There are several forms of three-dimensional (3-D) light microscopy but all utilize the principle of optical section recording, i.e. the 3-D image is a sequence of two-dimensional (2-D) images called optical sections. The optical sections are particular focal planes formed within the thick specimen and usually correspond to the conventional image projections recorded in a light microscope, referred to as x,y projections. The optical sections are recorded for a sequence of focus- or z-positions. This “stack” of 2-D images is the data set for the 3-D image. If quantitative analysis is to be performed on the 3-D images, the choice of the z-dimension increment between 2-D images is especially important, and its value may be more or less critical depending on the analysis algorithm used. A reasonable starting value for this dimension is the depth-of-field of the objective lens, but the actual value may have to be smaller to optimize the image analysis or larger to decrease the influence of photobleaching. The most photostable dyes should be selected and the specimen should be mounted in index-of-refraction matching media with an antioxidant.

The image resolution in all three-dimensions is determined by the 3-D point-spread-function (psf), and as a rough rule of thumb the z-resolution is degraded by a factor of 3 relative to the x,y resolution. To achieve or at least approach isotropic resolution the 3-D image can be deconvolved. Figure 1 shows the 2-D maximum value projection of a 3-D image of a cultured glial cell dual labeled for actin and vinculin before and after deconvolution. The actin fibers and vinculin focal contacts are more clearly resolved after deconvolution. Although a single cultured cell might traditionally be considered a thin object, it is really a thick object if the desired spatial resolution is less than the thickness of the cell. It is desirable to image as deep into a thick object as possible to maximize the tissue volume sampled. However, it has been shown that the image signal decreases with depth into the specimen. Figure 2 demonstrates this effect in a 3-D image of the nuclei of the rat hippocampus that have been labeled with the fluorescent Schiffs reagent acriflavine. In the x,y projection, it is not clear why some nuclei are dimmer than others, but the x,z projection shows that the dimmer ones tend to be deeper in the section. It has been shown that this depth dependent signal attenuation follows the form of an exponential function.

This presentation will describe a common core set of widely applicable image analysis techniques for automated quantitative analysis of volumetric microscope image data. Volumetric (as distinct from stereoscopic) three-dimensional (3-D) Microscopy is a rapidly maturing field offering the ability to image thick (compared to the depth of field) specimens using a variety of instrumentation techniques, and producing arrays of brightness values in three spatial dimensions. Also well developed are methods to correct the acquired images for a variety of physical effects including blur and attenuation.

Commonly, what is of interest is the best-possible visualization of thick specimens. The next step, increasingly being considered in view of growing computational resources, and progress in image analysis techniques, seeks to quantify many of the processes and effects being studied. In some mainstream fields, such quantitation is essential. For instance, various assays for substance testing in pharmaceutical and chemical industries involve quantitative end points. As an illustration, the Draize assay for ocular irritancy testing of drugs and biochemical products for human use requires counting of live and dead cells that stain differently. Another example is the mouse lymphoma test that requires a 3-D counting of bacterial colonies. Neurobiological assays require morphometry, as well as quantification of changes in neurons as a function of time and various applied stimuli such as drugs, heat, and radiation. Angiogenesis assays require quantification of changes in vascular morphometry. Computerized image analysis is a powerful tool for extracting quantitative data from 3-D images for statistical analysis.

One class of biological structures that has always presented special difficulties to scientists interested in quantitative analysis is comprised of extended structures that possess fine structural features. Examples of these structures include neuronal spiny dendrites and organelles such as the Golgi apparatus and endoplasmic reticulum. Such structures may extend 10's or even 100's of microns, a size range best visualized with the light microscope, yet possess fine structural detail on the order of nanometers that require the electron microscope to resolve. Quantitative information, such as surface area, volume and the micro-distribution of cellular constituents, is often required for the development of accurate structural models of cells and organelle systems and for assessing and characterizing changes due to experimental manipulation. Performing estimates of such quantities from light microscopic data can result in gross inaccuracies because the contribution to total morphometries of delicate features such as membrane undulations and excrescences can be quite significant. For example, in a recent study by Shoop et al, electron microscopic analysis of cultured chick ciliary ganglion neurons showed that spiny projections from the plasmalemma that were not well resolved in the light microscope effectively doubled the surface area of these neurons.

