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PubMed1984

Cytochemical analysis of calcium distribution in the superficial pineal gland of the Mongolian gerbil.

The potassium pyroantimonate-osmium method was employed to determine the distribution of cations in the superficial pineal gland of the Mongolian gerbil. The reaction product in the glands was presumed to be calcium following the use of both the chelator EGTA and x-ray microanalysis. A gradient of reaction product was found in the pineal with the highest concentrations occurring in and around the pineal concretions (PC). The periphery of mature PC demonstrated a heavy precipitate as did the surrounding pinealocytes. PC-associated pinealocytes contained reaction product in many organelles, including small vesicles that appeared to be opening in the direction of the concretions. The vacuoles of the pinealocytes contained various concentrations of precipitate. The results obtained in the present investigation have led to a hypothesis on the formation of pineal concretions: a pinealocyte vacuole is formed, calcium is transported to the vacuole, the cation content of the vacuole increases, and the cations precipitate into an immature concretion. Fibrillar material and an amorphous substance located within the pinealocyte vacuole appear to form cation containing vesicles that contribute to the periphery of the developing PC, resulting in the continued growth of the concretion.

Animals↗
PubMed1983

Quantitative X-ray microanalysis of semi-thick cryosections.

Methodological aspects of quantitative X-ray microanalysis of semi-thick cryosections (2--6 micrometers) of biological soft tissue were investigated. The preparation of a low background specimen holder is described. Scanning and scanning transmission images of the sections could be obtained, allowing identification and separate analysis of nuclei and cytoplasm. Parallel observations of histochemically stained adjacent sections in the light microscope allowed correlation of the microanalytical data with tissue morphology and histochemistry. Quantitative analysis could be carried out with the help of a standard: a gelatin/glycerol matrix containing mineral salts in known quantities, frozen and sectioned in the same way as the specimen. Mass loss under the electron beam was found to be comparable in specimen and standard. Comparison of various theoretical models for quantitative analysis showed that the 'P/B-method' (determination of the background intensity under the characteristic peak) is the most suitable for semi-thick sections. Factor determining the choice of accelerating voltage were analyzed. The usefulness of this specimen type is illustrated in some biological applications (human oral mucosa, rat salivary gland).

Animals↗
PubMed1990

Limitations of ZAF correction factors in the determination of calcium/phosphorus ratios: important forensic science considerations relevant to the analysis of bone fragments using scanning electron microscopy and energy-dispersive x-ray microanalysis.

A series of calcium phosphate standards having calcium/phosphorus (Ca/P) molar ratios of 0.50, 1.00, 1.50, and 1.67, respectively, was prepared for bulk specimen analysis using scanning electron microscopy (SEM) and energy-dispersive X-ray microanalysis (EDXA). The standards were mounted on carbon planchettes as either pure crystals or crystals embedded in epoxy resin. Ten different samples of each embedded and non-embedded standard were analyzed in a JEOL 100 CX electron microscope interfaced with a Kevex 8000 EDXA system using a lithium-drifted silicon detector and a multichannel analyzer. The Ca/P ratios were determined by calculating both net peak intensities without matrix corrections and atomic kappa-ratios using the MAGIC V computer program with ZAF correction factors for quantitative analysis. There was such extensive absorption of phosphorus X-rays in standards embedded in an epoxy matrix that the observed Ca/P ratios were statistically compatible with four different standards ranging in theoretical Ca/P ratios from 1.0 to 1.67. Although the non-embedded crystals showed a greater separation in the Ca/P ratios, both methods of preparation produced serious flaws in analysis. Direct application of the discovery of this caveat to the identification of suspected bone fragments for forensic science purposes is discussed.

Bone and Bones↗
PubMed2002

In situ ageing of fine beech roots (Fagus sylvatica) assessed by transmission electron microscopy and electron energy loss spectroscopy: description of microsites and evolution of polyphenolic substances.

