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PubMed2000

Heavy metal cytotoxicity studied by electron probe X-ray microanalysis of cultured rat hepatocytes.

Cytotoxicity of the heavy metals gold, mercury, thallium and lead was studied by measuring the intracellular element distribution of cultured rat hepatocytes by energy dispersive electron probe X-ray microanalysis of freeze-dried cryosections in a scanning transmission electron microscope. Exposure of the cells to aqueous solutions containing heavy metal ions in concentrations reaching a critical concentration caused increase of intracellular sodium and chloride content accompanied or followed by decrease of intracellular potassium content. Thus, the intracellular potassium/sodium ratio drastically decreased from control values of approximately 10 to values below 1 before changes of cell morphology became visible. In experiments with gold or mercury the decrease of the potassium/sodium ratio was preceded by transient cytoplasmic increase of sulfur and phosphorus. Heavy metal concentrations exceeding the critical concentration also caused an increase of cytoplasmic calcium concentration and finally decay of the cell structure. Cytotoxicity of heavy metals was found to increase in the order Pb, Au, Tl, Hg. Cytotoxic effects by Au, Tl or Hg in moderate concentrations were reduced by simultaneous addition of Zn or Pb to the culture medium. The results obtained prove electron probe X-ray microanalysis of cryosections as a sensitive probe of cell viability.

Animals↗
PubMed1988

Density gradients for isolation of mononuclear blood cells for magnesium analysis by electron probe X-ray microanalysis.

We present a mononuclear blood cell (MBC) isolation method for magnesium analysis by electron probe X-ray microanalysis. We compare the inorganic elemental composition of individual MBC isolated by either arabinogalactan (Stractan) or conventional Ficoll-Hypaque density gradients. We find that the MBC isolated with Stractan have the expected cellular element composition, but MBC isolated with Ficoll-Hypaque are contaminated with iodine and sodium. We discuss the source and significance of iodine and sodium contamination of Ficoll-Hypaque isolated MBC and recommend the Stractan separation method for electron probe X-ray microanalysis.

Adult↗
PubMed1979

Studies on the rat liver following iron overload. Electron probe x-ray microanalysis of acid phosphatase and iron.

Previous electron-microscopic studies on the liver have shown that following excessive administration of iron to experimental animals, small particles believed to represent ferritin and/or hemosiderin (electron-dense iron-containing particles [IPs]) accumulate in membrane-bound bodies--many with a lysosome-like structure--in liver parenchymal and Kupffer cells. Further identification of the IP-containing bodies has been facilitated by the application of histochemical techniques for the demonstration of acid phosphatase. The results have shown that reaction product was deposited over organelles similar in appearance to the IP-containing ones, indicating that they were lysosomes. However, the granular nature of the reaction product makes it difficult or impossible to decide whether IPs are present simultaneously with reaction product in the organelle. In order to clarify this qualitative aspect, x-ray microanalysis has been utilized to identify iron and lead (reaction product) in the various structures thought to represent lysosomes. The results indicate that all IP-containing bodies also show the presence of reaction product, and thus can be regarded as lysosomes. However, in the parenchymal cells there may exist a small population of iron-deficient lysosomes (only lead could be shown). The latter may correspond to "primary lysosomes."

Acid Phosphatase↗
PubMed1999

Use of SIMS microscopy and electron probe X-ray microanalysis to study the subcellular localization of aluminium in Vicia faba roots cells.

Received January 4, 1999; Accepted March 25, 1999 Secondary ion mass spectrometry (SIMS), electron probe X-ray microanalysis (EPMA) and transmission electron microscopy (TEM) were used to study the tissular distribution and subcellular localization of aluminium (Al) precipitate in roots of Viciafaba. The broad bean plant, grown in nitrate solution with 193 microM Al3+ at pH 4.8, for 15 days showed Al deposits in the roots. Al accumulation was not detected in the stems nor in the leaves. Al was found mainly localized on the root's surfaces and within the cell walls of the cortical cells. Al signal was not detected in the vascular tissues. Two weeks exposure to Al caused ultrastructural changes in cortical cells and sometimes a complete disruption of these cells. Deposition of Al in form of insoluble complexes associated with phosphorus, appeared as electron opaque materials in the vacuoles of disrupted cortex cells and in the intercellular inclusions. The leaves turned yellowish at the end of 15 days exposure. The use of electron microprobe, to investigate the same tissues as the ones investigated by SIMS, provided complementary results on aluminium allocation.

Aluminum↗
PubMed2001

Distribution analysis of epoxy groups in polymers by derivatization-electron probe X-ray microanalysis.

