Quantitative Electron Probe X-ray Microanalysis Research Articles

Evaluation of the viability of cultured corneal endothelial cells by quantitative electron probe X‐ray microanalysis

Construction of artificial organs and tissues by tissue engineering is strongly dependent on the availability of viable cells. For that reason, the viability and the physiological status of cells kept in culture must be evaluated before the cells can be used for clinical purposes. In this work, we determined the viability of isolated rabbit corneal endothelial cells by trypan blue staining and quantitative electron probe X-ray microanalysis. Our results showed that the ionic content of potassium in cultured corneal endothelial cells tended to rise initially, but significantly decreased in cells in the fifth (and final) subculture, especially in comparison to cells in the fourth subculture (P < 0.001). However, the concentration of sulfur was higher in the fifth subculture than in the fourth subculture (P < 0.001), with a nonsignificant increase in sodium in the fifth subculture (P = 0.031). These data imply a remarkable decrease in the K/Na ratio from the fourth to the fifth subculture. Our microanalytical results, along with the morphological differences between cells in the last two subcultures, are compatible with an early phase of the preapoptotic process in the fifth subculture, and suggest that cells of the first four subcultures would be better candidates for tissue engineering.

Quantitative energy-dispersive electron probe X-ray microanalysis of individual particles

An electron probe X-ray microanalysis (EPMA) technique using an energy-dispersive X-ray detector with an ultrathin window, designated low-Z particle EPM, has been developed. The low-Z particle EPMA allows the quantitative determination of concentrations of low-Z elements, such as C, N, and O, as well as higher-Z elements that can be analyzed by conventional energy-dispersive EPMA. The quantitative determination of low-Z elements (using full Monte Carlo simulations, from the electron impact to the X-ray detection) in individual environmental particles has improved the applicability of single-particle analysis, especially in atmospheric environmental aerosol research; many environmentally important atmospheric particles, e.g. sulfates, nitrates, ammonium, and carbonaceous particles, contain low-Z elements. The low-Z particle EPMA was applied to characterize loess soil particle samples of which the chemical compositions are well defined by the use of various bulk analytical methods. Chemical compositions of the loess samples obtained from the low-Z particle EPMA turn out to be close to those from bulk analyses. In addition, it is demonstrated that the technique can also be used to assess the heterogeneity of individual particles.

F42 Quantitative Energy-Dispersive Electron Probe X-ray Microanalysis of Individual Particles—Invited

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Changes in intracellular sodium, chlorine, and potassium concentrations in staurosporine‐induced apoptosis

Ion gradients across the plasma membrane, fundamentally K(+), play a pivotal role in the execution phase of apoptosis. However, little is known about other monovalent anions (Cl(-)) or cations (Na(+)) in apoptosis. In addition, the relationship between changes in total ion composition and morphological and biochemical events are poorly understood. We investigated simultaneous changes in sodium (Na), chlorine (Cl), and potassium (K) concentrations in stauroporine-induced apoptosis by quantitative electron probe X-ray microanalysis (EPXMA) in single cells. Apoptotic cells identified unequivocally from the presence of chromatin condensation in backscattered electron images were characterized by an increase in intracellular Na, a decrease in intracellular Cl and K concentrations, and a decrease in K/Na ratio. The ouabain-sensitive Rb-uptake assay demonstrated a net decrease in Na(+)/K(+)-ATPase activity, suggesting that increases in Na and decreases in K and the K/Na ratio in apoptotic cells were related with inhibition of the Na(+)/K(+)-ATPase pump. These changes in diffusible elements were associated with externalization of phosphatidyl serine and oligonucleosomal fragmentation of DNA. This alteration in ion homeostasis and morphological hallmarks of apoptosis occur in cells that have lost their inner mitochondrial transmembrane potential and before the plasma membrane becomes permeable.

