bluuebunny/crossref_metadata_2025 huggingface dataset:
The Dual Nipkow Disk Confocal Microscope and High Speed Confocal Imaging: New Designs, New Cameras, and New Capabilities.
Authors: Peeters G A , Buchin MP
Published: July 2020

The introduction of a dual Nipkow disk scanning head should facilitate confocal microscopic study of live specimens. The use of micro-lenses in this design improves the Z-axis resolution, scan speed and overall optical efficiency of the original Nipkow disk design. Advantages of this device include the capability for high speed scanning (360 scans/sec), real time viewing, superimposed transmitted light and confocal imaging, together with reliable optical alignment, and a compact modular scan head that attaches to the video port of existing microscopes. When used with low light & light amplified cameras, lower power densities can be used to illuminate live specimens resulting in longer experiments being successfully completed.

The schematic diagram shows the optical path and specifications of the new scan head. A pre-aligned collimated light beam illuminates the upper micro-lens disk array. Each lens within the array focuses light through a small pinhole aperture on the lower Nipkow disk providing confocal multi-point scanning of the specimen. Fluorescent light emitted from the specimen passes back through the pinhole apertures to the dichroic mirror located between the two rotating disks. The dichroic mirror reflects the confocal image into the detector path. The confocal image can be observed directly through a 10x ocular or relayed to the camera port. The camera is chosen to achieve the desired combination of speed, dynamic range and spatial resolution. The acquisition, display, and recording aspects of the system are matched to the camera and are readily available. Limitations of the system include a fixed Z-axis resolution determined by the objective lens and pinhole size.
A Confocal Microscope with Spectrophotometry Detection
Authors: Calloway C B
Published: July 2020

The Leica TCS SP Confocal Microscope combines spectrophotometric detection with confocal microcopy. The result is a multi-channel confocal imaging spectrophotometer that significantly increases the flexibility and efficiency of the detection system.

In conventional point scanning confocal microscopes excitation light from laser(s) is delivered to the scan head via fiber optic, passed through a pinhole, reflected by a primary dichroic to the scanning system, and scanned onto the surface of the specimen. Light emitted from the specimen is descanned and passed to the detection system. The detection pinhole(s) are placed either between the primary dichroics and the detection system as in Leica confocal microscopes or closer to the end of the detection system. In the detection system emitted light is separated using a combination of dichroics, mirrors, and barrier filters before being passed to the photomultiplier detectors.

The Leica TCS SP Spectral Confocal microscope has the same beam path as the filtered system up to and including the detection pinhole. The detection system including the secondary dichroics and the barrier filters is replaced by the spectrophotometer detection system (SP). The light emitted from the focal plane that passes through the detection pinhole has both intensity and spectral information. In the SP system this light is passed through a prism. The prism splits the emitted light into a spectrum from 400 to 750 nm. The spectrum is directed at a PMT for detection. The physical dimension of this spectrum is such that the entire spectrum can be imaged onto the window of the PMT.
A New Generation, Fast 3D Fluorescence Microscope using Wavefront Coding Optics
Authors: Cogswell Carol J , Arnison Matthew R , Dowski Edward R , Tuckert Sara C , Thomas Catheyt W
Published: July 2020

We are developing a “new-generation” fluorescence microscope that will allow very fast (milliseconds) acquisition of fully three-dimensional (3D) images for a wide spectrum of biological applications. This new system will overcome the slow image acquisition constraint of existing confocal and widefield deconvolution microscopes (the two most commonly used instruments for 3D fluorescence imaging) that has prevented them from being used for investigations of live-cell dynamics in three dimensions. Our new microscope incorporates the innovative techniques of optical wavefront coding, pioneered by W. T. Cathey and E. R. Dowski, University of Colorado. With this new system, as compared to the normal sequential plane-by-plane image acquisition requirement of confocal and widefield microscopes, we need acquire only a single CCD camera image to obtain an equivalent extended-depth-of-focus (EDF) rendering of a thick specimen, and a minimum of only two images for a 3D stereo view that has full depth.

