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PubMed2001

Heterogeneity assessment in individual CaCO3-CaSO4 particles using ultrathin window electron probe X-ray microanalysis.

In our previous studies, it has been demonstrated that both the excitation interactions between electrons and the atoms of the matrix and the matrix and geometric effects of electron-induced X-ray signals can be described by Monte Carlo simulation for low-Z elements, such as carbon, nitrogen, and oxygen, in individual atmospheric microparticles. In addition, by the application of a quantification method, which employs Monte Carlo simulation combined with successive approximations, at least semi-quantitative specification of the chemical compositions could be done. This has enlarged the scope of electron probe X-ray microanalysis (EPMA) for the single particle analysis of atmospheric environmental aerosol particles. In this work, we demonstrate that the heterogeneity of individual particles, even of micrometer size, can be characterized by the application of EPMA. X-ray photons obtained with different primary electron beam energies carry information on the chemical compositions for different regions in the particles. Artificially generated heterogeneous CaCO3-CaSO4 individual particles were measured at different accelerating voltages, and it was found that the Monte Carlo calculation is a powerful technique to extract the information on the heterogeneity of the particles that is contained in the measured X-ray data. Our approach can even estimate the thickness of the surface CaSO4 species by the application of the Monte Carlo calculation. A preliminary result for carbon-coated glass particles is also presented. The complexity involved in the analysis of real world particles is briefly mentioned with a result for heterogeneous SiO2 particle.

Journal Article↗
PubMed1981

[Element standards for quantitative X-ray microanalysis of biological specimens in the scanning transmission electron microscope].

Quantitation in biological X-ray microanalysis usually depends on the reference to one or more element standards. This paper deals with the general characteristics of such standards and gives a survey of the various methods of standard preparation reported in literature. Their advantages and disadvantages are briefly discussed. Practical experiments were carried out to prepare a potassium, calcium, and sulfur standard for X-ray microanalysis in the scanning transmission electron microscope.

Calcium↗
PubMed1987

Comparative studies of elemental composition on ejaculated fowl, bull, rat, dog and boar spermatozoa by electron probe X-ray microanalysis.

1. Comparative analyses of the concentrations of bulk and trace elements on the head, midpiece and tail regions of the ejaculated fowl, bull, rat, dog and boar spermatozoa were performed using X-ray microanalysis with scanning electron microscopy. 2. Although the pattern of distribution of elements on the surface of the three different subcellular regions was, in general, similar among all the species, there were substantial shifts in absolute concentrations. 3. Concentration of magnesium on the dog spermatozoa was significantly higher (about 2 times) than those of the other species. 4. Zinc, copper, iron and manganese concentrations were higher on fowl spermatozoa compared with those of the other species studied. 5. The Na-to-K ratios on the midpiece ranged from 1.46 (rat) to 2.26 (dog).

Animals↗
PubMed1994

Mitochondrial localization and characterization of 99Tc-SESTAMIBI in heart cells by electron probe X-ray microanalysis and 99Tc-NMR spectroscopy.

As the development of targeted intracellular magnetic resonance contrast agents proceeds, techniques for the quantitative analysis of the subcellular compartmentation and characterization of metallopharmaceuticals must also advance. To this end, the subcellular distribution and chemical state of hexakis (2-methoxyisobutyl isonitrile) technetium-99 (99Tc-SESTAMIBI), the ground state of the organotechnetium radiopharmaceutical used for the noninvasive evaluation of myocardial perfusion and viability by scintigraphy, has been determined by a novel application of electron probe X-ray microanalysis (EPXMA) and 99Tc-NMR spectroscopy. In cryopreserved cultured chick heart cells equilibrated in 36 microM 99Tc-SESTAMIBI, EPXMA imaging of mitochondria yielded a respiratory uncoupler-sensitive characteristic 99Tc X-ray peak representing 32.0 +/- 2.9 nmoles Tc/mg dry weight, while EPXMA of cytoplasm or nucleus showed no peak significantly greater than the threshold detectability limit of approximately 1 nmole/mg dry weight. Technetium-99 NMR spectroscopy of heart cells equilibrated with 99Tc-SESTAMIBI showed a single peak at -45.5 ppm with no evidence of significant line broadening or chemical shift compared to aqueous chemical standards, indicating that the majority of the complex exists unbound within the mitochondrial matrix. These data quantitatively demonstrate the localization of this lipophilic cationic organometallic complex within mitochondria in situ, consistent with a sequestration mechanism dependent on membrane potentials. Furthermore, this study establishes the general feasibility of combined EPXMA and NMR spectroscopy for the direct subcellular localization and characterization of metallopharmaceuticals, techniques that are readily applicable to MR contrast agents.

Animals↗
PubMed2007

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.

Animals↗
PubMed1981

Ion distribution in roots of barley seedlings measured by electron probe x-ray microanalysis.

