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We need only to consider our own bodies as an example to realize the complexity of biological material. Our body is three-dimensional and composed primarily of light elements, most of which are organized into a mixture of periodic and aperiodic structures. These structures range from simple molecules and macromolecules to complex heteropolymers, all bathed in an aqueous solution of ions and electrolytes. We are thermodynamically unstable, live at ambient temperatures and pressures, and are sensitive to ionizing radiation.

Chapter 9 presented the procedures for performing quantitative electron probe x-ray microanalysis for the casef an ideal specimen. The ideal specimen surface is flat and highly polished to reduce surface roughness to a negligible level so that electron and x-ray interactions are unaffected by geometric effects. Such a highly polished surface has a short-range surface topography (sampled at distances less than 1 μm) that is reduced to an amplitude of a few nanometers and the long-range topography (sampled at distances greater than 100 μm) that is reduced to 100 nm or less. These ideal specimens satisfy three “zeroth” assumptions that underlie the conventional EPMA technique: 1. The only reason that the x-ray intensities measured on the unknown differ from those measured on the standards is that the compositions of specimen and standard are different. Specifically, no other factors such as surface roughness, size, shape, and thickness, which can be generally grouped together as “geometric” factors, act to affect the intensities measured on the unknown. 2. The specimen is homogeneous over the full extent of the interaction volume excited by the primary electron beam and sampled by the primary and secondary x-rays. Because x-rays of different excitation energies are generated with different distributions within the interaction volume, it is critical that the specimen has a uniform composition over the full region. If a thin surface layer of different composition than the underlying bulk material is present, this discontinuity is not properly considered in the conventional matrix correction analysis procedure. 3. The specimen is stable under the electron beam. That is, the interaction volume is not modified through loss of one or more atomic or molecular species by the electron beam over the time period necessary to collect the x-ray spectrum (EDS) or peak intensities (WDS). Biological and polymer specimens are likely to alter composition under electron bombardment.

This chapter outlines a variety of sample preparation procedures for imaging and x-ray analysis of hard materials in the SEM. Several special and relatively new techniques, such as the use of focused ion beams for preparation of cross sections of various materials, are also discussed.

From the basic description in Chapter 4, the SEM image formation process can be summarized as a geometric mapping of information collected when the beam is sequentially addressed to an x–y pattern of specific locations on the specimen. When we are interested in studying the fine-scale details of a specimen, we must understand the factors that influence SEM image resolution. We can define the limit of resolution as the minimum spacing at which two features of the specimen can be recognized as distinct and separate. Such a definition may seem straightforward, but actually applying it to a real situation becomes complicated because we must consider issues beyond the obvious problem of adjusting the beam diameter to the scale of the features of interest. The visibility of a feature must be established before we can consider any issues concerning the spatial scale. For a feature to be visible above the surrounding general background we must first satisfy the conditions contained within the threshold equation (4.26). For a specified beam current, pixel dwell time, and detector efficiency, the threshold equation defines the threshold contrast, the minimum level of contrast (C = ΔS/S max) that the feature must produce relative to the background to be visible in an image presented to the viewer with appropriate image processing.

Obtaining a low-magnification (

This chapter outlines a variety of sample preparation procedures for performing scanning electron microscopy and x-ray microanalysis of polymer materials. Typical specimen preparation methods will be described and shown in a range of applications that are encountered every day in academic and industrial laboratories. The reader new to the study of polymer materials is directed to Chapter 12 of the Enhancements section on the accompanying CD, which provides background material for this chapter. The topics found on the CD are an introduction to polymer materials, E12.1, and polymer morphology, E12.2, and a description of some typical processes used to produce materials from polymers, E12.3. The book by Sawyer and Grubb (1996) is comprehensive, providing details of specimen preparation methods, dozens of practical examples, and references that describe both the structure and the microscopy of polymers.