While the resolution provided by the electron microscope has yet to be matched or replaced by light microscopic methods, one drawback of electron microscopic analysis has always been the relatively small sample size and limited 3D information that can be obtained from samples prepared for conventional transmission electron microscopy. Reconstruction from serial electron micrographs has provided one way to circumvent this latter problem, but remains one of the most technically demanding skills in electron microscopy. Another approach to 3D electron microscopic imaging is high voltage electron microscopy (HVEM). The greater accelerating voltages of HVEM's allows for the use of much thicker specimens than conventional transmission electron microscopes.

Not everyone can afford to own and operate a scanning electron microscope. Everyone can, however, access images and instruments in a variety of ways, depending on the objective. Remote access capability is a result of recent developments in desktop computers, software, and the World Wide Web. Some of these developments and some suggestions for future resource development will be reviewed here.

With the arrival of commercially available, digitally controlled SEMs in the 1980s, it became possible to control microscope operation with a desktop computer and the appropriate software. By connecting a desktop computer to the microscope via the Internet or dedicated data lines such as ATMs, remote control of microscopes became a reality. Although analog microscopes are not as readily controllable over the Internet, their video data stream is easily captured, digitized, and dispatched to remote observers using low-cost consumer video conferencing programs. These approaches enable users at one location to observe their samples in a microscope located elsewhere. However, transfer rates over the Internet fluctuate wildly depending on network traffic, at times making remote access or control almost impossible. Dedicated data lines ensure a relatively stable transfer rate, but at a significantly higher cost.

The kaolinite-mullite reaction series by heating has been controversial since the proposal of a new transformation mechanism. Recently energy-filtered electron diffraction data obtained from the specimen furnace-heated at 920 °C indicated that the spinel-type phase coexisted topotactically with metakaolinite. On the other hand, mullite first appeared from the specimen furnace-heated at 940 °C without showing any crystallographic relationship to the parent metakaolinite.

Direct beam heating experiments of the 920 °C-heated specimens using an energy-filtering TEM supported the previous results and ideas. Fig. 1 displays topotactic coexistence of metakaolinite and the spinel-type phase (spots in hexagonal arrangement) before beam heating. Electron beam heating gave rise to a noticeable change in the diffraction pattern, that is, the formation of mullite rings and enhancement of the diffuse intensity around the direct beam shown in Fig. 2. Comparison of EDS data for the specimen before beam heating and after beam heating (Fig. 3) indicates considerable increase of Si and O peaks by beam heating.

Aragonite (CaCO3) is one of the principal carbonate minerals utilized by metazoans to form shells, tests and other skeletal elements. It may also be inorganically precipitated under certain geological conditions. Aragonite is unstable in meteoric diagenetic environments (relative to its polymorph calcite), and is prone to dissolution and/or replacement by other minerals (usually calcite). The mobilization of Ca2+ and CO32− during aragonite alteration is largely responsible for early diagenetic cementation and the formation of limestone from unconsolidated carbonate sediment. Our study has been investigating the very earliest stages of aragonite alteration. To date, we have limited our work to bivalves of the genera Donax, Chione and Austrovenus collected from, respectively, southern Alabama, southwestern Florida and eastern North Island, New Zealand. Chione and Austrovenus are closely related genera which should permit direct comparison of results. The Florida and New Zealand study locations were chosen because they were near fossil localities containing identical Tertiary-aged specimens that had been subjected to more prolonged alteration. Our results confirm that initial aragonite alteration in meteoric environments is driven by dissolution. Outer surfaces begin to dissolve very rapidly (laboratory experiments suggest that this may occur after only a few months of exposure to meteoric water); leading to a chalky consistency along the outer extremities of the shell. Simultaneous selective leeching of some trace elements (notably Sr2+) also occurs. More prolonged dissolution results in progressive loss of mass and Sr2+ as well as penetration of the chalky “zone” into the interior of shells. Highly altered shells consist almost entirely of chalk.