An analytical method, referred to as "derivatization-electron probe X-ray micro-analysis (XMA)", has been developed to determine the distribution of a small amount of the functional groups in a polymer. The suitable conditions for the derivatization reaction with epoxy groups, which contribute to the hardening reactions of polymers, were investigated. It was found that epoxy groups in polymers were derivatized selectively using gas-phase esterification with hydrochloric acid (HCI). The most suitable amount of HCl in a 50 ml vial was 300 microl. After setting a sample in the vessel without directly contacting the reagent, by reacting the reagent and the sample at 25 degrees C for 1 h, the highest reaction yield and selectivity were obtained. By derivatization-XMA using this reaction condition, the measurement of the distribution of epoxy groups in the polymer became feasible. Actual applications to a depth analysis of epoxy groups in the hardened acrylic coating and epoxy resin proved that this method is useful for the characterization of polymers and for the study of the hardening reaction of polymers.

Journal Article↗
PubMedDate not supplied

In situ cryofixation of kidney for electron probe X-ray microanalysis.

Cell physiological and pathophysiological studies often require information about the elemental composition of intracellular organelles in situ. Electron probe X-ray microanalysis (EPXMA) is one of the few methods by which intracellular elemental content and distribution can be measured simultaneously. While several cryofixation techniques for EPXMA have been utilized on isolated cells, few have been applied successfully to whole tissue in vivo or in situ. A recently developed, commercial, portable, metal-mirror device was used for preserving kidney in situ to determine the intracellular element distribution in proximal tubule cells. Kidneys of male rats were exposed, cryofixed, and analyzed for organelle elemental contents by EPXMA imaging. In addition, some portions of the frozen tissue were prepared for conventional transmission electron microscopy. Proximal tubules were preserved with intact brush borders and open lumens. The quality of preservation of tubule cell organelles varied inversely as a function of depth from the point of first contact with the mirror surface; the best preservation was within 15 microns, while the poorest preservation was deeper than 30 microns. Analysis of EPXMA images from the best-preserved regions revealed that proximal tubule cell cytoplasmic K/Na was approximately 6, cytoplasmic Cl was low relative to other subcellular compartments, and mitochondrial Ca levels were 1.8 nmole/mg dry weight; these observations indicate that the cells were physiologically viable at the time of cryofixation. The advantages of in situ cryofixation by this metal-mirror method include acquisition of organelle elemental content data in vivo, ease of use, reproducibility, portability, applicability to other tissues, and suitability for pathophysiological studies.

Animals↗
PubMed1997

Electron probe X-ray microanalysis of otoconia in guinea pig inner ear: a comparison between young and old animals.

Using the electron probe X-ray microanalysis technique, the elemental composition of otoconia was analysed in both young and old normal pigmented guinea pigs. Calcium concentration in both the utricular and the saccular otoconia was lower in the old animals, which indicates that the loss of calcium from otoconia and/or decrease in calcium ion uptake may occur in the old animals. The present study has established that calcium and other elements (P, S, Cl and K) in the otoconia of the young animals are related via a linear function, indicating that P, S, Cl and K are present in the mineral phase of the otoconia. The associations of Ca-P, Ca-K and Ca-S are not maintained in the old animals. It has been suggested that the aging phenomenon may influence the ionic metabolism in the vestibular end organs resulting in the altered mineral composition of otoconia.

Aging↗
PubMedDate not supplied

Determination of iron to phosphorus ratios of iron storage compounds in patients with iron overload: a chemical and electron probe X-ray microanalysis.

The amounts of phosphorus and iron in various isolated ferritin preparations were investigated by: chemical analysis on ferritin samples and electron probe X-ray microanalysis on ferritin particles from the same preparations. A high correlation was found between iron to phosphorus ratios obtained by both methods. Further investigation by electron probe X-ray microanalysis on lysosomes of hepatic cells of patients with idiopathic and secondary hemochromatosis revealed lysosomal iron to phosphorus ratios which were very similar in all parenchymal cells but different from ratios obtained in Kupffer cells. Lysosomal iron to phosphorus ratios in hepatocytes did not change after intensive phlebotomy treatment. It is postulated therefore that, during phlebotomy, iron and phosphorus are concomitantly lost from the hepatic lysosomes.

Bloodletting↗
PubMed1976

Silicon-containing granules of rat liver, kidney and spleen mitochondria. Electron probe X-ray microanalysis.

