Quantitative Energy-dispersive X-ray Microanalysis Research Articles

Quantitative Energy-Dispersive X-Ray Microanalysis of Chlorine in Cement Paste

AbstractEnergy dispersive X-ray spectroscopy (EDS) is a microanalysis technique for material characterization that can provide accurate quantification of elemental composition while maintaining high spatial resolution. EDS microanalysis studies on cementitious materials can face several challenges essentially due to the complex chemical and mineralogical characteristics of cement hydration products. Furthermore, EDS microanalysis is widely carried out in “standardless analysis” mode, which relies on the internal standards of the microanalysis software. This can lead to highly erroneous analysis results because standardless analysis typically normalizes total to 100% without taking chemically bound water into account. In the case of quantifying chlorine content of a cement paste, such normalization is unacceptable because it can lead to overestimation. For accurate determination of elemental concentrations, X-ray spectra collected from minerals or glasses with known chemical compositions are necessary as r...

Quantitative chemical analysis of ocular melanosomes in stained and non-stained tissues

Energy-filtered Analytical Electron Microscopy (AEM) was used to image the ultrastructure and determine quantitatively the chemical composition of rat melanosomes of the choroid and the Retinal Pigment Epithelium (RPE). For the first time, the effect of staining in elemental analysis of melanosomes was investigated. Detection limits and accuracies of the applied methods were determined.Compared to previous work applying only quantitative Energy Dispersive X-ray microanalysis (EDX) in the TEM (Eibl, O., et al., 2006. Micron 37, 262), here we present a combined quantitative EDX and Electron Energy Loss Spectroscopy (EELS) analysis, including N. This yields the fraction of eumelanin and pheomelanin in melanosomes by the S/N mole fraction ratio. Melanosomes of the sepia ink sac, used as eumelanin standard, showed an S/N mole fraction ratio of <0.004. Thus, they consist primarily of eumelanin as reported by degradation analysis. In contrast, melanosomes of the rats contained mixed melanin with significant amounts of pheomelanin (S/N 0.02) in the RPE and the choroid. Consistent with the previous publication, it was shown that oxygen mole fractions are especially large in melanosomes (7–10at.%) compared to other cell compartments, e.g. 2–4at.% oxygen in the cytoplasm. In the melanosomes of non-stained tissue, the oxygen mole fraction clearly correlated with the Ca mole fraction.EDX spectra used for quantitative analysis had about 15,000 net counts under the oxygen peak, which is necessary to obtain (i) a small statistical error for oxygen and (ii) optimum minimum detectable mole fractions for S, Ca and transition metals. The precise determination of the oxygen mole fraction in melanosomes is important for understanding metabolism. Therefore, a detailed analysis was carried out on the possible errors affecting quantification.While O, S, and N mole fractions yielded similar results in stained and non-stained ocular melanosomes of rats, transition metals can only be determined reliably in non-stained tissues. High-precision EDX analysis of melanosomes yielded minimum detectable mole fractions of less than 0.04at.% for Cu and Zn, these elements were present in melanosomes with mole fractions of about 0.3at.% and 0.1at.%, respectively. Zn is of great importance for metabolism and for age related macular degeneration. Its mole fraction in melanosomes of rats is large enough to be detected and to be quantitatively analyzed by EDX spectroscopy. Ultrastructural information can now be correlated to the elemental composition. This is important to better understand the physical and chemical properties of melanosomal metabolism and turnover.

Understanding the chemical and mineralogical properties of the inorganic portion of MSWI bottom ash

This paper investigates the changes of mineralogical composition of bottom ash in the environment. The chemical and mineralogical bulk composition was determined by X-ray fluorescence (XRF) and X-ray powder diffraction (XRPD) Rietveld method. Single bottom ash particles were investigated by optical microscopy, scanning electron microscopy with quantitative energy-dispersive X-ray microanalysis (SEM/EDX) and electron probe micro analysis (EPMA). SEM/EDX and EPMA are valuable complement to bulk analysis and provide means for rapid and sensitive multi-elemental analysis of ash particles. The fresh bottom ash consists of amorphous (>30 wt.%) and major crystalline phases (>1 wt.%) such as silicates, oxides and carbonates. The mineral assemblage of the fresh bottom ash is clearly unstable and an aging process occurs by reaction towards an equilibrium mineral phase composition in the environmental conditions. The significant decrease of anhydrite and amorphous contents was observed in the aged bottom ash, leading to the formation of ettringite, hydrocalumite and rosenhahnite under atmospheric conditions. In the water-treated sample, the calcite contents increased significantly, but ettringite was altered by the dissolution and precipitation processes in part, to produce gypsum, while the remaining part reacted with chloride to form hydrocalumite. Gypsum and other Ca based minerals may take up substantial amounts of heavy metals and subsequently control leaching behaviour of bottom ash.