Capability and Uncertainty of Standardless Procedures for Quantitative Electron Probe X-ray Microanalysis

Capability and Uncertainty of Standardless Procedures for Quantitative Electron Probe X-ray Microanalysis

Barriers to Quantitative Electron Probe X-Ray Microanalysis for Low Voltage Scanning Electron Microscopy.

Low voltage x-ray microanalysis, defined as being performed with an incident beam energy ≤5 keV, can achieve spatial resolution, laterally and in depth, of 100 nm or less, depending on the exact selection of beam energy and the composition of the target. The shallow depth of beam penetration, with the consequent short path length for x-ray absorption, and the low overvoltage, the ratio of beam energy to the critical ionization energy, both contribute to minimizing the matrix effects in quantitative x-ray microanalysis when the unknown is compared to pure element standards. The low beam energy restricts the energy of the atomic shells that can be excited, forcing the analyst to choose unfamiliar shells/characteristic peaks. The low photon energy shells are subject to low fluorescence yield, so that the peak-to-continuum background is reduced, severely limiting detectability. The limited resolution of semiconductor energy dispersive spectrometry results in frequent peak interference situations and further exacerbates detection limits. Future improvements to the x-ray spectrometry limitations are possible with x-ray optics-augmented wavelength dispersive spectrometry and microcalorimeter energy dispersive spectrometry.

Biomass Burning in Southern Africa: Individual Particle Characterization of Atmospheric Aerosols and Savanna Fire Samples

Ambient atmospheric aerosols and savanna fireparticulate emission samples from southern Africa werecharacterised in terms of particle classes and theirnumber abundance by electron probe X-ray microanalysis(EPXMA). About ten particle classes were identifiedfor each sample. The major classes werealuminosilicates and sea salts for ambient coarse(2–10 μm equivalent aerodynamic diameter (EAD))samples, and K-S and S-only particles for ambient fine(<2 μm EAD) samples. The K-S particles are oneof the major products of biomass burning. The EPXMAresults were found to be consistent with the resultsfrom bulk analyses on a sample by sample basis. Forsavanna fire fine samples, quantitative EPXMA revealedthat many particles had a composition of simple saltssuch as KCl. Some particles had a deviatingcomposition in the sense that more ionic species wereinvolved in sustaining the balance between cations andanions, and they were composite or mixed salts.Because of extensive processing during the atmospherictransport, the composition of the K-S particles in theambient samples was different from K2SO4,and such particles were enriched with S. The finepyrogenic KCl particles and the fine sea-saltparticles were much depleted in chlorine.

Assessment of homogeneity of candidate reference material at the nanogram level and investigation on representativeness of single particle analysis using electron probe X-ray microanalysis

Particulate samples of a candidate reference material are evaluated on their homogeneity from bottle to bottle using electron probe X-ray microanalysis technique. The evaluation on the homogeneity is done by the utilization of the Kolmogorov–Smirnov statistics to the processing of the quantitative electron probe X-ray microanalysis data. Due to a limitation, existing even in computer controlled electron probe X-ray microanalysis, in terms of analysis time and expenses, the number of particles analyzed is much smaller compared to that in the sample. Therefore, it is investigated whether this technique provides representative analysis results for the characteristics of the sample, even though a very small portion of the sample is really analyzed. Furthermore, the required number of particles for the analysis to insure a certain level of reproducibility, e.g. 5% relative standard deviation, is determined by the application of the Ingamells sampling theory.

Electron probeX-ray microanalysis for the assessment of homogeneity of candidate reference materials at the nanogram level

A new approach to the assessment of homogeneity for powder samples of candidate reference materials with the help of electron probeX-ray micro-analysis (EPMA) is proposed. It is based on the utilisation of the Kolmogorov—Smirnov statistics coupled with the Akaike Information Criterion in the processing of the quantitative EPMA data. The evaluation of three IAEA candidate reference materials with the described approach is discussed.