The distribution of ions, particularly K and Na, was studied in roots of barley seedlings grown on various ionic solutions. Analyses were made by means of electron probe x-ray microanalysis using frozen, fractured bulk specimens. By this technique, it was demonstrated that there can be variability in the ratio K/Na measured in the vacuoles of cortical cells, with this ratio often being lower in epidermal cells of the root than in the inner cortex. A sharp difference in the K/Na ratio was also found between cells of the endodermis and those of the adjacent cortex, and generally higher ratios of K/Na occurred in the stele than in the cortex. Estimation of the concentrations in the cytoplasm was at the limit of resolution of this technique, but it can be shown that the K/Na ratio in the cytoplasm was higher than that in the vacuole. In low salt roots, the K concentration in the cytoplasm was higher than that in the vacuoles. The results with the x-ray microprobe confirm other measurements based on flux analysis or analysis of small samples of the root.

Journal Article↗
PubMed1988

Intracellular site of Sr2+ and Ba2+ accumulation in frog twitch muscle fibres as determined by electron probe X-ray microanalysis.

Strontium and barium can substitute for calcium at different levels of the excitation-contraction-relaxation cycle. The problem of sequestration of these ions in cellular microcompartments may be settled only by direct evidence obtained with analytical methods. Isolated frog twitch muscle fibres were perfused with increasing concentrations of potassium in Ca-free solution supplemented with Sr2+ (10 mmol/l) or Ba2+ (5 mmol/l). After equilibration in a Ca-free Ringer with Sr2+ or Ba2+ for 30 to 60 min the fibres were frozen in liquid propane (at 80 K) to immobilise ions. Ultrathin (150 nm) cryosections were cut at 170 K, freeze-dried, carbon-coated and analysed in an electron microscope equipped with an X-ray spectrometer. The ultrastructure of the superficial layer of the fibres was satisfactorily preserved. The terminal cisternae (t.c.) of the sarcoplasmic reticulum (SR) were dark and contained various amounts of Sr or Ba in addition to Ca. In Sr loaded fibres the longitudinal SR occasionally showed electron dense content with significant amounts of Ca; no Sr was present. The results suggest that t.c. is the common sequestering compartment for Ca, Sr and Ba. Essentially the same distribution pattern of Sr was found following precipitation of Sr with a solution containing digitonin and Koxalate.

Animals↗
PubMed2003

Detection of distribution of copper inside and outside of lysosomes in cultured hepatolenticular degeneration fibroblasts by electron probe X-ray microanalysis.

OBJECTIVE: To observe the distribution of copper in the subcellular structure for the understanding of primary pathogenesis of hepatolenticular degeneration (HLD). METHODS: Skin fibroblasts taken from HLD patients were cultured as an in vitro model of HLD, and the control cells taken from healthy volunteers were cultured in the same way. The distribution of copper inside and outside of lysosomes in fibroblasts was detected by quantitative electron probe X-ray microanalysis. The relationship between the subcellular location of copper and the genotype of the patients, and relationship between the distribution of copper and the course of the disease were analyzed. RESULTS: The content of Cu2+inside lysosomes of HLD cells (14.6+/-2.1 mmol/kg) and of heterozygote cells (11.6+/-0.6 mmol/kg) was higher than that of normal cells (4.5+/-1.2 mmol/kg) (P<0.01). The content of Cu2+outside lysosomes of HLD cells (17.5+/-4.2 mmol/kg) and of heterozygote cells (12.0+/-0.9 mmol/kg) was higher than that of normal cells (4.7+/-1.2 mmol/kg) (P<0.01). The distribution of copper in the subcellular structure was correlated with disease courses of HLD patients. With the progression of the disease, more copper was deposited in lysosomes (r=0.85, P<0.01). The content of copper in the diffused cytoplasmic compartment in HLD cells was correlated with that of sulfur (r=0.86, P<0.05), but not in heterozygote and normal cells. CONCLUSIONS: In the early stage of HLD, copper is accumulated outside lysosome, which is paralleled with increase of metallothionein-like proteins (copper and sulfur-binding proteins). With the development of the disease, more copper is deposited inside lysosome than outside lysosome. We conclude that the up-regulation expression of copper and sulfur-binding proteins and copper accumulation in lysosomes may play an important role in lowering the ATP7B gene mutation-induced toxic effects of free copper on the cell.

Adolescent↗
PubMed1991

Electron probe X-ray microanalysis of calcium and phosphorus distribution in developing hamster tooth germs in vitro and in vivo.

The aim of the present study was to investigate the spatial distribution of Ca and P in dentin and enamel of developing first (M1) and second (M2) maxillary hamster molars (age: 3-5 days) in comparison with cultured molars. For culturing the germs were dissected from 3-day-old hamsters and incubated for 1 and 2 days, respectively, in a modified BGJb medium. Electron probe X-ray measurements were carried out on 3 regions extending in a vertical axis from cusp tip over cusp middle to cusp base next to the cervical loop region. Neither the in vivo nor the in vitro group was statistically different in the Ca and P concentration in the regions of dentin. In both groups the measurements in enamel showed a gradient with an increase in Ca and P from enamel surface towards dentin-enamel junction and a gradient with an increase from cusp base towards cusp tip. Direct comparison of the in vivo group with the in vitro group did not demonstrate a statistical difference between the mineral content of the 4-day-old germs and the 1-day culture germs, respectively the 5-day-old germs and the 2-day culture germs. The results indicate a high correspondence between the mineralization process of in vitro and in vivo tooth germ development.