Quantitative Energy Dispersive X-ray Microanalysis of Electron Beam-Sensitive Alloyed Nanoparticles

An energy dispersive X-ray spectrometry (EDXS) method is developed to evaluate the composition of alloyed nanoparticles (NPs) where one of the alloying elements is removed under the electron beam during microanalysis with a transmission electron microscope (TEM). The method is demonstrated for alloyed Au-Ag NPs of a diameter ranging from 6 to 20 nm produced by laser evaporation of a water-suspended Ag-Au powder mixture of varying composition. Series of EDXS spectra are recorded for 30 NPs from samples with five different Ag:Au ratios revealing Ag depletion from NPs during electron irradiation. By studying the evolution of NPs composition as a function of dose, the initial Ag content for each NP is extrapolated. The rate of Ag depletion is discussed in terms of sputtering and knock-on damage. On average, approximately one Ag atom is lost from the NP for each Ag L X-ray detected. To assess the limitations of microanalysis in these sensitive nanoscale structures, the concept of detectability limit is adapted to our method. This benchmark is then evaluated for Ag in Au-Ag NPs of various sizes and acquisition times. This study should be regarded as a guide for the design of analytical TEM measurements of beam-sensitive NPs.

Characterization of municipal solid waste incineration (MSWI) bottom ash by scanning electron microscopy and quantitative energy dispersive X-ray microanalysis (SEM/EDX).

Scanning electron microscopy (SEM) with energy-dispersive X-ray microanalysis (EDX) is frequently used for morphological and qualitative chemical characterization of different materials. The applicability of this method for phase identification, is, however, often underestimated. The application of SEM/EDX for the characterization of different phases in fresh and altered municipal-waste incinerator bottom-ash samples with high lateral resolution is presented. Polished thin sections were prepared from the samples, but fresh fracture surfaces were also used. The EDX analyses were performed by using the correction procedures of a conventional standardless ZAF correction, a peak-to-background ZAF correction, and a correction method for light-element analysis. Because of their highly reactive properties the bottom-ash SEM samples require a special method of preparation. The method facilitates nondestructive preparation of the sensitive bottom-ash alteration phases (e.g. cement phases, hydroxides, salts) and their microstructures.

Morphological and Chemical Characterization of Calcium-Hydrate Phases Formed in Alteration Processes of Deposited Municipal Solid Waste Incinerator Bottom Ash

During a study which investigates the exothermal heating of municipal solid waste incinerator bottom ash (MSWI) in a landfill samples were taken at a solid waste incinerator in southern Germany operating at ∼1000 °C. The chemical and mineralogical bulk composition was determined by X-ray fluorescence (XRF) and X-ray diffraction (XRD). Single bottom ash particles were investigated by optical microscopy and scanning electron microscopy with quantitative energy-dispersive X-ray microanalysis (SEM/EDX). The fresh bottom ash consists of ash (42%), melting products (40%), metallic components (8%), usually aluminum, iron, and copper, and residual parts (10%). The main phase of the bottom ash is glass (∼ 40%) with relics (e.g. quartz) and quench phases (e.g. gehlenite). The main crystalline phases are silicates (e.g. gehlenite, augite, diopside, quartz), oxides (e.g. magnetite, spinel, hematite), carbonates (e.g. calcite, metal-carbonates), and salts (e.g. chlorides and sulfides). In the deposited bottom ash endo...