Isolated rat liver mitochondria containing granule aggregates (25-75 nm in diameter) and small (5-10 nm) electron opaque granules were examined by electron probe X-ray microanalysis. The granule aggregates gave an intense Si signal, while the small granules gave both Si and P signals. Isolated mitochondria of rat liver, spleen and kidney, subjected to detergent solubilization and differential centrifugation, produced two granule fractions: (1) a 10,000 g fraction consisting predominantly of granule aggregates (25--75 nm) composed of smaller granules (5--10 nm in diameter), and (2) a 10,000--30,000 g fraction of non-aggregated small granules (5-10 nm). Thin sections of isolated granule aggregates gave Si X-ray signals similar to those obtained from in situ granules. In addition S, Cl, Mg, Cr and Fe X-ray signals were observed. Cr occurred only in the large kidney granules, while Fe occurred in both fractions of the spleen and kidney granules. The presence of Si in the granules was confirmed by chemical analysis of the isolated granules and in vivo radiolabeling of the granules with 31Si and 68Ge. Contamination within the electron microscope was eliminated by a liquid nitrogen anticontamination device.

Animals↗
PubMed1996

Electron probe X-ray microanalysis of the elemental composition of the pineal gland of young adults and aged rats.

Fractures of deep-frozen and freeze-dried pineal glands were analysed for elemental composition by means of X-ray microanalysis with a scanning electron microscope. The results from young adults (3 months old) were compared with those from aged animals (24 months old); significant increases in S, Ca, Al, Si, and Fe were observed in aged animals when compared to young adults. There were no significant differences with Na, Mg, Cl, K, Ti, Cr, Mn, Ni, Cu, Zn, whereas a decrease of P was observed in aged animals when compared to young adults. Whether the changes observed in elemental composition have a direct effect on the activity and production of metalloenzymes and the overall physiology of the pineal gland are discussed.

Aging↗
PubMed2005

The distribution of light elements in biological cells measured by electron probe X-ray microanalysis of cryosections.

The intracellular distribution of the elements carbon, nitrogen, and oxygen was measured in cultured rat hepatocytes by energy dispersive electron probe X-ray microanalysis of 100-nm-thick freeze-dried cryosections. Electron irradiation with a dose up to 106 e/nm2 caused no or merely negligible mass loss in mitochondria and in cytoplasm. Cell nuclei lost carbon, nitrogen, and-to a clearly higher extent-oxygen with increasing electron irradiation. Therefore, electron doses less than 3 x 105 e/nm2 were used to measure the subcellular compartmentation of carbon, nitrogen, and oxygen in cytoplasm, mitochondria, and nuclei of the cells. The subcellular distribution of carbon, nitrogen, and oxygen reflects the intracellular compartmentation of various biomolecules. Cells exposed to inorganic mercury before cryofixation showed an increase of oxygen in nuclei and cytoplasm. Concomitantly the phosphorus/nitrogen ratio decreased in mitochondria. The data suggest mercury-induced production of ribonucleic acid (RNA) and decrease of adenosine triphosphate (ATP). Although biomolecules cannot be identified by X-ray microanalysis, measurements of the whole element spectrum including the light elements carbon, nitrogen, and oxygen can be useful to study specific biomolecular activity in cellular compartments depending on the functional state of the cell.

Animals↗
PubMed1977

Electron probe x-ray microanalysis of frozen-hydrated biological specimens.

A technique is described for preparing frozen-hydrated bulk samples of biological specimens for electron probe X-ray microanalysis. The method allows reproducible quantitative analyses to be made. Specimens are rapidly frozen, transferred to a vacuum evaporator, fractured under high vacuum at - 180 degrees C and coated with 20 nm of chromium. Transferal to the cryostage of a scanning electron microscope is accomplished without exposure to the atmosphere and without the specimen temperature rising above -120 degrees C. Analyses are made at a temperature of -145 degrees C. Contamination by frost does not occur. Etching and charging of the specimen are eliminated. Specimen charging is shown to be related to temperature. It can be eliminated at low temperature by coating with carbon, aluminium or chromium but consistent elimination could only be achieved with chromium. The chromium coat does not appear to have an absorption effect on quantitative analysis.

Animals↗
PubMed1985

Elemental analysis of individual rat blood platelets by electron probe X-ray microanalysis using a direct quantification method.

