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PubMed1982

Mass determination of thin biological specimens for use in quantitative electron probe X-ray microanalysis.

In the quantitative electron probe X-ray microanalysis of thin specimens the total mass thickness of a specimen is often required to express the elemental content as fractions of the total mass. In the present paper three methods for the measurement of mass thickness of thin specimens are reviewed and compared as to their applicability for use in quantitative X-ray microanalysis. The methods are based on the use of the continuum X-ray intensity, the backscattered electron intensity, and the transmitted electron intensity, respectively. From experimental results, it is concluded that the mass measurement method, based on the transmitted electron intensity, gives more accurate mass values than does the continuum X-ray method. The use of an independent mass measurement method is advantageous when low temperature oxygen plasma ashing is applied to lower X-ray analysis detection limits. In this way, elements present in ashed specimens can be expressed as fractions of the original specimen mass.

Animals↗
PubMed2000

Effect of 10% carbamide peroxide bleaching gel on enamel microhardness, microstructure, and mineral content.

The effect of 10% carbamide peroxide on human enamel subsurface layers was examined. Microhardness, microstructure, and mineral content were studied in such a manner that control and test side were located on the same tooth. Longitudinally sectioned samples were obtained from six teeth and Vickers microhardness of the enamel was measured. Enamel microstructure was analyzed using a scanning electron microscope and subjected to electron probe microanalysis for chemical analysis of calcium (Ca) and phosphorus (P). The concentration of Ca in the bleaching gel was measured spectrophotometrically and the concentration of P was measured photometrically. Results showed that a bleaching gel of 10% carbamide peroxide did not significantly affect the microhardness of the enamel. Scanning electron microscopic analysis showed local changes in enamel microstructure similar to those of initial caries. Electron probe microanalysis showed lowered concentrations of Ca and P; in addition the Ca:P ratio was lowered. There was some Ca and P in the bleaching gel after use. It is concluded that 10% carbamide peroxide causes local microstructural and chemical changes in enamel that are likely not clinically significant.

Adolescent↗
PubMed1989

Subcellular calcium content in cardiomyopathic hamster hearts in vivo: an electron probe study.

In the Syrian cardiomyopathic hamster heart, abnormal cellular calcium regulation, resulting in cellular calcium overload, is believed to play a role in the pathogenesis of cardiac hypertrophy and failure. Alternatively, the primary abnormality may be coronary vasospasm, resulting in reperfusion-induced necrosis. According to the latter hypothesis, only those cells that suffer an ischemic insult would contain elevated calcium levels. To determine whether a generalized elevation in myocytic calcium exists in myopathic hamster hearts, we measured cellular and subcellular calcium concentrations by electron probe microanalysis in cryosections of 50-day and 96-day myopathic and control hearts, rapidly frozen in vivo. Total calcium content of ventricular homogenates from each group was also measured by atomic absorption spectrophotometry. No significant differences in subcellular calcium were found by electron probe microanalysis among 50-day and 96-day myopathics and their age-matched controls. In 50-day myopathic and control hearts, mitochondrial calcium was 0.7 +/- 0.2 and 0.9 +/- 0.2, respectively, and A-band calcium was 3.0 +/- 0.4 and 2.6 +/- 0.4 mmol calcium/kg dry wt(+/- SEM). Results from 96-day animals were similar. Localized regions of elevated calcium were found only at sites of necrotic foci: in Na+-loaded cells (mitochondria: 4.7 +/- 1.3 (SEM) mmol/kg dry wt), in dying cells (mitochondria: 72 +/- 22 (SEM) mmol/kg dry wt) or as extracellular deposits (7-10 mol/kg dry wt). Total calcium content of hearts from myopathic hamsters, as determined by atomic absorption spectrophotometry, was also 13 times (50-day) and 50 times (96-day) higher than controls. These results demonstrate that there is a marked heterogeneity in cellular calcium content in myopathic hamster hearts, but the data do not support the hypothesis of a generalized cellular calcium overload.

Animals↗
PubMed1991

Morphologic features and nuclide composition of infarction-associated cardiac myocyte mineralization in humans.