Root biomass is quantitatively and qualitatively important in most ecosystems, but its contribution to the pool of organic matter in the soil is not clear. This work was designed to specify root ageing on an ultrastructural scale by transmission electron microscopy combined with microanalysis by electron energy loss spectroscopy. This approach is very suitable for studying the soil/plant interface, and for semi-quantitative analysis of the evolution of polyphenolic substances during root evolution. Three root segments were studied according to a gradient of root senescence: the apical and basal segments of the mycorrhiza and the mycorrhiza-carrier root. Each segment contained a certain proportion of senescent cells, some of which were of fungal origin, and this proportion increased as the root aged. In the three segments, the soil/plant interfaces were differentiated, and the micro-organisms observed in situ were described. Senescent root cells contained many polyphenolic substances and our results showed that these substances were, according to the root segment, differently associated with Ca, N and Si. When all these ultrastructural data are correlated with more global data, they can be usefully applied to root cell physiology, microbiology and pedology. This approach makes it possible to specify the evolution of organic matter in situ in soils whatever its origin.

Cellular Senescence↗
PubMed1987

Concentrations of elements in rat thymocytes measured by X-ray microanalysis.

Elemental concentrations of rat thymocytes in vivo were studied by X-ray microanalysis of freeze-dried sections. Cells from different regions, the subcapsular zone, the cortex and the medulla were studied in thymic tissue from a number of animals. Generally thymocytes situated in the medulla had higher concentrations of K compared to those in the subcapsular zone. The concentration of Na in the nucleus was constant in the medulla in all animals but some variation in this element was seen between animals in the subcapsular zone. The distribution of K/Na ratio in individual thymocytes was different in each region of the thymus. Cells with low K/Na ratio (less than 5) were predominant in the subcapsular zone, whereas cells with higher values for K/Na ratio were found in the cortex and medulla. The subcapsular zone is the region where mitotic cells are mostly situated. The finding of thymocytes with higher concentrations of Na and low K/Na ratios in this region is in accord with in vitro studies on thymocyte stimulation.

Animals↗
PubMed2000

Effects of dorsal root transection on morphology and chemical composition of degenerating nerve fibers and reactive astrocytes in the dorsal funiculus.

The morphology and chemical (elemental) composition of the dorsal funiculus of the rat spinal cord were examined 1 and 7 days after unilateral transection (rhizotomy) of the L4 and L5 dorsal roots, using light and electron microscopy as well as X-ray microanalysis. Changes were observed only in the dorsal funiculus on the side of injury and included disintegration of the axonal cytoskeleton, enlargement of axonal mitochondria, and widening of the myelin lamellae of the injured axons. X-ray microanalysis demonstrated a significant increase in intraaxonal sodium at 1 day after injury. This increase was abolished at 7 days, but at this stage there was a significant lowering of potassium in axons and myelin sheaths and of phosphorus in myelin as well as a marked increase in calcium in the axoplasm of the degenerating axons. The nonneuronal cell compartment, largely composed of astrocytes, showed elevated sodium, chlorine, and calcium and lowered potassium levels. The changes in chemical composition paralleled an increase in immunoreactivity for the calcium-binding Mts1 (S100A4) protein, which is exclusively expressed by white matter astrocytes. The influx of calcium is likely to play a crucial role in the loss of axonal integrity after rhizotomy, while the alterations in potassium, and perhaps also phosphorus, may contribute to activation of the nonneuronal cells, including the up-regulation of Mts1 expression in astrocytes.

Animals↗
PubMed1980

Calcification of cultured urticaria pigmentosa skin. An electron optical study of the calcium-apatite deposition.

Mast cells participate in experimental calcinosis. Skin from patients with urticaria pigmentosa (cutaneous mastocytosis) calcifies in vitro. In the present report, the nature of the calcium deposit was studied by electron optical techniques. The cultured mast cells of urticaria pigmentosa skin were surrounded by a large number of round bodies with an average diameter of 200-300 nm. Most of the round bodies were membrane bounded, while a few contained lamellar structures. These bodies may represent matrix vesicles as seen in calcifying hard tissues and/or may be remnants of mast cell granules. The calcified deposits were located on the round bodies, collagen fibrils and elastic matrix. Electron diffraction and X-ray microanalysis demonstrated that the deposits consisted of crystalline calcium apatite.

Apatites↗
PubMed1997

X-ray microanalysis and phagocytotic activity of cultured retinal pigment epithelial cells in hypoxia.