X-Ray microanalysis of the secretory granules in goblet cells of mouse intestinal tracts: Changes with age

Changes in the elements in goblet cell secretory granules in three portions of the intestinal tract — duodenum, proximal colon, and distal colon — in mice from 14 days to 18 months after birth were studied by quantitative electron probe X-ray microanalysis on quick-frozen and freeze-dried cryosections by calculating the ratio of each element. In the analysis of five elements, the peaks decreased in the order of sulfur (S), potassium (K), chlorine (Cl), phosphorus (P), and calcium (Ca) in the duodenum at 1 month; in the proximal colon, the peaks declined in the order of K, S, Cl, Ca, and P at 3 months; and in the distal colon, the peaks declined as S, K, Ca, Cl, and P at 3 months. The highest average ratio of S was obtained in the duodenum at 1 month, and this value then declined with age. In the proximal colon, the average ratio of K was the highest at 3 months, then declined afterward with age. The highest average ratio of S was at 3 months, and the highest average ratio of K was at 1 month in the distal colon. The ratio of peak counts to the background was calculated for each type of granule. Significant differences were found in the ratios of S and K between the proximal and distal colon. As for S, the duodenum and the distal colon had the highest ratios and the proximal colon had the second highest. Concerning K, the proximal colon had the highest and the duodenum and the distal colon the second highest ratio. The secretory granules of goblet cells in the three portions of the intestinal tract were shown to contain different amounts of each element. Especially, the amount of S differed most from the proximal to the distal colon.

Calcium in secretory vesicles of neurohypophysial nerve endings: quantitative comparison by X-ray microanalysis of cryosectioned and freeze-substituted specimens.

The calcium content of individual secretory vesicles in rat neurohypophysial nerve endings was measured by quantitative electron probe X-ray microanalysis. Directly frozen control and potassium-depolarized isolated endings were analysed using two presumably equivalent preparative techniques: (1) freeze-substitution in presence of oxalic acid followed by sectioning of resin-embedded pellets; or (2) direct cryosectioning of the frozen pellets followed by freeze-drying in the column of the microscope. In the pellets of stimulated endings, both approaches revealed an increase in the calcium content of neurosecretory vesicles. This increase was statistically more significant in the specimens prepared by cryosectioning, probably because in this case the contribution of 'dead' nerve endings could be eliminated on the basis of excessive cytoplasmic sodium and chloride. The results demonstrate that an increase in cytosolic calcium can lead to an increase in intravesicular calcium, and that when this occurs, it occurs within a subpopulation of vesicles in a given nerve ending. In addition, measured intravesicular calcium was dispersed over a wide range of concentrations, as predicted by the hypothesis of intravesicular calcium priming. When the vesicular calcium content was averaged per nerve ending, a relatively wide distribution of concentrations was again observed, indicating that some nerve endings respond more strongly to the stimulation than others.

“Standardless”, quantitative electron probe x-ray microanalysis with energy-dispersive spectrometry: What is the distribution of errors?

Conferences on quantitative electron probe X-ray microanalysis are typically highlighted by presentations which seek to advance the state of analytical science in this field through improvements in knowledge of the fundamentals of electron/X-ray-specimen interactions, inter-element (matrix) corrections, corrections for irregular specimen geometry, etc. However, in light of recent developments in computer-assisted, energy dispersive X-ray spectrometry analytical systems, we need to re-evaluate what is actually taking place in the everyday application of this analytical technique. The central paradigm in quantitative electron probe X-ray microanalysis, as established in the earliest work of Castaing and built upon by generations of contributors, is the measurement of the ratio of the intensity of an element in the unknown to the intensity for the same element in a standard under constant conditions of beam energy, known dose, and spectrometer performance. The set of such “k-values” determined for all constituents in the unknown is then converted into a set of concentrations through the use of matrix correction factors calculated through one of several schemes, e.g., “ZAF”, ϕ(ρz), etc. The great power of electron-excited X-ray microanalysis derives from the simplicity of the standards required.

"Standardless" quantitative electron probe microanalysis with energy-dispersive X-ray spectrometry: is it worth the risk?

"Standardless" procedures for quantitative electron probe X-ray microanalysis attempt to eliminate the need for standardization through calculation of standard (pure element) intensities. Either "first principles" calculations, which account for all aspects of X-ray generation, propagation, and detection, or "fitted standards" calculations, which use mathematical fits to measured intensities from a limited set of pure standards, can form the basis for standardless analysis. The first principles standardless analysis procedure embedded in the National Institutes of Health/National Institute of Standards and Technology comprehensive X-ray calculation engine and database, Desktop Spectrum Analyzer, has been tested against spectra measured on NIST standard reference materials, research materials, and binary compounds. The resulting distribution of errors is broad, ranging from -90% to +150% relative. First principles standardless analysis can thus lead to unacceptably large errors.

Quantitative electron probe X-ray microanalysis of calcium and phosphorus in human lumbar yellow ligaments

The elemental changes of biopsy specimens obtained from the human lumbar yellow ligaments were studied by quantitative electron probe X-ray microanalysis on both glutaraldehyde-fixed Epon-embedded sections and unfixed freeze-dried cryosections. Much higher soluble amounts of Ca and P in the unmineralized fibrocartilage zone were found in specimens obtained from older patients than in those from younger ones.

X-ray microanalysis of the secretory granules in the intestinal goblet cells of aging mice

The elemental changes in the goblet cell secretory granules of the mouse jejunum and ascending colon during postnatal development and aging were studied by a quantitative electron probe X-ray microanalysis on quick frozen and freeze-dried cryosections. In the jejunum, sulfur showed the highest peaks, followed by those for K, Cl, Ca, P and Na, respectively. In the colon, on the other hand, potassium showed the highest peaks, followed by those for S, Cl, Ca, P, Na respectively. Sulfur in the jejunum was at its lowest on postnatal day 14, as its highest at 1 month, and then gradually decreased with aging.

Electron probe microanalysis of gentamicin-induced changes on ionic composition of the vestibular gelatinous membrane

Gentamicin-induced changes in ionic composition in the otolithic membrane of adult OF1 mice were evaluated in the gelatinous layers of the saccule and utricle by quantitative electron probe X-ray microanalysis. The otolithic membranes were plunge-frozen and freeze-dried to prevent the redistribution of elements. Quantitative analysis was carried out with an energy dispersive detector using the peak-to-background ( P B ) ratio method and different salts dissolved in dextran as standards to calibrate the P B ratio against the concentration of the elements P, S and K in the microprobe. Gentamicin selectively decreased the concentrations of P ( P < 0.001) and S ( P < 0.01) in the gelatinous membrane of the saccule, and had no effect in the utricle. The concentration of K also increased in the utricular gelatinous membrane ( P < 0.05). The mechanism of ototoxicity in the gelatinous membrane is unknown, but the ability of aminoglycosides to block calcium channels may induce disturbances in the ionic equilibrium of the endolymphatic fluid, and thus affect the biochemical composition of the gelatinous membrane. This technique can be useful to evaluate the distribution of ions in the process of drug-induced ototoxicity.

Gentamicin Ototoxicity in Otoconia: Quantitative Electron Probe X-ray Microanalysis

Chronic gentamicin ototoxicity was evaluated in the otolithic membrane of adult OF1 mice at the otoconial layer of the saccule and utricle by quantitative electron probe X-ray microanalysis of Ca and K. The otolithic membranes were plunge-frozen and freeze-dried. The analysis was carried out with an energy dispersive detector using the peak-to-back-ground ratio method and different inorganic salts of Ca and K as standards to calibrate the microprobe. Ca and K in the otoconia are related via a linear function in both the saccule and the utricle. This association is not maintained after exposure to gentamicin, which suggests that this aminoglycoside antibiotic interferes with the Ca-K equilibrium in the otoconia. A dose of 200 mg/kg gentamicin twice a day for 5 days did not affect Ca in the mineral phase of the otoconia, but did increase K in both saccular (p < 0.05) and utricular (p < 0.01) otoconia. These increases in K may reflect a modification in the composition of the endolymph, resulting from cellular damage at the plasma membrane.

Microprobe analysis of technetium-mibi accumulation in cultured heart cell mitochondria: Correlation of structure and membrane potential

Hexakis (methoxyisobutylisonitrile)99mTechnetium (Tc-MTBI) is a gamma emitting radiopharmaceutical currently in clinical use for evaluating myocardial perfusion. Because Tc-MIBI exists as a lipophilic cation with a single positive charge, it has been shown to concentrate in mitochondria in response to membrane potential in a Nernstian fashion similar to rhodamine 123. Use of electron probe x-ray microanalysis (EPXMA) for localization and quantitation of the element technetium in mitochondria and calculation of membrane potential allows correlation for the first time between mitochondrial structure and membrane potential in intact cells.Heart cells from 11-day-old chick embryos were cultured into spherical aggregates 50 -100 μm in diameter after Ebihara et al. Aggregates were then incubated for 60 minutes in HEPES balanced buffer salt solution containing either 0, 36,72, or 144 μMTc99-MIBI as well as 20 mCi metastable Tc99m-MIBI. Some aggregates were prepared for conventional fixation and subsequent electron microscopic evaluation, while other aggregates were plunge frozen in liquid propane and cryosectioned for quantitative EPXMA.

Quantitative electronprobe X-ray microanalysis on the calcium and phosphorus distribution and calcium transport in epiphyseal growth plate

Quantitative electronprobe X-ray microanalysis on the calcium and phosphorus distribution and calcium transport in epiphyseal growth plate

Quantitative mapping of skeletal muscle fibers by x-ray microanalysis: Effect of long-time storage of freeze-dried cryosections

Electron probe X-ray microanalysis (EPXMA) is uniquely capable of performing quantitative topochemical analyses with high spatial resolution of elemental concentrations in cryosections from quickfrozen biological material. EPXMA requires very expensive instrumentation and maintenance, and data acquisition is very time consuming. For economic reasons, it is most desirable that any EPXMA facility serve several users who, while waiting for their turn, may need to store cryosections for a time. Storage of cryosections is associated with loss of structure and, under certain circumstances, also with changes in elemental distribution after short periods of time. We have re-examined several cryosections by EPXMA mapping that had been stored under vacuum for very long periods of time.Materials and Methods:Single, intact semitendinosus muscle fibers from R. temporaria were quick-frozen as previously described. The cryosections were freeze-dried in a Balzers FDU 101 and carbon-coated before external transfer to plastic desiccators in which they were stored under vacuum. Quantitative EPXMA maps were obtained as previously described (Figs.1A-F).Results and Conclusions:In the maps [Ca] was very high in the JSRs and, occasionally, (Fig. 1D) seen in hot spots that may correspond to spotty accumulations of negatively charged electron-dense material often seen in the free SR away from JSR. Overall, only small variations in elemental distribution or concentration were observed after storage for over one year; the significance of these variations (especially with such elements as Na) is difficult to assess because of the small number (4) of muscle fibers examined. The lighter region in the center of Fig. 1B is a result of slight etching due to extended static probes that were taken from this area in addition to mapping. JSR [Ca] remained remarkably constant after one year of storage. Freeze-dried cryosections revealed some smearing of structural detail, ranging from minimal to moderate, especially in the myofibrillar compartment (cf. Figs 1A,B). Our findings show that, when handled properly, carbon-coated cryosections can be stored over extended periods of time with minimal loss of structure or changes in elemental distribution. Thus, quantitative EPXMA image maps after long storage present a rather accurate representation of the original. Proper storage of freeze-dried cryosections is a viable procedure for the effective participation of independent research groups in multiple-user EPXMA facilities, access to which may occasionally require prolonged storage periods.