Our microscope system uses a special-purpose optical element to uniformly “code” the information from all planes throughout the specimen volume onto a single CCD camera image. Specimen-independent digital processing is then used to “decode” this raw image. In effect, the coded raw image is blurred by a special type of aberration which produces an image that is nearly independent of focus. The system then uses a fast, non-iterative, digital filtering algorithm to remove this special blur so that a large volume of the specimen image appears sharply focused all at once.
A Multicolor Femtosecond Lightsource for (Multiphoton) Confocal Fluorescence Microscopy
Authors: Potma Eric , Kahya Nicoletta , de Boeij Wim P , Wiersma Douwe A
Published: July 2020

Recent advances in fluorescence microscopy add to the versatility of this optical technique and intensify its significance as an indispensable tool in biological research. Especially the use of multiphoton excitation offers the microscopist many advantages like inherent optical sectioning, reduced out-of-focus bleaching and higher penetration depths into the sample. In this regard, the commercial availability of pulsed solid-state lightsources like the Ti:Sapphire laser, that provide short pulses needed in the nonlinear multiphoton process, have paved the way for the routine implementation of multiphoton microscopy in the biologists laboratory. Although the spectral range of the commonly used Ti:Sapphire laser allows the application of two-photon fluorescence microscopy on chromophores that absorb in the range of 350-450 nm, a lasersource that enables the two-photon excitation of molecular probes at even shorter wavelengths (<350nm) would be highly beneficial.

In this contribution we present a visible femtosecond optical parametric oscillator (OPO, Figure 1) that is ideally suited to excite molecular species at 285-335 nm by means of a two-photon process. Femtosecond pulses with durations as short as 30 fs can be generated within a tuning range from 570 to 670 nm. A cavity dumper incorporated in the laser cavity provides variable pulse repetition rates (single shot to 82 MHz).
Fluoronanogold as a Probe for High Resolution Correlation Between ImmunoFluorescence and Electron Microscopy
Authors: Takizawa T , Robinson J M
Published: July 2020

[Introduction] Immunocytochemical labeling of cryosections, especially immunofluorescence microscopy using semi-thin (0.5-μm) cryosections, has been a powerful technique for detection of cellular antigens in situ and has been widely employed in cell and molecular biology studies. In many cases, immunofluorescence provides sufficient resolution and sensitivity to answer the question being addressed. However, in certain cases the increased resolution of the electron microscope using ultrathin (90-nm) cryosections may be required to define more precisely the localization of specific molecules. Recently, a unique fluorescent ultrasmall immunogold probe, FluoroNanogold (FNG), has been developed for use as a secondary antibody in immunocytochemical applications. It consists of a Fab' fragment of an antibody to which a 1.4-nm gold particle and fluorochromes are conjugated. FNG permits correlative microscopic observation of a sample stained in a single labeling procedure by multiple optical imaging. Recently, we have shown FNG immunocytochemistry on ultrathin cryosections to be valuable for high-resolution correlation of immunofluorescence and immunoelectron microscopy. In the present study, we have examined the utility of FNG as a secondary antibody for immunolabeling of myeloperoxidase (a marker protein for the azurophillic granules) in ultrathin cryosectioned human neutrophils.

[Materials and Methods] Purified human neutrophils were fixed with paraformaldehyde, embedded in gelatin, infiltrated with sucrose, cut as ultrathin cryosections, and then collected on formvar film-coated nickel EM grids as described previously. Grids containing ultrathin cryosections were incubated with antimyeloperoxidase and then incubated with FNG.
Oxidative Injury by Oxygen and Nitrogen Radicals in Diabetic Retinopathy in the BBZ/WOR Rat: CytoChemical and ImmunoCytochemical Studies
Authors: Ann Ellis E , Grant Maria B , Guberski Dennis L
Published: July 2020

Previous studies have documented NADH oxidase as a source of the oxygen radicals, superoxide (O2) and hydrogen peroxide (H2O2), at sites of disruption of the blood retinal barrier (BRB) in a rat model of noninsulin dependent diabetes (NIDDM). Excess production of nitric oxide (NO) by inducible nitric oxide synthase (iNOS) has been implicated in the complications of diabetes. The effects of NO depend on its microenvironment and one mechanism for its role in complications of diabetes is interaction with oxygen radicals to form the toxic species, peroxynitrite (ONOO−). Since NO is a highly labile molecule, its potential localization is best achieved by immunocytochemical studies of NOS. The activity of iNOS can be detected by localization of nitrotyrosine, a marker indicative of the formation and activity of ONOO−.