PURPOSE: To investigate the influence of the functional and morphological changes induced in retinal pigment epithelial (RPE) cells by retinal ischemia, we evaluated the phagocytotic activity, the concentration of various elements, and ultrastructure in cultured RPE cells in hypoxia. METHODS: The concentrations of oxygen in incubators were adjusted to 20, 10, and 1% by the addition of nitrogen for 72 hr. To observe phagocytotic activity and its relationship to actin filaments, the filaments of RPE cells incubated with fluoresbrite carboxylate YG microspheres were stained with rhodamine phalloidin. Some of the specimens were subjected to X-ray microanalysis by scanning electron microscope after being fixed, freeze-dried, and coated with carbon to investigate the cytoplasmic concentration of elements. A part of the latter specimens was also observed by transmission electron microscope after being embedded in epon and cut into ultrathin sections to see the ultrastructural changes inside cell. RESULTS: Lowering oxygen concentrations from 20% to 1% swelled RPE cells and decreased the number of fluoresbrite carboxylate YG microspheres phagocytized by RPE cells. Phagocytosis of a large amount of latex beads (30 microl) for 24 hr in 1% oxygen caused a disruption of RPE cells. Na, S, and P were detected in RPE cells cultured in 20% oxygen. Reducing the oxygen concentration from 20 to 10 or 1% significantly decreased Na and increased S. Mitochondria were observed in RPE cells in 20 and 10% oxygen, but many vacuoles were observed in the cytoplasm in 1% oxygen. CONCLUSION: Hypoxia as low as 1% oxygen induced malfunction of phagocytosis and the fragility of RPE cells. We could speculate the imbalance of the electrolytes such as Na or a decrease of antioxidants such as glutathione containing S as a reason of disturbance of cell viability.

Actins↗
PubMed1993

Dynamics of soldering reactions: microscopic observations.

This paper provides a summary of some in situ, high-resolution studies of solder spreading reactions on microelectronic circuit metallizations. Experiments are described that focus on the use of the environmental scanning electron microscope, or ESEM. Those experiments have been complemented by studies using optical hot-stage microscopy and have been supplemented by additional analytical tools such as energy dispersive X-ray microanalysis, Auger and ESCA to evaluate chemical processes. Two general results from dynamic scanning electron microscope observations are that 1) molten solder alloys undergo a segregation process during spreading in which a "precursor" film spreads in advance of the bulk solder and 2) the spreading front, which may be enriched in Sn from Pb-Sn or Bi-Sn solders, or In from Pb-In solders, spreads along high-reactivity features of the metallization surface as a reacting "precursor" film. A third observation from these tests is that, in unconfined geometries, the reactive metallization, if not sufficiently thick, can be dissolved by the solder before wetting is complete, leading to dewetting of the solder. Both the kinetics and extent of spreading of these films and the relationship of these phenomena to the commonly measured contact angle and wetting forces are currently being examined by a range of complementary techniques. Information gathered in these studies shows that process temperature as well as composition, reactivity, and relative amounts of the solder and metallization species should all be factors of interest to the those responsible for control of soldering processes.

Alloys↗
PubMed1997

Ion transport in cultured pig tracheal submucosal gland acinar cells studied by X-ray microanalysis.

The submucosal glands of the airway may contribute more to the airway fluid than the surface epithelium. The cellular mechanisms underlying the regulation of electrolyte and water transport in airway submucosal glands are, however, still poorly understood. Therefore, we attempted to establish a cell culture system to facilitate study of this regulation. Acinar cells were isolated by enzymatic disaggregation from pig tracheal submucosal tissue and cultured on a plastic substrate coated with human placental collagen. The fine structure of the cells in confluent culture was studied by conventional transmission electron microscopy. The elemental content in resting cells and stimulated cells grown on a permeable substrate was studied by X-ray microanalysis. The cultured cells retained structural characteristics (microvilli, secretory granules and desmosomes) of in situ epithelia. The total intracellular Cl and K concentrations significantly decreased after stimulation with the cholinergic agonist acetylcholine, the predominantly alpha-adrenergic agonist norepinephrine, or the beta-adrenergic agonist isoproterenol. Both ionomycin and 8-bromoadenosine 3':5'-cyclic monophosphate (8-bromo-cAMP) caused a marked decrease of the intracellular Cl, K and Na concentrations. Cl- secretion induced by acetylcholine was inhibited by Cl- channel blockers anthracene-9-carboxylic acid and 5-nitro-2-(3-phenylpropyl-amino)-benzoic acid (NPPB), but Cl- efflux induced by 8-bromo-cAMP was blocked only by NPPB. The intracellular Cl and Na content significantly increased and the cellular K content markedly decreased after treatment with ouabain. These results indicate that the cultured acinar cells maintained the structural and functional characteristics of in situ tissue and that this system is suitable for studying aspects of ion and water transport by the airway submucosal gland cells.

Animals↗
PubMed1981

Levels of dinoflagellate chromosome-bound metals in conditions of low external ion availability: an x-ray microanalytical study.

Cells of the dinoflagellate Glenodinium foliaceum were able to grow and divide in artificial (AE50) liquid culture medium diluted down to 10% of its normal concentration. X-ray microanalysis, with on-line computation of mass fractions, was used to determine the quantitative occurrence of bound (insoluble) elements in the chromosomes of cells grown in normal, 30 and 10% medium. In normal medium, chromosomes contained high levels of phosphorus (nucleic acid), sulphur (protein) and a variety of bound divalent metals, including Ca, Fe, Ni, Cu and Zn. With a decrease in the level of external ions (30 and 10% dilutions), the level of sulphur showed a significant decrease, but the mass fractions of insoluble phosphorus and total divalent metals did not fall. The presence of these metals in dinoflagellate chromatin does not, therefore, simply involve intracellular deposition in response to high external levels. The occurrence of divalent metals showed variation with medium concentration in terms of the proportions of different metals present, the nature of the metal--nucleoprotein associations, the metal concentrations per unit mass of chromatin and the number of metal atoms per 100 nucleotides. None of the above characteristics is therefore constant in an individual species.

Animals↗
PubMed1979

Ultrastructure and x-ray microanalysis of macrophages exposed to cadmium chloride.

Macrophages have a direct role in the inflammatory response to cadmium exposure. Cadmium is not only an important air pollutant, but is also one component of cigarette smoke. To study the effects of soluble cadmium on macrophages, two model systems were chosen:rabbit alveolar macrophages (RAMs) obtained by pulmonary lavage and peritoneal macrophages elicited by intraperitoneal injections of 10(7) viable M. bovis, bacillus Calmette Guérin (BCG MACs). Macrophages were maintained in standard tissue culture medium from 4 to 30 hours with concentrations of cadmium chloride (CdCl2) ranging from 0 to 1.0 mM. Attached macrophages and RAMs in suspension were studied by conventional transmission electron microscopy (TEM), scanning electron microscopy (SEM), and energy dispersive x-ray microanalysis (EDX). In addition to routine techniques for TEM and SEM, preparatory procedures included snap freezing in liquid propane, and either cryoultramicrotomy or freeze-substitution with 1% osmium tetroxide in acetone. By TEM many macrophages exhibited laminated nuclear inclusions at doses as low as 0.05 mM CdCl2) (at 20 hrs) and as early as 4 hrs (at 1.0 mM CdCl2). Sections of cells fixed by freezing exhibited the same nuclear inclusions as well as mitochondrial densities that were not visible with any other preparative technique. Cadmium was demonstrated in the nuclear inclusions and mitochondrial densities by EDX in snap frozen cells. Lesser amounts of cadmium were also detected diffusely in treated cell cytoplasm and nuclei. Cadmium was only detected by EDX in cells fixed by freezing. These studies document the localization of Cd in nuclear inclusions and mitochondria providing morphological support for the biochemical findings of other laboratories. In addition, the value of fixation by freezing over conventional chemical fixation is illustrated.

Animals↗
PubMed1996

Hard tissue of teeth and their calcium and phosphate content in Ambystoma mexicanum (Urodela: Ambystomatidae).