Low dietary Mg results in Ca loading of cardiac myocytes, which increases the likelihood of myocyte calcification in the event of acute myocardial infarction (AMI), and possibly increases myocyte vulnerability to necrosis. Bloom and Peric-Golia1 previously reported an autopsy study of cases from the Washington, D.C. area (a region with low levels of Mg in the drinking water), demonstrating AMI-associated mineralization in myocytes with histologically normal nuclei and cross striations, as well as in obviously necrotic myocytes. The authors have re-examined mineralized myocytes from the same autopsy material, using electron probe microanalysis, light microscopy, and transmission electron microscopy. Microprobe analysis identified Ca and P as the nuclides composing the inorganic phase of the mineral deposits. Ultrastructurally, all Ca deposits, regardless of size or intracellular location, were composed of aggregates of needlelike hydroxyapatite crystals. The mildest form of intracellular Ca deposition was observed as small Ca deposits limited to some mitochondria of myocytes, which demonstrated intact nuclei and regular sarcomere pattern. More advanced stages of intracellular calcification, in the form of Ca deposits associated with mitochondria, Z-band regions and nuclei, were observed in other myocytes that also retained intact nuclei and sarcomeres. Massive Ca deposits were associated with myocytes which showed morphologic features of advanced necrosis, including loss of nuclei, disruption of sarcomere structure and masses of cellular debris. These observations support the theory originally proposed by Bloom and Peric-Golia1 suggesting that Ca loading of myocytes, possibly related to Mg deficiency in humans, increased vulnerability of the myocytes to subsequent AMI-associated necrosis and dystrophic calcification. In addition, the light microscopic impression of calcification of otherwise normal myocytes is contradicted by the electron microscopic identification of hydroxyapatite crystals free in the sarcoplasm, a condition unlikely to be compatible with viability. Lastly, the fact that all Ca deposits were in the form of hydroxyapatite supports the view that they were formed in a Mg-poor environment, which favors conversion of the more common amorphous form of Ca phosphate into the needlelike crystals of hydroxyapatite.

Calcium↗
PubMed1979

Metal binding by intestinal mucus.

Electron probe microanalysis offers distinct advantages for the study of intestinal mucus. This technique permits analysis of metal binding in situ, requires only a small amount of tissue, allows several experiments to be performed with one animal, and can resolve variations in binding that may occur in different portions of the intestine. We have used electron probe microanalysis to examine the metal binding capacity of intestinal mucus in situ. We have exposed portions of excised intestine to various concentrations of several metals, rapidly frozen the tissue and freeze dried it. After anhydrous embedding, thick sections were cut and analyzed on silicon discs or carbon coated copper grids. Qualitative analysis shows two distinctive patterns of distribution. The results of this work show clearly that at least three divalent cations are bound by mucus, that mucus exhibits different affinities for different metals, and that binding of metals is not uniform throughout mucus.

Animals↗
PubMed1995

Elemental composition of lamellar bodies from fetal and adult human lung.

Previous studies in the rat using electron probe microanalysis have suggested that most of the Ca2+ in alveolar space is probably derived from secreted lamellar bodies (LB). However, the LB Ca2+ content in cultured rat type II cells is low and unaltered by dexamethasone supplementation. In this study, we examined LB Ca2+ content in adult human lung and cultured explants of fetal lung treated with hormones to promote type II cell differentiation. Lung tissue of 20 to 24 wk of gestation was cultured for 4 to 6 days without hormone and with dexamethasone (10 nM), triiodothyronine (2 nM), 8-bromo-cyclic adenosine monophosphate (0.1 mM), or their combinations. The cultured tissue samples were processed for light and electron microscopy or rapidly frozen and stored in freon-22 under liquid nitrogen. Thin cryosections from the frozen samples were prepared and examined using the electron probe microanalysis. Human lung tissue up to 24 wk of gestational age had no detectable LB before explant culture. Explants cultured for 4 days without hormone supplementation (control) had no detectable LB, single-hormone treatments resulted in small LB, and combination treatments resulted in the formation of many large LB in explant type II cells. Despite such morphologic changes, LB Ca2+ in both control and hormone-treated explants was low (overall mean, 4.1 +/- 0.6; P < 0.05) compared with LB of in situ rat and adult human lung (30 +/- 2 and 27 +/- 1.5 mmol/kg dry wt, respectively) and was similar to that found in cultured rat type II cells. LB phosphorus and sulfur under all explant culture conditions were comparable. These observations indicate that human and rat LB elemental compositions are similar and that optimal in vitro conditions with respect to LB Ca2+ metabolism have not been established.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1984

Interrelations among Na and K content, cell volume, and buoyant density in human red blood cell populations.