The elemental content of individual rat blood platelets and their dense granules was determined by electron probe X-ray microanalysis using a direct quantification method with microdroplets as standards. The quantification procedure was a modification of the 'direct mass' method involving a correction for differences in electron beam intensity in the analysis of standards and specimens. Whole air-dried platelets had a mean magnesium content of 12 X 10(-8) nmol (SE = 1 X 10(-8) nmol, n = 68) and a mean calcium content of 3.2 X 10(-8) nmol (SE = 0.3 X 10(-8) nmol, n = 68). A good correlation was found between the magnesium content and the phosphorus content of the dense granules of the platelets (r = 0.95). There was also good correlation (r = 0.77) between the number of dense granules per platelet and the magnesium content of the platelets.

Animals↗
PubMedDate not supplied

Electrolyte redistribution in cystic fibrosis fibroblasts studied by electron probe X-ray microanalysis.

The elemental composition of fibroblasts from cystic fibrosis (CF) patients and from healthy controls was compared by means of electron probe X-ray micro-analysis. Significantly lower sodium levels (p < .01) and higher calcium levels (p < .02) were found in CF fibroblasts, indicating a disturbance of ion regulation in these cells. Treatment with Staphylococcus aureus protein A and gammaglobulin (proteins that may influence the ciliotoxic CF factor) did not change the elemental composition of the fibroblasts.

Adolescent↗
PubMed1980

Distribution of calcium and other elements in cryosectioned Bacillus cereus T spores, determined by high-resolution scanning electron probe x-ray microanalysis.

The distribution of a number of key elements in Bacillus cereus T spores was determined by high-resolution scanning electron probe X-ray microanalysis. To circumvent the redistribution of soluble or weakly bound elements, freeze-dried cryosections of spores, which had been rapidly frozen in 50% aqueous polyvinyl pyrrolidone, were employed. The sections were examined by using a modified Philips EM400 electron microscope fitted with a field emission gun, scanning transmission electron microscopy attachment, and a computer-linked energy-dispersive X-ray microanalysis system. X-ray maps for selected elements and the corresponding electron image were produced simultaneously by scanning the cryosections with a fine electron beam in a raster pattern, using the scanning transmission electron microscopy attachment. The results indicated that almost all of the calcium, magnesium, and manganese, together with most of the phosphorus, was located in the core region. An unexpectedly high concentration of silicon was found in the cortex/coat layer. Granules containing high concentrations of calcium, manganese, and phosphorus were demonstrated in spores containing reduced levels of dipicolinic acid. Spot mode analyses, in which a stationary beam was located over the region of interest in the spore cryosection, confirmed the results obtained with the scanning mode and also provided a more accurate quantitation of the elemental concentrations on a dry weight bases.

Bacillus cereus↗
PubMed1985

Electron probe x-ray microanalysis studies on the ionic environment of nuclei and the maintenance of chromatin structure.

We have demonstrated the application of electron probe X-ray microanalysis to studies aimed at determining the role of the ionic environment in the maintenance of a stable chromatin structure during interphase. Our results indicate that X-ray microanalysis can be used to accurately measure the concentration of ions, such as Na+ and K+ within subcellular compartments. We also present evidence from our X-ray microanalysis studies to show that only a portion of the K+ in the interphase nucleus is in a free ionic state and that the rest of the intranuclear K+ is adsorbed to intranuclear macromolecules. The stable structural state of interphase chromatin was correlated with the maintenance of a normal range in the chemical activities of monovalent ions in the oocyte nucleus. Finally, we demonstrated that the chemical activity of the monovalent ions probably increases beyond this normal interphase range as the chromatin in the nucleus condenses into discrete chromosomes during the first meiotic division in the amphibian oocyte. The above observations support the hypothesis of Kellermayer et al. (1984) that the maintenance of chromatin structure in the nucleus of living cells requires a balance between the presence or absence of mobile nuclear proteins and the chemical activity of monovalent cations. It also suggests that an elevation of the chemical activity of the monovalent cations above a normal range will result in the destabilization of interphase chromatin structure. It is hypothesized that an elevation of monovalent cations may be directly involved in the structural and functional changes in chromatin which are known to take place at the time of meiosis and mitosis.

Animals↗
PubMed1985

Iodine distribution in the thyroid follicles of the hagfish, Eptatretus burgeri and lamprey, Lampetra japonica: electron-probe X-ray microanalysis.

In the thyroid follicles of species of cyclostomes, a hagfish and a lamprey, the distribution of stable iodine was examined by electron-probe X-ray microanalysis. A high concentration of stable iodine, heterogeneously distributed, was observed in the follicular cells of hagfish thyroid follicles. In the lamprey a low concentration of iodine was seen in the follicular lumina. The relative values for stable iodine determined in this way corresponded to values obtained by a chemical analytical method.

Animals↗