The wall of the pulp cavity, fracture faces and the demineralized surfaces of teeth from larvae and adults of Ambystoma mexicanum were investigated by scanning electron microscopy (SEM). Calcium and phosphate contents were determined by microanalysis. The apical part of the tooth (crown, tooth apex) contains dentin canals. In the larva, these do not reach the enamel-dentin border but end below this border in front of a denser hard substance, possibly enameloid. The pedicel in the adult and the basal portion of the tooth in the larva (base) are without dentin canals. These parts of the teeth are characterized by longitudinally arranged collagen fibres as visualized on the demineralized surfaces. These observations indicate a congruency in early-larval and adult teeth between base and pedicel as well as apex and crown. This partition is also confirmed by the calcium and phosphate values which were identical in larvae and adults. Highest values are found in enamel and lowest values in the tooth-bearing bone. Calcium and phosphate content show a clear difference between dentin and the basal part of the tooth (pedicel and base). The ring-like dividing zone in the adult tooth is less well mineralized.

Ambystoma mexicanum↗
PubMed2001

The polyphosphate bodies of Chlamydomonas reinhardtii possess a proton-pumping pyrophosphatase and are similar to acidocalcisomes.

Acidocalcisomes are acidic calcium storage compartments described initially in trypanosomatid and apicomplexan parasites. In this work, we describe organelles with properties similar to acidocalcisomes in the green alga Chlamydomonas reinhardtii. Nigericin and NH(4)Cl released (45)Ca(2+) from preloaded permeabilized cells, suggesting the incorporation of a significant amount of this cation into an acidic compartment. X-ray microanalysis of the electron-dense vacuoles or polyphosphate bodies of C. reinhardtii showed large amounts of phosphorus, magnesium, calcium, and zinc. Immunofluorescence microscopy, using antisera raised against a peptide sequence of the vacuolar type proton pyrophosphatase (H(+)-PPase) of Arabidopsis thaliana which is conserved in the C. reinhardtii enzyme, indicated localization in the plasma membrane, in intracellular vacuoles, and the contractile vacuole where it colocalized with the vacuolar proton ATPase (V-H(+)-ATPase). Purification of the electron-dense vacuoles using iodixanol density gradients indicated a preferential localization of the H(+)-PPase and the V-H(+)-ATPase activities in addition to high concentrations of PP(i) and short and long chain polyphosphate, but lack of markers for mitochondria and chloroplasts. In isolated electron-dense vacuoles, PP(i)-driven proton translocation was stimulated by potassium ions and inhibited by the PP(i) analog aminomethylenediphosphonate. Potassium fluoride, imidodiphosphate, N,N'-dicyclohexylcarbodiimide, and N-ethylmaleimide also inhibited PP(i) hydrolysis in the isolated organelles in a dose-dependent manner. These results indicate that the electron-dense vacuoles of C. reinhardtii are very similar to acidocalcisomes with regard to their chemical composition and the presence of proton pumps. Polyphosphate was also localized to the contractile vacuole by 4',6-diamidino-2-phenylindole staining, suggesting, with the immunochemical data, a link between these organelles and the acidocalcisomes.

Acids↗
PubMed1981

Application of scanning electron microscopy to x-ray analysis of frozen-hydrated sections. II. Analysis of standard solutions and artificial electrolyte gradients.

New specimen handling and analytic techniques for the application of x-ray microanalysis to studies of cell and organ biology have been recently described (Saubermann et al., 1981, J. Cell Biol. 88:257-267). Based on these techniques, absolute quantitative standardization has been established through x-ray analysis of frozen-hydrated and then dried sections of independently measured standard solutions of elements. These experiments demonstrate that the specific techniques employed have a probable error of less than 10%. Artificial electrolyte gradients established in gelatin were subjected to analysis to determine whether there was elemental displacement under non-membrane-limited conditions at the temperatures employed for sectioning (-30 degrees to -40 degrees C). No significant difference was observed between such gradients in serial sections cut at -30 degrees and -80 degrees C. Similarly, no additional ice-crystal-damage artifact was found in sections cut at -30 degrees C when compared with sections cut at -80 degrees C. Thus, in terms of ice-crystal size, gradient maintenance, and compartmental differentiation, cryosectioning at -30 degrees to -40 degrees C was not associated with redistribution incompatible with 1- to 2-micrometers spatial resolution, and absolute measurements of elemental concentration were practical within regions of this size.

Chlorides↗
PubMed1995

Backscattered electron imaging and energy-dispersive X-ray microanalysis studies of evidence for calcium salt heterogeneity in fifteen gallstones from an elderly human.

We examined 15 variably-sized gallstones, taken from an elderly male, by backscattered electron imaging and energy-dispersive X-ray microanalysis to learn the structural and distribution patterns of gallstone calcium (Ca-) salts. Of the 13 cholesterol-rich stones, nine stones had peripheral concentric layers of Ca-carbonate, whereas 2 stones had peripheral layers of Ca-phosphate. No Ca-salts were detected from 2 cholesterol-rich stones. The 2 stones containing Ca-phosphate had no Ca-salt cores, whereas the stones containing Ca-carbonate were separated into 3 different types: two stones with a Ca-carbonate core, four stones with several Ca-bilirubinate cores of glass-like structure, and 3 stones lacking Ca-salt cores. A closer view of the Ca-salt layers, which may be occasionally coexistent with Ca-bilirubinate, mainly showed either laminate deposits or numerous globules with a few laminae. Of the 2 cholesterol-poor stones, one had dispersed particles mainly of Ca-phosphate, and the other had loosely dispersed particles with small amounts of Ca-phosphate, bilirubinate, and/or palmitate. Some relationship between the size and Ca-salt species of these gallstones was suggested. Gallstones collected from the same individual showed a considerable heterogeneity of Ca-salts.

Aged↗
PubMed1994

Data handling in quantitative microanalysis in biology.

In cell biology, electron probe X-ray microanalysis can reveal the distribution of chemical elements inside a single cell. The full description of a biological system (cell population, tissue) requires a great number of spot measurements. In quantitative analysis, the measurements are subject to experimental errors of several types; moreover, the relations between the resulting values are usually more interesting than the absolute concentrations. Nevertheless, the proper evaluation of quantitative values can discover information more on the object of study. A system of simple statistical tests is suggested here which can solve several problems. Some concentration values can be far from the statistical average due to errors in measurement; therefore, a statistical test of plausibility of the measured values is carried out. In the compartments (e.g., nucleus, cytoplasm or other selected areas), the distribution of an element can be nonhomogeneous, and hence a statistical test of homogeneity of the element distribution in specified areas is provided. The tests continue with a test for correlation, in which the concentrations of a given element in a pair of specified areas are compared. These test proceed step-by-step for all elements of interest. Subsequently, the relations of concentrations in all possible pairs of elements in the area in question are calculated. Moreover, cells within a population can be different from the point of view of elemental concentration; a statistical test of homogeneity of the cell population is provided. In the case of nonhomogeneity, the concentration values and/or cells within a population are clustered into homogeneous groups. The evaluation is carried out automatically, with a simple program. The system of programs, in which the program for evaluation is incorporated, is included semi-on-line in the EDAX9900 system, where the measurement and evaluation are carried out in sequence. The results for a population of Streptomyces aureofaciens are shown as an example.

Analysis of Variance↗
PubMed1987

Image analysis and X-ray microanalysis in cytochemistry.

When cytochemical reaction products are homogeneously distributed within an organelle, point analyses suffice for the quantitative approach. However, quantitative analysis becomes tedious, when the elements in the reaction product are inhomogeneously distributed. Problems arise when elements from two reaction products have to be related to each other, or to endogenous cytological products (ferritin, haemosiderin, calcium, electron dense markers), either topographically or in concentration. When analyzing inhomogeneous/heteromorphical reaction product-containing organelles special attention has to be paid to measure and relate both volume and concentration. In this paper a relative simple structure (eosinophil granules) is chosen to demonstrate that the acquisition of the requested morphometrical plus chemical information and their integration is possible. The following points will be covered to acquire the morphometrical and chemical information: a). How to estimate the total cell cross-sectioned area. b). How to estimate the total cross-sectioned area of all reaction product-containing particles inside that cell. The ratio of these two areas will provide the requested information about the particle volume fraction. By using the X-ray detector in addition: c). How to acquire the chemical information at the requested resolution, within a reasonable total acquisition time d). How to integrate the morphometrical and chemical data per organelle, by matrix analysis in a reduced scan area. e). How to acquire quantitative chemical information, by the use of cross-sectioned standards. f). How to make this acquisition method independent from changes in the instrumental conditions during the acquisition.

Animals↗