This study establishes a method for determining the concentration of Na and K in single red blood cells from electron probe microanalysis of a cell's Na and K content. To this end, red blood cells were separated into subpopulations according to their buoyant density by means of bovine serum density gradient centrifugation. Cell water and Na + K contents were then determined in each fraction by conventional analytic methods with cell volume estimated from measurements of hematocrits and cell number. It was found that an inverse relationship obtains between the mean cell volume and buoyant cell density since cells increased in size as density decreased. Although the amount of hemoglobin per cell was found to slightly increase as cell density decreased, hemoglobin concentration showed the inverse relationship, indicating that buoyant cell density differences are primarily the result of differences in hemoglobin concentration. In confirmation of Funder and Wieth (Funder, J., Wieth, J.O. 1966. Scand. J. Lab. Invest. 18:167-180) cell water and cell volume was found to vary directly with the summed content of Na + K. Finally, by means of electron probe microanalysis of single cells, the cellular concentration of hemoglobin was found to vary inversely with the Na + K content, providing a quantitative basis for directly estimating cell volume, and thus ionic concentration, with this technique.

Body Water↗
PubMed1995

Decreased inotropic response to beta-adrenergic stimulation and normal sarcoplasmic reticulum calcium stores in the spontaneously hypertensive rat heart.

Cardiac hypertrophy in the spontaneously hypertensive rat has been shown to be accompanied by a diminished inotropic response to beta-adrenergic stimulation. This diminished response has been attributed to abnormalities in various components of the beta-adrenergic signaling system. There is also evidence that regulation of intracellular Ca2+ cycling may be altered in the hypertrophied heart of the spontaneously hypertensive rat. We proposed that the diminished response to beta-adrenergic stimulation may reflect abnormalities in Ca2+ cycling, specifically alterations in the ability of the sarcoplasmic reticulum to effectively release and resequester Ca2+. We have used the unique combination of functional measurements on isolated, isometrically contracting papillary muscles from hearts of 26-week-old spontaneously hypertensive rats and their Wistar-Kyoto controls, together with electron probe microanalysis measurements of sarcoplasmic reticulum Ca2+ content in the same muscles after rapid freezing, to determine the availability of Ca2+ for activation of contraction, following beta-adrenergic stimulation. We observed a significant decrease in the inotropic response to beta-adrenergic stimulation in papillary muscles from the spontaneously hypertensive rats, as compared with Wistar-Kyoto controls, however in these same muscles, frozen during relaxation, there was no evidence of an accompanying decrease in the size of the sarcoplasmic reticulum Ca2+ store. In an additional group of muscles which were frozen during contraction, the amount of Ca2+ remaining in the sarcoplasmic reticulum after stimulated release was also not different in the two strains. These results indicate that the decreased inotropic response to beta-adrenergic stimulation in hypertrophied hearts of the spontaneously hypertensive rat is unlikely to be due to decreased availability of Ca2+ for activation of contraction. Additionally, to determine whether there is intracellular Ca2+ overload in the cardiac muscle cells of hearts of spontaneously hypertensive rats, we measured the amount of Ca2+ in mitochondrial and at the level of the myofilaments by electron probe microanalysis. These results indicate that intracellular Ca2+ overload does not accompany cardiac hypertrophy in the spontaneously hypertensive rat. This study therefore shows no correlation between altered intracellular Ca2+ cycling and the decreased inotropic response to isoproterenol in the spontaneously hypertensive rat at 26 weeks of age.

Actin Cytoskeleton↗
PubMed1988

Microanalytical study of thorium 232 deposits in bone marrow and liver.

Analytical microscopy was used to study the distribution and chemical composition of thorium deposits in bone marrow and liver after injection of thorium dioxide and thorium nitrate. Thorotrast (thorium dioxide) was identified as being localized in bone marrow macrophages of a patient who had undergone cerebral arteriography forty two years ago. Large thorotrast deposits were also present in liver cells. We show that non-colloidal thorium (thorium nitrate) injected in rats concentrates in a non soluble form in bone marrow macrophages, hepatocytes and Kupffer cells. These deposits of thorium associated with phosphorus can be explained by the formation of thorium phosphate in lysosomes and we demonstrate that they remain in tissue for a long time. Microanalysis was performed with ion microscopy, and electron probe microanalysis by X ray spectrometry, which can identify and localize thorium and associated elements at cellular or intracellular level.