Low-temperature specimen preparation is not to be considered solely in the context of hydrated biological systems, although much of what will be discussed here will be directed toward these types of samples. Low temperatures are an essential prerequiste for studying naturally frozen materials such as snow and ice and frozen foods such as ice cream (Fig. 14.1). The technology is central to the preparation of aqueous systems such as paints, suspensions, emulsions, solutions, soils, clays, muds, and cements; of any nonaqueous liquid systems such as oils and organic liquids; and even of gases, vapors, and volatile materials. In addition, as has been shown in Chapter 12, low temperatures may play an important part in the preparation of plastics, polymers, and elastomers. The common physical parameter of these diverse samples is that they all can be solidified, provided the temperature is below their melting point. Once solidified they can then be further manipulated for subsequent examination and analysis in electron beam instruments.

As discussed in Chapters 5 and 6, the x rays emitted from a specimen bombarded with the finely focused electron beam of the scanning electron microscope (SEM) or electron-probe microanalyzer (EPMA) can be used to identify which elements are present (qualitative analysis). With the proper experimental setup and data-reduction procedures, the measured x rays can also be used to quantitatively analyze chemical composition with an accuracy and precision approaching 1%. This chapter provides an overview of the basic principles and techniques used for determining chemical composition, on the micrometer scale, with the SEM and EPMA. Our intention is to provide the conceptual basis for an understanding of the x-ray microanalytical data-reduction procedures that today are almost always incorporated into black-box computer-based, integrated analysis systems with which the analyst interacts as a user. As a user, the analyst depends on the knowledge and skill of programmers to have devised an accurate, robust analytical procedure from the diverse approaches available in the literature. Despite the apparent disconnection which has arisen between the analyst as user and the underlying physics as incorporated into the algorithms of the software, it is nevertheless extremely important to grasp the underlying physical principles to become a sophisticated analyst rather than a mere user.

The specimen can be thought of as an electrical junction into which flows the beam current i B. The phenomena of backscattering of the beam electrons and secondary electron emission represent currents flowing out of the junction, i BSE and i SE, respectively. For a copper target with an incident beam energy of 20 keV, η is approximately 0.3 and δ is approximately 0.1, which accounts for 0.4, or 40%, of the beam current. The remaining beam current must flow from the specimen to ground to avoid the accumulation of charge in the junction (Thevenin’s current theorem). The balance of the currents is then given by $$ \eqalign{ & \sum {{i_{in}}} = \sum {{i_{out}}} \cr & {\rm{ }}{i_B} = {i_{BSE}} + {i_{SE}} + {i_{SC}}, \cr} $$ (15.1) where i SC is the specimen (or absorbed) current. For the example of copper, i SC = 0.6i B. Thus, even with a conducting specimen such as a metal, an electrical connection must be established to conduct this substantial current from the specimen to ground (typically the specimen stage is wellgrounded). Because all materials (except superconductors) have the property of electrical resistivity ρ, the specimen has a resistance R ( R = ρ l / A, where l is the length of the specimen and A is the cross section). The passage of the specimen current i SC through this resistance will cause a potential drop across the specimen, V = i SC R. For a metal, ρ is typically of the order of 10-6 ohm-cm, so that a specimen 1 cm thick with a crosssectional area of 1 cm2 will have a resistance of 10-6 ohm, and a beam current of 1 nA (10-9 A) will cause a negligible potential of about 10-15 V to develop across the specimen.

The electron beam generates x-ray photons in the beam–specimen interaction volume beneath the specimen surface. X-ray photons emerging from the specimen have energies specific to the elements in the specimen; these are the characteristic x-rays that provide the SEM’s analytical capabilities (see Fig. 6.1). Other photons have no relationship to specimen elements and constitute the continuum background of the spectrum. The x-rays we analyze in the SEM usually have energies between 0.1and 20 keV. Our task in this chapter is to understand the physical basis for the features in an x-ray spectrum like that shown in Fig. 6.1.

Valid Conventions throughout the Book. 1. The History of the Electron Biprism. 2. Principles and Theory of Electron Holography. 3. Optical Characteristics of a Holography Electron Microscope. 4. Practical Electron Holography. 5. Quantitative Electron Holography. 6. The Reconstruction of Off-Axis Electron Holograms. 7. Electron Holography of Electromagnetic Fields. 8. On Recording, Processing and Interpretation of Low Magnification Electron Holograms. 9. High Resolution Off-Axis Electron Holography. 10. Off-Axis Stem Holography. 11. Focus Variation Electron Holography. 12. Applications of Electron Holography. 13. Electron Holography using Diffracted Electron Beams (DBH). 14. Electron Holography at Low Energy. 15. A Plus or Minus Sign in the Fourier Transform? Bibliography. Index.

Chemical analysis in the scanning electron microscope and electron microprobe is performed by measuring the energy and intensity distribution of the x-ray signal generated by a focused electron beam. The subject of x-ray production has already been introduced in the chapter on electronbeam-specimen interactions (Chapter 3), which describes the mechanisms for both characteristic and continuum x-ray production. This chapter is concerned with the methods for detecting and measuring these signals as well as converting them into a useful form for qualitative and quantitative analysis.

Nonconducting samples invariably need some sort of treatment before they can be examined and analyzed under optimal conditions in electron-beam instruments that rely on an emitted signal to provide information. The treatment is necessary to eliminate or reduce the electric charge that builds up rapidly in a nonconducting specimen when it is scanned by a beam of high-energy electrons. Figs. 4.63a–c and 4.64a–c show examples of pronounced and minor charging as observed in the SEM. In addition to charging phenomena, which result in image distortion, the primary beam also causes thermal and radiation damage, which can lead to a significant loss of material from the specimen. In many situations the specimen may acquire a sufficiently high charge to decelerate the primary beam, and the specimen may even ultimately act as an electron mirror (see Fig. 4.62).

Success or failure in obtaining comprehensive structural or chemical information from organic, polymeric, biological, and hydrated specimens examined by electron-beam instrumentation depends critically on the way the samples are prepared. This general statement is nowhere more important than in scanning electron microscopy and x-ray microanalysis.

The versatility of scanning electron microscopy and of x-ray micro-analysis is derived in large measure from the rich variety of interactions that the beam electrons undergo in a specimen. These interactions can reveal information on the specimen’s composition, topog­raphy, crystallography, electrical potential, local magnetic field, and other properties. The electron—specimen interactions can be divided into two classes.

Specimen preparation for SEM and EPMA is in many ways still as much of an art as a science. This chapter outlines a variety of procedures used to prepare inorganic materials for SEM and EPMA, and supplemented by laboratory exercises (Lyman et al., 1991), will provide the micros- copist and analyst with the starting point for most specimen-preparation problems for inorganic materials. This chapter specifically addresses the preparation of metals; ceramic and geological materials; sands, soils, and clays; electronic devices and packages; semiconductor materials; and particles and fibers. In addition, specimen preparation for both microstructural and microchemical characterization are discussed.

This chapter will consider the formation and interpretation of SEM images. One of the most surprising aspects of scanning electron microscopy is the apparent ease with which SEM images of three- dimensional objects can be interpreted by any observer, including young children with no prior knowledge of the instrument. This aspect of the SEM is often taken for granted, and yet it is one of the most important reasons for the great utility and wide acceptance of the instrument. SEM images are routinely presented in textbooks and popular scientific articles with little or no mention of the type of microscopy employed in preparing the image or of the complex way in which the image was constructed. It can safely be assumed that the reader will automatically perceive the true nature of the specimen without any instruction on the origin of the image. For this to be true, the SEM imaging process must in some way mimic the natural experience of human observers in visualizing the world around them. Such a situation is somewhat surprising in view of the unusual way in which the image is formed, which seems to differ greatly from normal human experience with images formed by light and viewed by the eye. In the SEM, high-energy electrons are focused into a fine beam, which is scanned across the surface of the specimen.