Atomic Force Microscopy (AFM) imaging of a komatiite specimen from the Morro do Ferro Greenstone Belt, M.G, Brazil, has revealed thin (0.2 - 2.0 micron in width), randomly oriented olivine crystals (fig. 1a,b), of prismatic shape. The length of such crystals is highly variable, ranging from less than 1 μm to more than 10 μm. The randomly oriented crystals typically occur in three or more distinct directions, indicating that the spinifex texture is reproduced at the micron-scale.

In addition, typical skeletal crystals were observed (figure 1c,d) with width of the order of one micron. Figure 1d is the sketch representation of the skeletal crystal shown on figure 1c Figures 1c,d also show that skeletal crystals sometimes branch out thinner crystals, indicating that nucleation also occurs at the surface of previously crystallized minerals, besides generating isolated new crystal seeds.

It is worth noting that the olivine crystals are characterized as low topographic regions on the AFM images. What distinguishes them from polishing scratches is the fact that scratches typically do not mimic crystal shapes, especially skeletal, or fabrics which are readily seen on different scales, i.e., petrographic sections or hand-specimens.

A number of cave systems host colorful deposits of what has been termed “corrosion residue” (CR), material that appears to be the breakdown product of bedrock minerals. The CR may be red, pink, orange, ocher, brown gray, or black and usually occurs in a variety of places within caves (ceilings, walls, tops and sides of boulders, etc.). Geologists have hypothesized that CR is the long-term result of upwelling corrosive air. However, discovery of evidence of microbial activity has led to a complimentary explanation that microbes could be active participants in the production of the corrosion residue. These deposits have been cursorily examined in several caves, including Jewel Cave (SD), Lechuguilla and Spider Caves (NM), and Cueva de Villa Luz (Tabasco, Mexico) using microscopy techniques of SEM and TEM, along with EDS and WDS analysis.

In all cases, the CR is a complex mixture of iron and/or manganese oxides, clays, quartz, and corroded bedrock material. In several cases, rare earth element (REE) phosphate minerals and other unusual minerals have been observed. The REE minerals are believed to result from recrystallization of apatite present in the bedrock; rare earth elements are commonly present in trace amounts within the apatite minerals. X-ray diffraction and bulk chemical analysis has shown that Fe3+ makes up the bulk if not all of the Fe-oxides in the CR while microprobe analysis of thin sections of the bedrock has found Fe2+ in dolomite as high as 700 ppm.

Within much of examined material, evidence of microbial life is present in the form of structures shaped like coccoid or filamentous bacteria. There is often a close association of presumptive bacteria with small dissolution pits in corroded fragments of bedrock minerals in the CR (Figure 1). In addition to the putative bacteria, the mineral residue often hosts small star-shaped minerals containing Fe-oxide (Figure 2).

In the early days of geochronology, attempts were made to date U-Th bearing rocks and minerals using the Pb-U-Th chemical method, but fundamental assumptions that all the lead was of radiogenic origin, and the system was closed, often were invalid. With the advent of the U-Th-Pb isotopic and common-lead methods of dating rocks and minerals, the chemical method was deemed clinically dead. However, several workers, for example, Montel et al., Suzuki and Adachi, have recently demonstrated new life for the chemical method. Using, the same theoretical basis as the chemical method, geologically reasonable dates often can be obtained by electron microprobe analysis of U-Th rich monazite (Ca-REE-U-Th phosphate). Other U- or Thrich minerals sometimes can be used to obtain microprobe U-Th-Pb dates, e.g., zircon, xenotime, uraninite.

Microprobe U-Th-Pb dates of uraninites from the McAllister Ta-Sn pegmatite, Coosa County, Alabama, are reported here. The McAllister pegmatite is a NW-SE dike-like body in the western part of the Rockford granite pluton. Polished grain mounts were made from screened table runs of heavy mineral separates. Wodginite (Mn, Sn, Nb, Ta-oxide), tantalite-columbite, zircon, cassiterite comprise most of the grains in the samples. Uraninite occurs as inclusions, generally <0.01 mm, in these grains (Figure 1).

U, Th, Pb concentrations were measured using a JEOL 8600 Superprobe. Operating conditions were 100 nA, 20 kV, and count times up to 200 seconds for peak plus background. The U Mβ, Th Mα, and Pb Mα peaks were measured. Standards were UO2, ThO2, and PbS.