Animals↗
PubMed1992

[Selective localization of neptunium-237 in nuclei of mammalian cells].

After injection in the rat of soluble neptunium salt, the distribution of this element was studied at the subcellular level by electron microscopy and electron probe microanalysis. Abnormal structures have been observed by electron microscopy in the nuclei of hepatocytes, and the same structures have also been observed in the nuclei of the proximal tubules cells of the kidney. These structures are formed of clusters of very small and dense particles, several nanometers in diameter. The clusters are localized in the central part of the nuclei and they are separate from nucleoli and heterochromatin. Electron probe X-ray analysis of this cluster have shown that they contain neptunium associated with phosphorus. In the cell containing neptunium inclusions, other non specific lesions are also observed (nuclear pycnosis, mitochondrial depletion).

Animals↗
PubMedDate not supplied

[An electron probe x-ray microanalysis of dental alloys].

Electron probe x-ray microanalysis of 3 commercial cobalt-chromium-based alloys, CCA (USSR), Biosil and Vakukast (FRG), has revealed that CCA is initially heterogenic, this being explained, probably, by elevated chromium content. Biosil and Vakukast are similar in composition and represent a matrix with eutectic insertions of intermetallides and a small amount of oxides. Physical and mechanical characteristics of these alloys are described.

Dental Alloys↗
PubMed1978

Penetration of ions from silicate cement restorations into Copalite--covered cavity walls.

This study aimed to asses the effect of silicate cement on Copalite -covered cavity walls in extracted human teeth. Class V cavities were prepared in 24 premolars and filled with silicate cement (Bio-Trey). Four cavities were unlined, the rest of the cavities were lined with 1 or 2 layers of Copalite before insertion of the restorations. After 6 months, 70-100 microns thick longitudinal sections of the teeth were studied by polarized light microscopy, microradiography and electron probe microanalysis. When imbibed in water or quinoline, a subsurface zone of altered birefringence was noticed in nearly all cavity walls. Nearly half of the cavity walls in the experimental groups showed a surface zone of increased radiopacity. In a few instances a subsurface radiolucent zone was present. By electron probe microanalysis F (0,4-3% by weight), Zn (1-4%) and Al (0,2-6%) were measured in the outer 10-60 microns of the cavity walls. The study shows that even with a double layer of Copalite, known to prevent microleakage, a desirable uptake of F and Al from silicate restorations into cavity walls can take place. Copalite does not prevent a phosphoric acid effect on the cavity walls.

Aluminum↗
PubMed1998

Expression of osteopontin in Meckel's cartilage cells during phenotypic transdifferentiation in vitro, as detected by in situ hybridization and immunocytochemical analysis.

The localization of osteopontin (OP) was examined in Meckel's cartilage cells that bipotentially expressed cartilage and bone phenotypes during cellular transformation in vitro. Cultured cells were analyzed by in situ hybridization, immunostaining followed by light and electron microscopy, electron microscopy, and electron probe microanalysis. The combination of ultrastructural analysis and immunoperoxidase staining indicated that OP-synthesizing cells were cells that were autonomously undergoing a change from chondrocytes to bone-forming cells at the top of nodules. Double immunofluorescence staining of 2-week-old cultures revealed that OP was first synthesized by chondrocytic cells at the top of nodules. After further time in culture, the distribution of OP expanded from the central toward the peripheral regions of the nodules. Electron probe microanalysis revealed that the localization of OP was associated with matrices of calcified cartilage and osteoid nodules that contained calcium and phosphorus. Immunoperoxidase electron microscopy revealed that, in addition to the intracellular immunoreactivity in chondrocytes and small round cells that were undergoing transformation, matrix foci of calcospherites and matrix vesicles, in particular, included growing crystals that were immunopositive for OP. An intense signal due to mRNA for OP in 3-week-old cultures was detected in nodule-forming round cells, while fibroblastic cells, spreading in a monolayer over the periphery of nodules, were only weakly labeled. These findings indicate that OP might be expressed sequentially by chondrocytes and by cells that are transdifferentiating further and exhibit an osteocytic phenotype, and moreover, that expression of OP is closely associated with calcifying foci in the extracellular matrix.

