drifted silicon detector. The measured intensities were corrected for background, line overlap, and absorption within the target and within the detector. The results are compared with those obtained by other investigators.

Electron probe analysis and biochemical characterization of electron-dense granules secreted by Entamoeba histolytica

The interaction of Entamoeba histolytica with collagen induces the intracellular formation and release of electron-dense granules (EDGs) containing collagenase activity which are important in the pathogenicity of this parasite. Purified EDGs contain at least 25 polypeptides with acidic pIs, nine gelatinase activities, small molecules, including inorganic phosphate (Pi), pyrophosphate (PP) and other elements, including Na, Mg, S, Cl, K, Ca and Fe as measured by scanning transmission electron microscopy. Six of these polypeptides with apparent molecular weights of 108, 106, 104, 97, 68 and 59 kDa and two protease activities with apparent molecular weights of 40 and 85 kDa were detected exclusively in the EDGs and were not observed in total trophozoite extracts. Actin was also detected in the EDGs. Therefore, EDGs are a complex of mainly cationic proteins, which contains numerous proteolytic activities, actin and small molecules such as P(i), PP and cations.

On the use of ionization cross sections in analytical electron microscopy

There are two approaches to the utilization of the ionization cross section, Q, for use in the determination of kA B factors for quantitative microanalysis in the analytical electron microscope. The first approach is to interpolate a value of Q from experimentally determined kA B factors at a fixed accelerating voltage (kV). The second approach uses a theoretical parameterization of Q generated by fitting the fundamental Bethe expression to selected experimental values of Q over a wide range of kV. This paper discusses the relative merits of the two approaches.

Quantitative X-Ray Microanalysis

With the EPMA and the SEM one can obtain quantitative analyses of ~1-µ3 regions of bulk samples using a nondestructive x-ray technique. For samples in the form of thin foils and sections of organic material, the size of the analyzed microvolume is reduced to about one tenth of the value for bulk samples. For metals and alloys the ZAF technique is usually employed. Pure element or alloy standards can be used and the surfaces of the samples and standards must be properly prepared and analyzed under identical operating conditions. For geological samples the a factor or empirical technique is usually employed. For this class of samples secondary x-ray fluorescence is usually not significant and oxide standards of similar atomic number as the sample are used. Biological samples are often adversely affected by the impinging electron beam. It is important to ensure that the standards are in the same form and matrix as the specimen. The purpose of this chapter is to describe in some detail the various methods by which quantitative analyses can be obtained for inorganic, metallic, and biological samples in the form of bulk specimens, small particles, thin films, sections, and fractured surfaces.

A sample preparation for quantitative determination of magnesium in individual lymphocytes by electron probe X‐ray microanalysis

We present a sample preparation method for measuring magnesium in individual whole lymphocytes by electron probe X‐ray microanalysis. We use Burkitts lymphoma cells in culture as the test sample and compare X‐ray microanalysis of individual cells with atomic absorption analysis of pooled cell populations. We determine the magnesium peak‐to‐local continuum X‐ray intensity ratio by electron probe X‐ray microanalysis and calculate a mean cell magnesium concentration of 39± 19 mmol/kg dry weight from analysis of 100 cells. We determine a mean cell magnesium concentration of 34 ±4 mmol/kg dry weight by atomic absorption analysis of pooled cells in three cell cultures. The mean cell magnesium concentrations determined by the two methods are not significantly different. We find a 10% coefficient of variation for both methods of analysis and a 30% coefficient of variation in magnesium concentration among individual cells by electron probe X‐ray microanalysis. We wash cells in ammonium nitrate for microanalysis or in buffered saline glucose for atomic absorption analysis. We find cells washed in either solution have the same cell viability (85%), recovery (75%), cell volume (555 μm3) and cytology. We air dry cells on thin film supports and show by magnesium X‐ray mapping that magnesium is within the cells. We conclude that: (a) our microanalysis cell preparation method preserves whole intact lymphocytes; (b) there is no systematic difference in results from the two methods of analysis; (c) electron probe X‐ray microanalysis can determine the variation in magnesium concentration among individual cells.