An understanding of the solid-phase thermodynamics and aqueous speciation of aluminum is critical to our ability to understand and predict processes in a wide variety of geologic and industrial settings. Boehmite (AlO(OH)) is an important phase in the system Al2O3-H2O that has been the subject of a number of structural and thermodynamic studies since its initial synthesis [1] and discovery in nature [2]. Unfortunately, it has long been recognized that thermogravimetric analysis (TGA) of both synthetic and natural boehmite samples (that appear well crystallized by powder XRD methods) yields significant excess water - typically losing 16-16.5 wt. % on heating as compared with a nominal expected weight loss of 15.0 wt. % [3,4]. The boehmite used in our experiments was synthesized hydrothermally from acid-washed gibbsite (Al(OH)3) at 200°C. Powder XRD and SEM examination showed no evidence of the presence a contaminant phase. The TGA patterns do not suggest that this is due to adsorbed water, so a structural source is likely. We therefore undertook to examine this material by TEM to clarify this phenomenon.

Boehmite is orthorhombic (a = 0.0285nm, b = 0.1224nm and c = 0.0365nm, Amam). The crystals were tabular with major surfaces normal to <010>. Simple powder dispersals onto holey carbon films typically resulted in b-axis orientations parallel to the electron beam. To view other orientations, specimens were mulled in M-Bond epoxy, pressed between plates of single-crystal Si (aligning the boehmite tablets parallel to the Si plates) while the epoxy cured. Electron transparent thin foils normal to the silicon plates were produced by argon ion milling techniques. Sample stability in the electron beam was dramatically improved by cooling to −140°C using an 2 cold stage.

This paper will cover the history of the first steps of electron probe microanalysis. I was not personally involved with the electron microprobe at the very beginning, although I have been fully involved in the field from 1955. Nevertheless I have interesting information about the heroic times - when, just after the World War, Raimond Castaing was preparing for his doctoral degree.

In January 1947, R. Castaing, graduate in physics, joined the Institute for Aeronautical Research (ONERA), recently created by the French government. In the Materials Science Department, he was very lucky to be endowed with two electron microscopes, very exceptional equipment - a real luxury in 1948. The first one, an American RCA instrument was devoted to metallurgical studies. The second one, build by a French company, was cannibalized by Castaing to produce a fine electron beam to perform point chemical analysis. The idea was suggested to him by Andre Guinier, a famous x-ray crystallographer (Guinier-Preston zones, discovered in 1939) as the best way to solve identification problems in metallography. Castaing was not so enthusiastic about this proposal; the story seemed to him too simple, so simple, he thought, that it was quite surprising that nobody had done it before; but he soon had to face many physical problems that made him understand why many people probably failed, especially lens aberrations that had to be corrected to produce the intense, fine beam of electrons. Another question soon arose about x-ray detection. It was easy to detect the continuous background, but not very informative. Finally Guinier lent Castaing one of his precious quartz crystals (specially cut and ground, of the Johanson type); it was adjusted on a small spectrograph that fitted the outside of the main column (safety rules were in their infancy!). In the first days of 1949, he was able to measure characteristic x-ray emission from a l-μm, 4 nA electron probe. Considering the poor conditions in France after the terrible war, the rapidity with which this result was obtained is quite remarkable.

The early days of the electron microprobe were characterized by the variety of designs emerging from different laboratories in Europe, the United States and the USSR. Notable amongst these was that of Castaing in 1954, which employed a magnetic lens in combination with an optical microscope for viewing the sample and positioning the electron probe on the desired point for analysis. The X-ray emission was analysed by two high resolution spectrometers having their axes in the same plane as the electron-optical axis, and with their foci accurately set to coincide with the point of impact of the electron probe. This was a design well suited to point analysis by high resolution X-ray spectroscopy and formed the basis of the first Cameca instrument (Fig 1a).

By contrast, work by Duncumb in the Cavendish Laboratory in Cambridge started with the object of scanning the electron probe over the sample, in order to image the surface in terms of its characteristic X-ray emission. This required a strong lens to give a high current into a finely focused electron probe (Fig. 1b). The first element maps were demonstrated in 1956, and led to the design of Cambridge Instruments’ Microscan, intended as a metallurgical instrument, in conjunction with D.A. Melford of Tube Investments Research Laboratories.