Animals↗
PubMed1989

Electronic core level microanalyses and microcopies in multipurpose apparatus.

Using an instrument equipped with two electron guns, an electron analyzer, and a Si(Li) diode detector, we developed microanalytical techniques based on inner-shell electron excitations by incident electrons and X-rays, that is, electron energy-loss spectroscopy (EELS) in the reflection mode; electron probe microanalysis (EPMA) and X-ray appearance potential spectroscopy (XAPS); electron-induced Auger electron spectroscopy (e-AES); X-ray photoelectron spectroscopy (XPS), X-ray absorption spectroscopy (XAS); X-ray induced AES (XAES), X-ray fluorescence analysis (XRF), and scanning X-ray radiography (SXR). The corresponding characteristic images (including X-ray microradiography and X-ray photoelectron microscopy) were obtained in the scanning mode. The principle of the apparatus is described. Each spectroscopy and microscopy is illustrated by an example. Their performance and limits are discussed.

Electron Probe Microanalysis↗
PubMed1992

Postvaccinal sarcomas in the cat: epidemiology and electron probe microanalytical identification of aluminum.

An increase in fibrosarcomas in a biopsy population of cats in the Pennsylvania area appears to be related to the increased vaccination of cats following enactment of a mandatory rabies vaccination law. The majority of fibrosarcomas arose in sites routinely used by veterinarians for vaccination, and 42 of 198 tumors were surrounded by lymphocytes and macrophages containing foreign material identical to that previously described in postvaccinal inflammatory injection site reactions. Some of the vaccines used have aluminum-based adjuvants, and macrophages surrounding three tumors contained aluminum oxide identified by electron probe microanalysis and imaged by energy-filtered electron microscopy. Persistence of inflammatory and immunological reactions associated with aluminum may predispose the cat to a derangement of its fibrous connective tissue repair response, leading to neoplasia.

Aluminum↗
PubMed1996

In vitro characterization of enamel epithelium and pulp cells in mouse tooth germs.

Mixed cell populations consisting of enamel epithelium and pulp cells obtained from 18-day embryonic mouse tooth germs were cultured in vitro. Epithelial cells and pulp cells were also cultured individually and examined. Isolated cells were cultured in alpha-MEM supplemented with 10% fetal bovine serum for up to four weeks and examined morphologically using histological procedures including immunostaining, light and electron microscopy, and electron probe microanalysis. The pulp cells proliferated and differentiated in the absence of epithelium, but the number of epithelial cells showed a strong dependence on the pulp cells. Pulp cells showing fibroblastic morphology in the mixed culture gradually became elliptical, and eventually transformed into spherical cells surrounded by a calcified extracellular matrix. Alkaline phosphatase (ALPase) activity was expressed on the pulp cells prior to calcification of the extracellular matrix, as shown by von Kossa's and alizarin-red staining. Calcification deposition, which is closely associated with thick banded type-I collagen fibrils, was shown to be composed of calcium and phosphorous using electron probe microanalysis. Type-I collagen immunoreactivity was detected on the extracellular matrix after two weeks of culturing. The present results show that the proliferation and differentiation of pulp cells, and subsequent calcification of the extracellular matrix occur in the presence or absence of epithelial elements, but that the proliferation of epithelial cells depends on the presence of pulp cells.

Ameloblasts↗
PubMed1997

Backscattered electron imaging of cultured cells: application to electron probe X-ray microanalysis using a scanning electron microscope.

We report a simple method to study the elemental content in cultured human adherent cells by electron probe X-ray microanalysis with scanning electron microscopy. Cells were adapted to grow on polycarbonate tissue culture cell inserts, washed with distilled water, plunge-frozen with liquid nitrogen and freeze-dried. Unstained, freeze-dried cultured cells were visualized in the secondary and backscattered electron imaging modes of scanning electron microscopy. With backscattered electron imaging it was possible to identify unequivocally major subcellular compartments, i.e. the nucleus, nucleoli and cytoplasm. X-ray microanalysis was used simultaneously to determine the elemental content in cultured cells at the cellular level. In addition, we propose some improvements to optimize backscattered electron and X-ray signal collection. Our findings demonstrate that backscattered electron imaging offers a powerful method to examine whole, freeze-dried cultured cells for scanning electron probe X-ray microanalysis.

Animals↗