An overview of the basic principles and techniques used to determine chemical composition, on the micrometer scale, with the SEM and EPMA was presented in Chapter 8. We outlined the approach to quantitation, the need for matrix corrections, and the physical origins of the matrix effects. The x-ray production process and the use of φ(ρz) curves to describe x-ray production were introduced. Finally, we discussed the three major matrix effects, atomic number (Z), absorption (A), and fluorescence (F) and showed, on a conceptual basis, how they are calculated. This chapter presents the more detailed theory and equations which can be used to determine the three major matrix corrections for quantitative analysis of flat polished specimens. The two commonly used correction schemes, the ZAF and φ (ρz) methods, will be described separately although the two schemes are, in many ways, closely related.

As discussed in Chapter 6, qualitative analysis is based on the ability of a spectrometer system to measure characteristic line energies and relate those energies to the presence of specific elements. This process is relatively straightforward if.

The purpose of this laboratory is to prepare samples of metals, ceramics, and geological and microelectronic specimens for examination and analysis in the transmission electron microscope (TEM) and analytical electron microscope (AEM). Material forms include bulk, thin films, and particles. To achieve suitable electron transparency, and meet various requirements for electron diffraction and microanalysis, the thin region of the specimen must be

The SE signal collection efficiency of the conventional E-T detector is limited by the asymmetry of the collection field resulting from the detector position and from the surface potential of rough specimens. Specimen biasing should be routinely applied to optimize signal collection for a given specimen and imaging situation. The voltage supply must be of extreme stability which can be provided by dry batteries such as 45-V farm batteries. Two batteries connected in series provide for an easy change of polarity. There is no rule to predict the effect of specimen biasing on signal collection. The bias modifies only the accelerating voltage of the probe and the collection field of the collector. Thus, SE components will still be collected. (Note: Only a grounded specimen grid which shields the specimen from all other biased surfaces allows establishment of a positive field between the grid and the specimen for the absorption of SE-I+II as described in experiments of Section 12.3.)

This laboratory is designed to give you a feel for the amount of care and time that must go into any measurement involving trace elements. The trace element concentration range is often defined as concentrations below 0.5 wt% for most elements and below 1 wt% for the light elements. The example used here is the measurement of small amounts of phosphorus (P) in FeNi alloys. More details on these techniques may be found in SEMXM, Chapters 7 and 8.

This laboratory demonstrates: (1) the two major types of contrast in SEM images, known as atomic number contrast and topographic contrast, (2) the factors affecting the quality of the image and how they ultimately limit the image resolution, and (3) the effects of electronic signal processing on the visibility of features in the image. More details and references may be found in SEMXM, Chapters 3 and 4.

This laboratory explores some of the principal phenomena observed at low beam energies. Images prepared at “conventional” beam energies, e.g., 15 keV and above, are compared with low-beam-energy images, e.g., 5 keV and below. The possibility of examining uncoated insulators by taking advantage of enhanced emission of secondary electrons at low energy is also examined.

This laboratory demonstrates: (1) election gun saturation and alignment; (2) the measurement of beam current, beam size, and beam convergence; (3) the concept of electron gun brightness; and (4) the effects of these parameters on depth-of-field and resolution. More details and references can be found in SEMXM, Chapter 2.

The purpose of this laboratory is to prepare samples of metallic, ceramic, polymeric, and biological specimens for examination and analysis in the SEM. The organization is such that under each type of material sample preparations are discussed for surface topography (e.g., fracture surface), microstructural analysis (e.g., phase morphology), and x-ray microanalysis. Special procedures for semiconductor devices, polymers, and biological samples are also considered. The objective is to provide a brief outline and enough general references to enable the reader to produce all of the specimens used in this workbook. The outlined methods should not be considered comprehensive, however, and the reader is strongly urged to consult the references listed. For further discussion, see SEMXM, Chapters 9–12.

There are two main objectives of this laboratory: (1) to study the differences observed in EDS and WDS x-ray spectra obtained from particles and rough surfaces as compared to bulk targets, and (2) to test the comparative accuracies of (a) conventional quantitative analysis methods, (b) the peak-to-background method, and (c) the particle ZAF method when applied to particles. More details may be found in SEMXM, Chapter 7.

The purpose of this laboratory is to demonstrate quantitative x-ray microanalysis as practiced on an electron column instrument equipped with an energy-dispersive spectrometer (EDS) and a computer-based multichannel analyzer (MCA). Although the difficult calculations of quantitative x-ray microanalysis are performed automatically in the MCA, the analyst must be aware of the responsibility to select operating conditions that optimize accuracy and precision. As with the WDS, increasing the accelerating voltage will improve the x-ray count rate and peak-to-background ratio and therefore the precision and sensitivity, respectively. However, increasing the beam voltage will also increase the absorption of lower-energy x-ray lines such as aluminum. More details may be found in SEMXM, Chapters 3, 5, and 7.

This first laboratory is designed to acquaint the beginning SEM operator with the steps for taking a micrograph. The steps are described without reference to a particular instrument. Please consult the manufacturer’s operation manual or an instructor before proceeding.

The objective of this laboratory session is to introduce the principal method of obtaining electron diffraction information from regions smaller than the limit of conventional selected area diffraction (SAD), about 0.5 μm in diameter. The technique is termed convergent beam electron diffraction (CBED). We will see that CBED patterns can be used to generate much more information than can be obtained from SAD patterns.

This laboratory is concerned with use of the digital computer to aid in the acquisition, display, and interpretation of various digital images obtained with the scanning electron microscope, e.g., secondary electron, backscattered electron, x-ray, etc. We will be concerned with all aspects of SEM imaging from the generation of the signal, when the electron beam interacts with the specimen surface, to the perception and interpretation of this information in the mind of the observer. Indeed, we will view all steps involved as an information channel which has certain imperfections and nonlinearities that must be examined. This aspect of microscopy is a rapidly developing area and consequently it is difficult to condense all of the important concepts into a single laboratory exercise. Nevertheless, the topics covered in the following experiments should give a sense of the power of this new tool for the microscopist. For more details see ASEMXM, Chapter 5, and references [1–4] at the end of the laboratory.

The light element regime traditionally defined as the elements Be through F (Z = 4 to 9) has posed many difficulties for the analyst. Poor detectability due to low count rates caused by insufficient beam current at low voltage and small spot sizes has made microanalysis very difficult Adequate matrix correction procedures are just being developed and quantification is difficult without very good standards. Contamination of the sample during analysis often makes the results questionable. These problems are slowly but surely being corrected in today’s modern electron microprobes. New WDS crystals and recent ultra-thin-window (UTW) have contributed to improved detectability; modern electron columns have much better low-voltage performance; and finally, today’s SEMs and microprobes are designed with anticontamination devices. However, with all these improvements, the measurement and subsequent quantification of light elements is far from routine. This laboratory will illustrate operational aspects of light element analysis background measurements, peak overlap problems, and quantitation techniques. More details may be found in SEMXM, Chapter 8.

This laboratory demonstrates the operation of a wavelength-dispersive x-ray spectrometer (WDS) fitted to a scanning electron microscope (SEM) or electron probe microanalyzer (EPMA). The WDS has important advantages over the energy-dispersive spectrometer (EDS) in terms of the peak-to-background ratio, improved elemental sensitivity, and better energy resolution of characteristic x-ray peaks to avoid peak overlaps. Comparisons of the major characteristics of the WDS and EDS detectors will be made. More detailed background information may be found in SEMXM, Chapters 5–8.

The aim of this laboratory is to introduce the principles and practice of quantitative x-ray microanalysis in the AEM using an energy-dispersive spectrometer (EDS). Since it is important to recognize the limitations of the technique as well as the relative ease of quantification, some effects due to spurious x-rays in the EDS spectrum will be identified in the first experiment. More details may be found in PAEM, Chapters 4 and 5.

The modern energy-dispersive x-ray spectrometer (EDS) coupled with a computer-based multichannel analyzer (MCA) provides a powerful analytical facility in the SEM lab. The purpose of this laboratory is to introduce the student to the wide range of analytical capabilities of the EDS/MCA system and to illustrate the basic appearance and characteristics of electron-excited x-ray spectra. Procedures for both qualitative and quantitative analysis will be examined. More detail on these subjects can be found in SEMXM, Chapters 6, 7, and 8.