Meanwhile, Long, also in Cambridge, was pioneering applications to mineralogy, and built an instrument for studying conventional slide-mounted rock samples, which could be viewed optically while analysis was in progress (Fig. 1c). This made use of a weaker probe-forming lens, with space for an inclined sample to be viewed in transmitted light. The slim design of the lens allowed it to be partially enclosed in the spectrometer, which received X-rays leaving the sample at a high angle to the surface - a benefit carried through into the Cambridge Geoscan.

The main initial source of information in the USA concerning the electron probe was a meeting at the Naval Research Laboratories in February 1958, followed in 1960 by a summer school at MIT organized by Prof. Norton and R. Ogilvie. Further visits to the USA and publications in English by the inventor of the instrument, R. Castaing, also contributed to the attention given here to the new technique. Since numerous research laboratories in the USA were keenly interested in the new device, the development of instruments and the contributions to its use were considerable. The relatively small number of active researchers in the field rendered personal connections and cooperation possible and attractive.

Before the availability of American-made commercial microprobes, many investigators built their own instruments. Most of them were not trained as instrument builders, and the usefulness of the resulting devices was somewhat limited. These improvisations in instrument construction ceased once commercial microprobe manufacturers entered the field. Yet, in the process, much was learned and the American investigators contributed significantly to the theory of microanalysis. We realized that, with the limited calculation facilities then available, the complex interactions leading to the x-ray signal had to be treated in simplified ways, and we described ‘approximations’ rather than ‘laws’ in our efforts to understand and quantitatively describe the physical facts. We also came to realize that even where the physical processes were well known, we often lacked the knowledge of the physical parameters required for quantitation and the effect of their uncertainties on the accuracy of the analytical results. Another area in which advances were needed, and soon achieved, was the application of the technique to a wide range of technical and scientific problems. The interaction between manufacturers and users greatly stimulated improvements in the instrument design.

In May of 1955 Professor Shinoda introduced the development of the electron probe microanalyzer (EPMA) in a review article published by Oyobutsuri (the Applied Physics journal written in Japanese), which showed a schematic diagram of an EPMA system. That paper, published in the most popular journal in Japan, was very probably the first paper which drew the attention of the people involved in the field of material science, in particular, the field of metallurgy, who were especially interested in EPMA. That paper stimulated the development of EPMA in Japan.

In 1957, the funding provided by the Grant-in-Aid for Developmental Research to the university research groups headed by Profs. Akutagawa, Gokyuu, and Abe at the University of Tokyo was used, in a cooperation with the Hitachi group headed by Drs. Tadano and Watanabe, to develop the EPMA in Japan. Their research results led to the funding of a 2 year Grant-in-Aid for Cooperative Research project (April 1960 to March 1962) called “The Study of X-ray Microanalyzer”, which was chaired by Prof. Sakaki and consisted of 37 members from various universities, national institutes and industries. Prof. Shinoda, who became the Chairman of that project in April of 1962, led that group for another year until March of 1963.

It was just after the start of the Cooperative Research project, in September of 1960, that Professor Castaing visited Japan and gave several talks as a representative of the French Mission Culturelle. Professor Castaing gave a talk at the Hitachi Central Research Laboratory on the 19th of September, another talk at the Nagoya University on the 24th of September, and also a talk at Osaka University on the 28th of September. During his stay in Japan from September 18th to October 4th he talked about his work on EPMA a total of six times by also giving two informal presentations. The visit of Professor Castaing gave a great push forward for the commercial development of EPMA instruments in Japan.