Quantitative Energy-Dispersive X-Ray Microanalysis of Calcium Dynamics in Cell Suspensions during Stimulation on a Subsecond Time Scale: Preparative and Analytical Aspects as Exemplified with Paramecium Cells

We analyzed preparative and analytical aspects of the dynamic localization of Ca2+ during cell stimulation, using a combination of quenched flow and energy-dispersive X-ray microanalysis (EDX). Calcium (or Sr, as a substitute) was retained as fluorides during freeze-substitution, followed by epoxide embedding. The quenched-flow used allowed analyses, during stimulation, in the subsecond time range. Sections of 500 nm were analyzed and no artificial Ca or Sr leakage was recognizable. We calculated a primary beam spread from 63 to 72 nm that roughly indicated the resolution of EDX/structure correlation. These values are quite compatible with the size of potential structures of interest, e.g., Ca stores (∼100-nm thickness) or cilia (∼250-nm diameter). We used widely different standards to calibrate the ratio of CaKα net counts in relation to actual [Ca]. Calibration curves showed a linear relationship and a detection limit of [Ca] = 2 mM, while [Ca] in cytosol was 3 mM and in stores was 43 mM, both in nonactivated cells. Eventually Sr2+ can rapidly be substituted for Ca2+ in the medium before and during stimulation, thus allowing one to determine Me2+ fluxes. With our “model” cell, Paramecium, we showed that, upon stimulation (causing rapid Ca2+ mobilization from subplasmalemmal stores), Ca was immediately exchanged for Sr in stores.

Low Voltage ED-X-ray Microanalysis of Bulk Organic Materials

An earlier investigation had shown that it is possible to perform quantitative energy dispersive x-ray microanalysis at primary beam voltages as low as 5kV on bulk biological material maintained in either the fully frozen hydrated or the dried state. This present study extends the earlier work and provides data which shows that the same type of analysis can be achieved at voltages as low as 3kV on organic samples composed primarily of hydrogen, carbon and oxygen which contain varying amounts of bromine, sulphur and chlorine. Such materials are equally beam sensitive as fully frozen hydrated biological samples and range in density from 1.1 to 2.2kg.m3. The samples fall into three main classes. 1. Chlorinated and/or sulphonated organic polymers. 2. A brominated organic sample. 3. Three experimental brominated plastics kindly supplied by Ms. B.S.Chuah, Melville Laboratory for Polymer Synthesis, Cambridge University. The remaining samples were purchased from Aldrich Chemicals at the highest purity available. The atomic weight percent and density was calculated for each of the eight specimens using the Electron Flight Simulation™ software programme.

Activity-Dependent Calcium Sequestration in Dendrites of Hippocampal Neurons in Brain Slices

Synaptic activity-dependent changes in the spatio-temporal distribution of calcium ions regulate important neuronal functions such as dendritic integration and synaptic plasticity, but the processes that terminate the free Ca2+ transients associated with these changes remain unclear. We have characterized at the electron microscopic level the intracellular compartments involved in buffering free Ca2+ transients in dendritic cytoplasm of CA3 neurons by measuring the larger changes in the concentrations of total Ca that persist for several minutes after neuronal activity. Quantitative energy-dispersive x-ray microanalysis of cryosections from hippocampal slice cultures rapidly frozen 3 min after afferent synaptic activity identified a subset of dendritic endoplasmic reticulum (ER) as a high-capacity Ca2+ buffer. Calcium sequestration by cisterns of this subset of ER was graded, reversible, and dependent on a thapsigargin-sensitive Ca2+-ATPase. Sequestration was so robust that after repetitive high-frequency stimulation the Ca content of responsive ER cisterns increased as much as 20-fold. These results demonstrate that a subpopulation of ER is the major dendritic Ca sequestration compartment in the minutes after neuronal activity.

Versatility of the application of thermal analysis to the simulation of gas desulphurisation

The sulphation process of pre-calcined limestone illustrates the application of programmed thermal analysis techniques using different gas sequences. The direct usage of commercial equipment (e.g. Derivatograph and Mettler thermoanalysers) is limited because the thermobalance unit must be additionally isolated to operate under a corrosive gas atmosphere. The TA methods are capable of predicting the behaviour of the potential sorbents towards SO 2 SO 3 in a larger scale installation. The thermal stability of the compounds from the CaSO system and the influence of additives on sulphation have been found useful in elucidation of the limestone sulphation mechanisms. The thermoanalytical investigations had to be supplemented by independent complementary techniques such as X-ray diffraction, scanning electron microscopy with backscattered electron image followed by quantitative energy dispersive X-ray microanalysis.

High magnesium concentrations in the dense bodies of human blood platelets from patients with atopic dermatitis and chronic myelogenous leukemia

Dense bodies of the normal human blood platelet contain high concentrations of phosphorus and calcium and very small amounts of magnesium, 69 mM/kg dry weight. Quantitative energy dispersive X-ray microanalysis of the platelet dense bodies on fresh air-dried smears of blood from patients with atopic dermatitis and chronic myelogenous leukemia disclosed higher concentrations of magnesium, 251 and 176 mM/kg, respectively, as well as large amounts of phosphorus and calcium. High magnesium content in the dense bodies is discussed in relation to large amounts of magnesium in the dense bodies from the blood of rats, mice and guinea pigs.

Ultrastructural localization and quantification of extracellular calcium binding sites in mouse vibrissa and human scalp follicles.

The purpose of these experiments was to determine if an extracellular calcium binding site gradient is evident in freshly dissected or cultured mouse vibrissa and human scalp follicles and to measure possible drug effects on this gradient. Mouse vibrissae were cultured with or without either minoxidil or pinacidil, and human scalp follicles were cultured with or without epidermal growth factor. Anagen vibrissa and scalp follicles were dissected and placed in culture for 4 h to 4 days, then fixed in a solution containing lanthanum chloride and prepared for either quantitative energy-dispersive X-ray microanalysis (X-ray) or qualitative transmission electron microscopy (TEM). Since lanthanum has a high charge density it displaces Ca2+ ions from anionic binding sites. TEM analysis revealed extensive accumulation of electron-dense lanthanum deposits in the intercellular compartment of differentiating cells in the hair shaft and inner root sheath in the apex of the follicular bulb. Sparse lanthanum precipitate was observed in the intercellular space of the proliferative cells at the base of the bulb. This gradient of lanthanum precipitate was evident in both freshly dissected and cultured vibrissa and scalp hair follicles, irrespective of treatment with drugs that grow hair or epidermal growth factor. X-ray microanalysis indicated that percent by weight of lanthanum was markedly higher in the apex compared to the base of the follicular bulb in vibrissa and scalp follicles. These qualitative and quantitative data demonstrate that an extracellular calcium binding site gradient exists in cultured vibrissa and scalp hair follicles, and that this gradient is not significantly affected by hair growth altering drugs including minoxidil or pinacidil, and epidermal growth factor.

Magnesium and sulfur in mast cell and basophil granules of lower vertebrates in relation with histamine

Mast cell and basophil granules of the vertebrate contain heparin or related sulfated proteoglycans. Histamine is also present in mammalian mast cells and basophils. However, no histamine is detected in mast cell granules of the amphibian or fish, while it is shown in those of reptiles and birds A quantitative x-ray microanalysis of mast cell granules of fresh frozen dried ultrathin sections of the tongue of Wistar rats and tree frogs disclosed high concentrations of sulfur in rat mast cell granules and those of sulfur and magnesium in the tree frog granules. Their concentrations in tree frog mast cell granules were closely correlated (r=0.94).Fresh frozen dried ultrathin sections and fresh air-dried prints of the tree frog tongue and spleen and young red-eared turtle (ca. 6 g) spleen and heart blood were examined by a quantitative energy-dispersive x-ray microanalysis (X-650, Kevex-7000) for the element constituents of the granules of mast cells and basophils. The specimens were observed by transmission electron microscopy (TEM) (80-200 kV) and followed by scanning transmission electron microscopy (STEM) under an analytical electron microscope (X-650) at an acceleration voltage of 40 kV and a specimen current of 0.2 nA. A spot analysis was performed in a STEM mode for 100 s at a specimen current of 2 nA on the mast cell and basophil granules and other areas of the cells. Histamine was examined by the o-phthalaldehyde method.

Evaluation of particle effects in bremsstrahlung and in quantitative energy-dispersive x-ray microanalysis

The magnitude and the spectral form of bremsstrahlung signal contains much information about the target and is significantly influenced by the surface irregularities. By computer modelling, it is possible to extract the information to perform more accurate quantitative x-ray microanalysis for specimens with special geometries. In this research, bremsstrahlung spectra obtained from electron beams striking at selected locations of spherical particles were investigated by Monte Carlo simulations and experimental observations. The effects of surface geometries and the x-ray path lengths in the specimens on the variation of bremsstrahlung spectra and peak-to-background ratios were studied.The Monte Carlo electron trajectory calculations were determined with a screened Rutherford cross-section and a plural scattering model. The continuum x-ray depth profile is computed using Kramer's bremmstrahlung cross-section. The cross-section for inner shell ionization proposed by Green and Cosslett were employed for calculating characteristic x-rays. Bethe stopping power was determined by using the Luo-Joy modification at low energies. The mass absorption coefficient was calculated using Heinrich expression. The detector efficiency was determined experimentally.

Is spectrometer resolution or system count rate performance more important in quantitative energy-dispersive x-ray microanalysis?

Recently, important improvements have been achieved in both the resolution of energy dispersive spectrometers (EDS) and their count rate performance. Figure 1 is a plot of the count rate performance versus resolution for one EDS system. This particular system was chosen for our study because data were available for 6 pulse processing times. It can be seen that there is a direct trade off between count rate performance and energy resolution. The purpose of this study is to examine the consequences of the various pulse processor conditions for quantitative analysis. A number of analytical problems were simulated including various combinations of acquisition times, degree of spectral overlap, and relative peak heights.We provide here one example from the study, the analysis of PbS, which offers a difficult spectral overlap of the Pb M lines and the S K lines. We used the recently available computer program DTSA (Desk Top Spectrum Analyzer), to simulate 1000 spectra for each of the pulse processor resolution conditions shown in Figure 1. A linear least squares procedure was used to determine the peak areas.

分析電子顕微鏡エネルギー分散型Ｘ線定量分析の手法と応用

分析電子顕微鏡エネルギー分散型Ｘ線定量分析の手法と応用

Quantitative energy dispersive X-ray microanalysis of eight elements in pancreatic endocrine and exocrine cells after cryo-fixation

Quantitative X-ray microanalysis of 8 elements was performed on ultrathin, freeze-dried sections of islets and pancreas pieces from non-inbred ob/ob-mice. Diffusion of elements was reduced to a minimum by rapidly freezing the tissue samples between nitrogen-cooled polished copper surfaces and avoiding the use of chemical fixatives and stains. The ultrastructural morphology was adequately maintained to allow measurements on secretory granules, mitochondria, cell nuclei, and cytoplasm free of these organelles. The distribution of the various elements between cellular compartments was similar in islet beta-cells and exocrine pancreas cells. However, the insulin secretory granules were outstanding in exhibiting the highest concentrations of zinc and calcium. In comparison with cytoplasm in the beta-cells, the insulin granules accumulated calcium 2-fold and zinc as much as 40-fold. As no correlation could be made for endoplasmic reticulum in the cytoplasmic measurements areas, the true accumulations above cytosol are likely to be even higher.

Techniques for cryoultramicrotomy of propane jet frozen biological samples

Ultra-rapid freezing followed by cryoultramicrotomy is essential for the preservation of diffusible elements in situ within cells prior to scanning transmission electron microscopy and quantitative energy dispersive x-ray microanalysis. For cells or tissue fragments in suspension and for monolayer cell cultures, propane jet freezing provides cooling rates greater than 30,000°C/sec with regions up to 40μm in thickness free of significant ice crystal formation. While this method of freezing has frequently been applied prior to freeze fracture or freeze substitution, it has not been widely utilized prior to cryoultramicrotomy and subsequent x-ray microanalytical studies. This report describes methods devised in our laboratory for cryosectioning of propane jet frozen kidney proximal tubule suspensions and cultured embryonic chick heart cells, in particular a new technique for mounting frozen suspension specimens for sectioning. The techniques utilize the same specimen supports and sample holders as those used for freeze fracture and freeze substitution and should be generally applicable to any cell suspension or culture preparation.

Standards for quantitative energy dispersive X-ray microanalysis of biological cryosections: validation and application to studies of myocardium.

The preparation of a comprehensive set of gelatin-glycerol-salt standards for quantitation of diffusible ions in ultrathin cryosectioned tissue is described. This set of standards serves two basic functions. The first is to calibrate the X-ray analytical system so that peak-to-continuum ratios obtained from test specimens may be converted to elemental concentrations. The second is to provide a known set of elemental concentrations which can be used to check the performance of the preparative and analytical steps involved in thin section biological X-ray microanalysis. The application of these standards and techniques for cryoultramicrotomy and analysis of myocardial tissue also is described.

Extracellular space and ionic distribution in rat ventricle.

Extracellular space and ionic distribution in rat ventricle.