Colloidal gold labeled immunocytochemical studies of iNOS and nitrotyrosine were done on sections of neural retina in eyes of obese, NIDDM BBZ/Wor rats in which NADH oxidase had been localized with cerium perhydroxide. Disruption of the BRB was documented by immunocytochemical localization of extravasated endogenous serum albumin. Age matched, nondiabetic control retinas were from eyes from BBDR/Wor rats.
Gold-Based Autometallography
Authors: Hainfeld James F , Powell Richard D , Stein Joshua K , Hacker Gerhard W , Hauser-Kronberger Cornelia , Cheung Annie L M , Schöfer Christian
Published: July 2020

Gold labels such as Nanogold® and colloidal gold are enlarged and visualized in the electron microscope or optically by the selective deposition of silver onto their surfaces. This process, known as autometallography (AMG), silver amplification or silver enhancement, is initiated by exposing the particles to a solution containing silver (I) ions and a reducing agent such as hydroquinone or npropyl gallate. Particles may be enlarged to between 30 and 100 nm in diameter, giving a distinctive black, punctate staining in the light microscope. Nanogold® labeling with silver amplification is one of the most sensitive methods available for histopathology applications such as in situ hybridization. With Catalyzed Reporter Deposition (CARD; also called Tyramide Signal Amplification, or TSA® ; NEN Life Sciences, Boston MA), it has been used to detect as few as 1-2 copies of viral DNA or RNA per cell. However, its uses are restricted by reactions of silver (I) with halides and other elements in tissues. Also, after signal development, self-nucleation and non-specific background deposition begin quickly, which can make end-point selection difficult or prevent incorporation into automated procedures.
Multiple Labeling for EM using Particles of Different Shape and Metal Composition
Authors: Meyer DA , Albrecht RM
Published: July 2020

Multiple labeling for electron microscopy (EM) is typically accomplished by using colloidal gold (cAu) particles of different sizes. The size distribution of each cAu preparation, which may vary by up to 15%, is the principal factor that limits the number of labels which can be used simultaneously. Furthermore, there is no effective way to use multiple labeling for quantitative, high resolution EM studies. Such analysis requires the use of a single Fab antibody fragment conjugated to a single cAu particle in order to ensure that each particle corresponds to only one antigenic site. Whole antibody molecules cannot be used for quantitative analysis because they are at least divalent, and some, such as dimeric IgA or pentameric IgM, have even more antigen-binding sites. Consequently, it is impossible to deduce whether the presence of one whole antibody molecule corresponds to the presence of one, two, or more targets. Particle diameters ranging from 3 to 5nm are optimal for quantitation because one Fab fragment adsorbs to one particle, more than one Fab fragment may adsorb to a single particle larger than 5nm, and, when smaller than 3nm, several particles may bind to a single Fab fragment.

We are evaluating parameters apart from particle size variation to accomplish multiple labeling for both qualitative and quantitative analyses. One method relies on electron energy loss spectroscopy (EELS) to distinguish particles of several metallic compositions, including Au, Ag, Pt, Pd, Rh, and Ru. EELS is performed using a LEO 912 energy filtering transmission electron microscope (EFTEM) with an in-column Omega spectrometer.
Localization of Actin Filaments in the Central Nervous System Using Phalloidin and Correlative Light and Electron Microscopy
Authors: Capani Francisco , Martone Maryann E , Deerinck Thomas J , Ellisman Mark H
Published: July 2020

Determination of the fine organization of actin networks is important for understanding different functional aspects of the central nervous system (CNS) such as plastic events. Early studies investigating the localization of actin filaments at the ultrastructural level relied on structural methods or antibodies raised against actin isoforms. More recently, many investigators have employed the actin-binding peptide phalloidin conjugated to various fluorescent molecules. By conjugating phalloidin to the fluorophore eosin, we have been able to localize f-actin at the electron microscopic level using photooxidation of diaminobenzidine (DAB) by eosin. The goal of this study is to provide a better description of the cellular and subcellular localization of actin filaments at the light and electron microscopy level based on the high binding affinity of phalloidin for actin and utilizing this novel method.

Tissues were obtained from male Sprague Dawley rats that were perfused transcardially under deep anesthesia with normal rat Ringer's solution at 35°C followed by fixative. The fixative contained 4 % paraformaldehyde and different concentrations of glutaraldehyde, ranging from 0.1- 2.5%. Sections of cerebellum, striatum and hippocampus which were cut at a thickness of 50-80 μm with a Vibratome were incubated with phalloidin-eosin in a solution of 0.05% in 0.5% cold water fish gelatin/50mM glycine-PBS (“working buffer”) for 2 hrs. As a control, phalloidin-eosin was omitted for one set of tissues.
Novel Methods in Fluorescence Sensing
Authors: Lakowicz Joseph R , Gryczynski Ignacy , Gryczynski Zygmunt , Tolosa Leah , Rao Govind , Dattelbaum Jonathan , Eichorn Lisa
Published: July 2020

Chemical sensing based on fluorescence is presently undergoing rapid development. As one example, fluorescence lifetime measurements are now being used for measuring cation and anion concentrations in clinical chemistry, cellular imaging and flow cytometry. Additionally, there is an increasing use of proteins as sensors because of their high affinity and specificity for ligands.

In this report we describe some recently developed approaches to fluorescence sensing. These new methods are based on the use of a fluorescence reference either within the sample or external to the sample, but within the optical path. Two types of references are used; microsecond lifetime metalligand complexes (MLCs) or oriented films of ns decay time fluorophores. A protein sensor for glucose was developed using the glucose-galactose binding protein (GGBP) from E.coli (Figure 1).
Analisis of PKA Binding to A-Kinase Anchoring Proteins Using Fluorescence Resonance Energy Transfer
Authors: Ruehr M L , Bond M
Published: July 2020

The cAMP dependent-protein kinase (PKA) signaling pathway plays a key role in the sympathetic regulation of muscle contraction in adult cardiac myocytes. This pathway may be regulated via compartmentalization of its components, whereby A-kinase anchoring proteins (AKAPs) tether PKA to specific subcellular areas to give each receptor binding event specificity. The purpose of this study is to investigate the binding kinetics between Ht31, a peptide containing the PKA binding portion of an AKAP from human thyroid, and the regulatory subunit of PKA (R).

Fluorescence resonance energy transfer (FRET) was used to monitor binding events between the type II regulatory subunit of PKA (RE) and Ht31 or Ht31P, a mutated form of Ht31 which does not bind RII. Each protein was fused to a derivative of the green fluorescent protein (GFP) so that the excitation-emission spectra of the two fluorescent proteins overlap.
A Tutorial of the Fib Lift-Out Technique for TEM Specimen Preparation
Authors: Giannuzzi Lucille A
Published: July 2020

The focused ion beam (FIB) instrument has been developed and exploited by the microelectronics arena for specimen preparation for both scanning and transmission electron microscopy (TEM). The inception [1] and subsequent development [2] of the FIB TEM lift-out (LO) technique has enabled electron transparent membranes of generally uniform thickness to be produced for TEM analysis. The primary advantage of the FIB technique is that site specific cross sections (or plan view sections [3]) may be fabricated quickly and reproducibly. The FIB LO technique has been used extensively in our laboratory for a wide range of materials [4] and biological applications [5] which are summarized in figure 1.

The FIB LO method consists of milling a series of trenches around an area of interest. Then the bulk sample is tilted up to ∼60 degrees to allow the beam to impinge on the lower portion of the specimen surface so that cuts can be made along the bottom edge and the lower 2/3 of the distance up one side of the specimen.
Bridging the Resolution Gap: Correlated 3D Light and Electron Microscopic Analysis of Large Biological Structures
Authors: Martone Maryann E
Published: July 2020

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.
Application of Electron Microprobe Scanning Techniques to Resolve Trace Element Variations in Biogenic Aragonite.
Authors: Haywick Douglas W , Bersch Michael G
Published: July 2020

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 CO3  2− 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.
Microstuctural Characterization of Water-Rich Boehmite (AlO(OH)): TEM Correlation of Apparently Divergent XRD and TGA Results
Authors: Anovitz LM , Allard LF , Porter W D , Coffey D W , Benezeth P , Palmer DA , Wesolowski DJ
Published: July 2020

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.
The Time of Pioneers
Authors: Philibert J
Published: July 2020

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.
Microprobe Design in the 1950’s - Some Examples in Europe
Authors: Duncumb P
Published: July 2020

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.
Advances in WDS Crystal Performance
Authors: Nielsen C H , Kawabe K , Yamada H , Okumura T , Saito M
Published: July 2020

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.
Castaing’s Electron Microprobe and its Impact on Materials Science
Authors: Newbury Dale E
Published: July 2020

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.
Quantitative Electron Probe Microanalysis: Fifty Years of Developments in the Application of Castaing’s “Z” and “A” Corrections
Authors: Armstrong John T
Published: July 2020

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:
Characteristic and Continuum Fluorescence in Electron Beam X-ray Microanalysis
Authors: Nockolds CE
Published: July 2020

Of the different aspects of electron probe microanalysis(EPMA)which were studied by Castaing during his doctorate the work on characteristic x-ray fluorescence was the most definitive. In his thesis, which was completed in 1951, Castaing established the physical and mathematical framework for a correction procedure for fluorescence which is essentially still used in EPMA today. Much of the effort since then has been in refining and improving the accuracy of the correction and extending the scope of the correction to a wider range of specimen types. The Castaing formula was developed for the case of a K x-ray from element A being excited by a K xray from element B (K-K fluorescence) and in 1965 Reed extended the range of the correction by including the K-L, L-L and L-K interactions. In the same paper Reed also introduced the expression from Green and Cosslett for the calculation of K intensities, which was believed to be more accurate than the expression used by Castaing. The original formula included a somewhat unrealistic exponential term to allow for the depth of the production of the primary x-rays and a number of workers have tried replacing this term with a more accurate expression, however, in general this has led to only small changes in the final correction. Reed also simplified the formula in order to make the calculation easier in the days before fast computers; in particular he replaced the jump ratio variable by two constants, one for the K-shell and one for the L-shell. Much later Heinrich showed that this simplification was no longer necessary and that the jump ratio could in fact be calculated directly.
Selecting Standards to Optimize Electron Microprobe Analysis
Authors: Essene EJ , Henderson CE
Published: July 2020

Quantitative analysis with the electron microprobe analyzer (EMPA) has yielded more accurate results over time as a result of improvements in ZAF and other correction routines, mass absorption coefficients, synthetic pseudocrystals for ultralight elements, computers, software programs, backscattered electron (BSE) and energy dispersive (EDS) X-ray detectors. Consequently, many geoscientists view EMPA as routine, and details of procedures, standards, and operating conditions are seldom provided in current publications. However, in overseeing a facility with many users, we have learned that acceptable analytical data are sometimes difficult to obtain even with established analytical procedures and a choice of several hundred standards. After novice users have mastered the routines of sample polishing, cleaning, coating, handling and machine focus, their choice of nonoptimal standards often prevents them from obtaining the most accurate results possible. Optimal analysis for geological problems requires choosing appropriate standards, selection of optimal operating conditions, as well as consideration of the possibility of omitted elements, peak and background overlaps, matrix absorption effects, beam damage and elemental migration, reintegration of heterogeneous materials, fluorescence effects, and variations in the oxidation state of iron.
Minimizing Errors in Electron Microprobe Analysis
Authors: Lifshin Eric
Published: July 2020

Back in the sixties our ability to do both qualitative and quantitative microprobe analysis was often restricted by many factors we now take for granted. Limitations of power supplies, electron optical systems and the mechanical aspects of specimen stages and crystal spectrometers often led to unacceptable drift in both beam position and x-ray intensity measurements. Detector electronics were relatively primitive and phenomena such as pulse distribution shifts with count rate could cause serious errors when pulse height analysis was incorrectly used for noise reduction. The ZAF method, the principal approach for quantitative analysis, was undergoing frequent modification to make it applicable to a broader range of specimens. There were also continuing disputes about which equations to use for a given situation and what experimentally determined constants worked best in the models. Data logging and specimen stage and spectrometer automation were in their infancy and data processing was done on calculators or in batch mode on central computers. Opportunities for errors in data entry abounded.
A Few Examples of Electron Microanalysis of Art Objects at the Boston Museum of Fine Arts
Authors: E. Ogilvie Robert
Published: July 2020

The purpose of this paper is to present a few examples of art objects that have been analyzed with the electron microprobe over the past 40 years at the Boston Museum of Fine Arts. The results of many examinations have led to the fact that the object was a forgery. However, the results of most examinations have proved that the object has the necessary structure and chemistry to be authentic. It should be pointed out, that an object can be proved to be a forgery. But you cannot prove authenticity. The materials used for the object may not fit the time frame for the period, therefore a forgery, or the materials may fit the time period, the structure and style may fit the work of a particular artist or craftsman and still be a copy.

In the early 1960’s a 15th century painting, a Portrait of a Lady, see FIG. 1, was brought to the laboratory at the Boston Museum of Fine Arts for examination.
Electron Probe Microanalysis of Bi-Sr-Ca-Cu Superconductors with Transition Metal Substitutions
Authors: McGee JJ , Obien JM , Wilson RR , Payne JE
Published: July 2020

Electron probe microanalysis (EPMA) is being utilized to characterize the composition of superconductors in the Bi-Sr-Ca-Cu-O system (BSCCO) that are doped with minor amounts of transition metal s. Specifically, the Fe Group (Fe, Ru, Os), the Co Group (Co, Rh, Ir), the Ni Group (Ni, Pd, Pt), the Zn Group (Zn, Cd, Hg), and the other elements in the Cu Group (Ag, Au) can be used as dopants. These first long period ions in their common valence states have similar ionic radii, so only small lattice distortions are expected when other first period ions are substituted for Cu. Our research involves the systematic study of the effect of transition metal substitutions (for Cu) on the properties of the two high temperature superconductors, Bi2Sr2CaCu2O8+δ (2212) and Bi2Sr2Ca2Cu3O10+δ (2223).

Substitution for Cu with transition metal ions enables one to see what effect the changing d and s character of the ions have on the superconducting properties of these compounds.
On the φ(ρz) Curves of Heterogeneous Materials
Authors: Gauvin Raynald , Lifshin Eric
Published: July 2020

Quantitative X-Ray microanalysis is a well established technique for the chemical analysis of homogeneous materials having a flat surface. Precision better than 2% can be routinely obtained if the analysis is performed used the so called ZAF where standards of known composition must be used for all the elements presents in the system under analysis. However, real materials have generally different phases and their composition in a specific phase is not necessary homogeneous. Also, real materials may have surfaces that are not flat and where it is inappropriate to polish them, like a fractured or a corroded surface. It is therefore of paramount importance to develop quantitative schemes for such materials. The first step is to understand the shape of the φ(ρz) curves of heterogeneous materials. This paper present some of these curves that have been obtained with the Monte Carlo program CASINO available for free at www.gme.usherb.ca/casino.
Status Report on Corning Standard Glasses 95IRV, 95IRW, and 95IRX
Authors: Carpenter PK
Published: July 2020

In 1971, Art Chodos and Arden Albee of Caltech contracted Corning Glass Works to produce synthetic glasses containing a number of elements at about 1 weight percent, to be used as electron microprobe trace element standards. Three glasses were produced, 95IRV, 95IRW, and 95IRX (informally known as Corning/Caltech glasses GLV, GLW, and GLX, respectively, in the microanalysis community), which contain the following elements in a Ca-Mg-Al borosilicate glass matrix:

95IRV K Ti Cr Fe Ce Hf

95IRW V Mn Co Cu Cs Ba La Th

95IRX Ni Zn Rb Sr Y Zr Pb U

The source materials for each oxide were weighed-in at 0.80 percent, and each glass was batched in 2 pound lots and drawn into a 0.25 inch cane that was cut into 9 rods of approximately 5 inch length. Initial distribution to end-users was of 1/8 inch slices from the beginning of each cane, as well as sufficient material for chemical analysis to be performed by volunteers. The glass standards were formed in much the same way as the NBS 612, 614, and 616-series trace element glasses, but used a different matrix composition to avoid problems with alkali migration under the electron beam.
Angle-Resolved X-Ray Depth Profiling: Interpretation of Angleresolved Profiles Using a Monte Carlo Approach
Authors: Wilkinson DK , Prutton M , Loveday DA
Published: July 2020