An x-ray spectrometer is actually a scanning monochromator because two conditions must be satisfied, namely: (1) nλ, = 2d sinθ and (2) θI{= θR to within the rocking curve of the crystal 2Δθ. Before Castaing (B.C.) there were four known types of focussing monochromators, namely: Johann, Johansson, Cauchois, and von Hamos. Castaing was fortunate in having Andre Guinier (well known for his work in x-ray crystallography) as his thesis advisor. Thus, he wisely chose to use a spectrometer of the Johansson type. Had he used a flat crystal spectrometer, the result would have been unsatisfactory and the history of microanalysis would have been significantly different. Many of the spectrometers that were used in the early instruments were either of the Johansson type, in which the crystal planes are curved to a radius of 2R and the surface has a radius of R, the focal circle radius. This is sometimes called exact focussing because rays in the plane of the focal circle converge exactly to a point focus. For simpler construction, the Johann crystal which has the surface parallel to the atomic planes (also curved to a radius of 2R) was sometimes used. While both types provide one-dimensional focussing, the Johansson type is preferred because the effective width of the crystal can be greater.

In addition to the type of geometry used, early instruments developed after Castaing (A.C) also had variations in the mechanism of the crystal movement. Many used a relatively simple mechanism with the crystal and detector moving about a fixed center for the focal circle as shown in Fig. 1A. Some later instruments used curved crystals that were fixed in position, and scanning was accomplished either by rotating the crystal as in Fig. 1B or by a mechanism that flexed the crystal and changed the radius of the focal circle as in Fig. 1C. The most satisfactory arrangement and the one subsequently adopted by most manufacturers of commercial microprobes utilized a linear motion of the crystal carriage with a provision for tilting the crystal so that the focal circle always passed though the x-ray source as shown in Fig. 1D. If the detector moves so that it remains on this focal circle, its motion is along a leaf-shaped curve. With this design it is easy to have a readout proportional to wave length.

EPMA... .it all starts with Bragg’s Law. It’s all about crystals. WDS crystals are really the rate-limiting step to the usefulness of the EPMA technique. As such they represent the focal point of as much research as any other component of the instrument. There is an inherent need to improve the performance of crystals to assure the survival of the technique. As electron columns permit smaller and smaller spot sizes and as the need to analyze smaller and smaller things increases the search for improved crystals intensifies.

There are generally two crystal parameters that beg for improvement: 1) Energy Resolution and 2) Intensity (especially for light elements). The resolution of crystals is considered adequate for most applications but it’s always useful to have more resolution to better deal with overlapping lines. It’s also becoming useful to measure valence states by observing relative shifts in peak positions. Intensity is the thing that one never gets enough of. It seems that more and more applications demand more intensity. Trace element analysis, particle analysis, heat sensitive sample analysis, light element analysis all scream for more intensity.

The Sepia years. 1951. Castaing’s thesis(1) : Genesis of a Non-Destructive and truly Quantitative Microanalysis method. 1958. The beginning of commercial microprobes : Early instruments had no computers and were lacking special analyzing crystals, but overall they were well designed. Modern features we are familiar with today, such as light element analysis, field emission gun, Energy Dispersive analysis, analysis of insulated material and scanning analysis, although not widely implemented, were discussed in scientific reviews even then(2) 1963. One of my early personal experience: The daring job of obtaining Castaing’s acceptance for his Cameca-buih EPMA at the University of Paris/Orsay.

From early models to present microprobes Early microprobes were developed, after WWII, in a world driven by metallurgy. They had few WD spectrometers, usually at low take off angle. However, the need for light element analysis and the fast growing use of EPMA in geology have sent the manufacturers back to the drawing board. Why ? The design of modern microprobes was a compromise between light optics, electron optics and higher take off angle X-ray spectrometry. There were three possible designs of X-ray path geometry : X-rays through the(final) lens, X-rays through the gap of the lens, X-rays outside the lens, hence three suppliers arose based on these concepts.

Present and future EPMA improvements. As in the initial era of EPMA newer applications will point the direction in which the electron microprobe of the future should evolve.