Aging↗
PubMed1988

Preparation of biological samples for transmission X-ray microanalysis: a review of alternative procedures to the use of sectioned material.

Although transmission X-ray microanalysis of biological material has traditionally been carried out mainly on sectioned preparations, a number of alternative procedures exist. These are considered under three major headings - whole cell preparations, analysis of cell homogenates and biological fluids, and applications of the technique to microsamples of purified biochemicals. These three aspects provide a continuous range of investigative level - from the cellular to the molecular. The use of X-ray microanalysis with whole cell preparations is considered in reference to eukaryote (animal) cells and prokaryotes - where it has particular potential in environmental studies on bacteria. In the case of cell homogenates and biological fluids, the technique has been used mainly with microdroplets of animal material. The use of X-ray microanalysis with purified biochemicals is considered in relation to both particulate and non-particulate samples. In the latter category, the application of this technique for analysis of thin films of metalloprotein is particularly emphasised. It is concluded that wider use could be made of the range of preparative techniques available - both within a particular investigation, and in diverse fields of study. Transmission X-ray microanalysis has implications for environmental, physiological and molecular biology as well as cell biology.

Animals↗
PubMed1977

Localization of gold in synovial membrane of rheumatoid arthritis treated with sodium aurothiomalate. Studies by electron microscope and electron probe x-ray microanalysis.

The localization of gold in the synovial membrane of rheumatoid arthritis patients treated with sodium aurothiomalate was examined and quantitative analysis of epon-embedded sections was carried out with a wavelength dispersive x-ray microanalyser. Gold was only detected in the lysosomes of synovial lining type A cells and subsynovial mononuclear cells in the form of filamentous deposits and highly electron-dense granules, the latter being few in number. The concentration of gold within the lysosomes containing the characteristic deposits and granules was equivalent to that in epon-embedded standard specimens of freeze-dried albumin in which 2-0-87-4 mg/ml of gold was included. In addition, sulphur was detected in the lysosomes containing the filamentous deposits, but the S/Au x-ray signal ratio was not equal to that detected in sodium aurothiomalate. The significance of the coexistence of gold with sulphur in lysosomes is discussed.

Aged↗
PubMed2003

Beryllium detection in human lung tissue using electron probe X-ray microanalysis.

Chronic berylliosis is an uncommon disease that is caused by the inhalation of beryllium particles, dust, or fumes. The distinction between chronic berylliosis and sarcoidosis can be difficult both clinically and histologically, as both entities can have similar presentations and exhibit nonnecrotizing granulomatous inflammation of the lungs. The diagnosis of chronic berylliosis relies on a history of exposure to beryllium, roentgenographic evidence of diffuse nodular disease, and demonstration of beryllium hypersensitivity by ancillary studies, such as lymphocyte proliferation testing. Additional support may be gained by the demonstration of beryllium in lung tissue. Unlike other exogenous particulates, such as asbestos, detection of beryllium in human lung tissue is problematic. The low atomic number of beryllium usually makes it unsuitable for conventional microprobe analysis. We describe a case of chronic berylliosis in which beryllium was detected in lung tissue using atmospheric thin-window energy-dispersive X-ray analysis (ATW EDXA). A woman with a history of occupational exposure to beryllium at a nuclear weapons testing facility presented with progressive cough and dyspnea and a nodular pattern on chest roentgenograph. Open lung biopsy showed nonnecrotizing granulomatous inflammation that was histologically indistinguishable from sarcoidosis. Scanning electron microscopy and ATW EDXA demonstrated particulates containing beryllium within the granulomas. This application of EDXA offers significant advantages over existing methods of beryllium detection in that it is nondestructive, more widely available, and can be performed using routine paraffin sections.

Adult↗
PubMed1993

Scanning electron microscopic study of the effect of citrate and pyrophosphate on calcium oxalate crystal morphology.

The effects of citrate and pyrophosphate on the morphology of calcium oxalate monohydrate crystals formed in a supersaturated calcium oxalate solution were examined by scanning electron microscopy. Within the normal range of urinary concentrations, citrate and pyrophosphate affected the morphology of the crystals. The length/width ratio of the crystals decreased at higher concentrations of citrate and pyrophosphate. Citrate seems to have a dual mechanism of inhibition of crystal growth (binding to the crystal surfaces) and of complexation (with calcium in the solution). Pyrophosphate is believed to bind preferentially to calcium in the solid phase rather than in the solution phase, as it affected crystal morphology at very low concentrations and was detected on the crystals by X-ray microanalysis. Pyrophosphate seems to bind to the monohydrate but not to the dihydrate.

Calcium Oxalate↗