A technique has been developed for the interpretation of composition depth profiles from angleresolved x-ray data using a Monte Carlo electron scattering simulation. Conventional methods for the interpretation of angle-resolved depth profiles used in the fields of x-ray photoelectron spectroscopy (XPS) and Auger electron spectroscopy (AES) assume that the outgoing signal is exponentially attenuated along its path. This assumption if not valid for angle-resolved x-ray techniques, as the x-ray signal is dependent on both the paths of the incident electrons and the path of the emitted x-rays. In this case, while the latter can be treated using an exponential attenuation, the path of the incident beam is more complex and corresponds to the well known “pear-shaped” interaction volume. In order to reliably interpret angle-resolved depth profiles in which the angle of the incident beam is varied, it is necessary to be able to obtain the distribution of x-ray emission within the sample for any angle of incidence.
Relative Variation of K- And L-Shell Ionization Cross Sections by Electron Impact
Authors: Llovet X , Merlet C , Fernández-Varea JM , Salvat F
Published: July 2020

Knowledge of inner-shell ionization cross sections by electron impact is needed for quantitative procedures in electron probe microanalysis (EPMA) and Auger electron spectroscopy (AES) The common practice is to use semi-empirical formulas, based on the asymptotic limit of the Bethe theory, which sometimes are used beyond their domain of validity. Experimental measurements of ionization cross sections are scarce and affected by considerable uncertainties, thus a mere comparison with experimental data does not permit to draw a definite conclusion abou the accuracy of the various formulas. In this communication, we present new measurements o the relative variation of K- and L-shell ionization cross sections deduced from the counting rate of characteristic x-rays emitted by extremely thin films of Cr, Ni, Cu, Te, Au and Bi bombardec by keV electrons.

The studied films were produced by thermal evaporation on backing self-supported 30 nm carbon films.
Do Ultra-Thin Polymer X-Ray Windows Leak Water Vapor?
Authors: Nielson A , Thorne J
Published: July 2020

Ultra-thin polymer x-ray windows have been developed for energy dispersive spectroscopy (EDS) that enable analysis of the elements lighter in mass than sodium while protecting the detector from light and gases. Windowless detectors produce the ultimate in detector sensitivity, however that sensitivity is lost when ice and other contaminants form on the detector. Polymer windows have had a problem with icing in the past, however modern ultra-thin polymer windows contain metalized layers to prevent the diffusion of water. Nevertheless, over the course of time it has been observed that some detectors with polymer windows that are kept continually cool with liquid nitrogen form a thin layer of ice that attenuates light element sensitivity. The source of this water has been hypothesized to be a gradual leak of water vapor through the polymer x-ray window. This hypothesis has been questioned on the basis that the windows were helium leak tight to 1 x 10−9 mbar L/sec and helium is a smaller molecule than water.
Expert System for EPMA
Authors: Fournier C , Merlet C , Staub PF , Dugne O
Published: July 2020

Analysis parameters for an electron microprobe are numerous, and the accuracy of the quantitative analysis is very sensitive to the selection of these experimental conditions. The expert system intends to optimize the choice of each analysis parameter as well as to automate the phases of a quantitative analysis on all kinds of materials. To summarise, the aim of the expert system is to simplify the procedures, improve the accuracy of results and control the analysis time. The figure 1 illustrates the various stages of the expert system.

The starting point of the expert system is an interactive questionnaire concerning the sample,( ie, what is already known), and about the expectations on the analysis,( ie, the accuracy of the results and/or the duration of the analysis required by the operator). Then, the expert system performs a semi-quantitative analysis on the sample. Based on the acquisition of a wavelength qualitative spectrum, this method is a way to obtain the sample composition in a short time with the advantages of the WDS system, and with a reasonable accuracy.