Early Development Automation of electron probe analysis began to flourish in the early 1970s spurred on by advances in computer technology and the availability of operating systems and programming languages that the individual researcher could afford to dedicate to a single instrument. By the end of the decade, most researchers and vendors in the microanalysis field had adopted the PDP-11 minicomputer, and languages such as FOCAL, FORTRAN and BASIC that ran on these computers. A good summary of these early efforts was given by Hatfield. The first use of the energy dispersive detector on the electron probe in 1968 added the need to control the acquisition, display and processing of EDS spectra. As a result, the 70’s were also a time when much attention was focussed on development of software for on-line data reduction and analysis. These efforts produced a suite of programs to provide matrix corrections and spectral processing, and automation of WDS data collection. The culmination of these development efforts was first reported in 1977 with the analysis of a lunar whitlockite mineral by simultaneous EDS/WDS measurement. This analysis determined the concentration of 23 elements, 8 by EDS and took a total of 37 minutes for data collection and analysis. In this paper, the authors noted the complementary use of the EDS and WDS (WDS for trace elements and severe peak overlaps, EDS for other elements and rapid qualitative analysis) in their automated instrument, a convention that remains common on the electron probe even today. Toward the end of the decade the analytical accuracy and precision achieved by automated analysis of bulk samples approached the limits of the instrumentation, with the exception of analysis of light element concentrations.

Two Decades of Improvements The explosive growth in digital electronics and microprocessors for data processing and control functions during the 80’s was rapidly applied to electron probe automation. Second and third generation automation systems included direct control of many microscope functions, beam position and imaging conditions. Motor positioning was more precise and far faster. As a result, the data collection and analysis of 23 elements reported in 1977 could be accomplished at least three times faster on a modern instrument.

A central theme of modern materials science has been the exploration of the relationship between the microstructure of a material and its macroscopic properties. Beginning in the late 19th century, the developing field of metallography permitted scientists to view the microstructure of metal alloys. Mechanical polishing followed by selective chemical etching produced differential relief on chemically distinct phases or at grain boundaries. With such specimens, reflection optical microscopy revealed structures with micrometer and even finer dimensions. The microstructural world that was found proved to be highly complex, and most alloys were observed to be chemically differentiated into two or more distinct phases. The answer to many materials science questions required knowledge of the specific composition of such fine scale phases. Castaing’s research was motivated by these considerations, as evidenced by the title of his first paper, “Application of electron probes to metallographic analysis”. The subsequent impact of Castaing’s electron probe microanalyzer (EPMA) has occurred across a broad range of the physical and biological sciences. Materials science has been one of the most active areas and chief beneficiaries, as well as a source of researchers who not only employed electron beam microanalysis to solve their problems but who also contributed innovations that advanced the microprobe field. For example, the abstracts of the First National Conference on Electron Probe Microanalysis contain numerous examples of advanced applications of the electron microprobe to materials science, including analysis of (1) refractory metal coatings (P. Lubllin and W. Sutkowski), (2) diffusion in the Ti-Nb system (D. Nagel and L. Birks), (3) Au-Al alloys (C. Nealey), (4) various steels (H. Nikkei), (5) Al-V-Mo-Ti alloys (R. Olsen), (6) corrosion of Ni-Co alloy (C. Spengler and R. Stickler), and (7) analysis of metal oxides and carbides (T. Ziebold). Integrated over 50 years, the impact of electron probe microanalysis on materials science has been so broad and varied, especially in its continued development and incorporation within scanning electron microscopy and analytical electron microscopy, that a suitably comprehensive review could consume the entire presentation time available for all topics to be covered in this conference! Instead, selected examples of critical applications will be presented to illustrate the impact in materials science.

Castaing, in his remarkable PhD thesis on the electron microprobe, not only detailed the construction of the prototype of what was to be the first commercial electron microprobe and demonstrated its application as a working tool in metallurgy, geochemistry and other materials sciences. In, addition, he also outlined the physical factors involved in the x-ray emission process and developed the general scheme of data correction for quantitative analysis that is still in use today.

In his thesis, Castaing outlined the nature of the processes involved in electron inelastic scattering leading to inner shell ionization and x-ray production (the so-called “stopping power” correction) and the loss of generated x-ray intensity due to elastic scattering of energetic electrons resulting in their emission from the sample (the so-called “backscatter” correction). He showed that the combination of these factors resulted in a correction primarily dependent upon the sample’s average atomic number (the so-called “atomic number” or “Z” correction), which for a thin specimen or one with minimal x-ray absorption and fluorescence would suffice in relating relative intensity to relative concentration. He noted that, in what is known as “Castaing’s first approximation”, the ratio of emitted x-ray intensity of a given line from element “A” in a specimen to that in a standard, KA = IA,spec/IA,std, can be related functionally to the mass concentration of the analyzed element: