Silicon-containing granules of rat liver,kidney and spleen mitochondria

Summary 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 10,000g 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.Supported by Grant GM-08229-13-15 from the National Institutes of Health, USPHSWe are grateful to the Perkin-Elmer Corporation and to their Western Branch Manager, Mr. Michael E. Mullen and Microscopist, Mr. Minoru Saito, for use of and assistance with the Hitachi H 500 transmission electron microscope with the scanning attachment, and to the Kevex Corporation for the use of the Kevex X-ray Spectrometer. We also wish to acknowledge Mary Louise Chiappino for her technical assistance in preparing the thin sections, the final micrographs and X-ray spectra photographs and Darlene Lum for technical assistance in the laboratory     

Silicification of bamboo (Phyllostachys heterocycla Mitf.) root and leaf

Bamboo is a silicon accumulating plant. In leaves, the major place of silicon (Si) deposition is the epidermis, with the highest concentration of Si in silica cells. In bamboo roots, the deposition of Si is found only in endodermal cell walls. The silicification of leaves and roots was examined in the economically important bamboo plant Phyllostachys heterocycla, using an environmental scanning electron microscope coupled with X-ray microanalysis, as well as gravimetric quantification. The content of Si on a dry weight basis measured by gravimetric quantification was 7.6% in leaves and 2.4% in roots, respectively. Moreover, quantification of EDX data showed high Si impregnation of the inner tangential endodermal walls. Si content in this part of the root endodermal cell walls was even higher than that in the outer leaf epidermal walls, where conspicuous deposition of Si often occurs in grass plants.     

Subcellular localization of mineral deposits in the roots of wheat (Triticum aestivum L.)

Summary Mineral distribution in the roots of wheat (Triticum aestivum L. cv. Wheaton) was investigated using X-ray microanalysis of bulk frozen hydrated roots in SEM and of freeze substituted sections in TEM. Results obtained using the two methods agreed reasonably well. A total often elements were detected: Na, Mg, Si, P, S, Cl, K, Ca, Mn, and Fe. Of these Si, P, Ca, and Mn were incorporated into biomineralized structures. Silica was deposited in the endodermal walls in the older parts of the root. Silicon was also detected in the large central metaxylem lumina in the basal zone of the root, and in the smaller peripheral metaxylem and the immediately contiguous pericycle and outer parenchyma cells bridging the small metaxylem vessels to the endodermal layer. In the basal zone of the root some of the inner cortical cells contained intracellular electron opaque deposits. These were associated with the cell walls, had non-opaque inclusions and microanalysis revealed that they consisted of calcium, phosphorus and manganese.Abbreviations A apical zone of root - M midzone of root - B basal zone of root - SEM scanning electron microscope - TEM transmission electron microscope     

Heavy metals in the terrestrial isopod Porcellio scaber latreille. I. Histochemical and ultrastructural characterization of metal-containing lysosomes

Different populations of metal-loaded and uncontaminated Porcelio scaber Latreille were studied. Combined light and electron microscopial methods as well as X-ray microanalysis were applied for localization and characterization of intracellular sites of metal deposition within the small cells of hepatopancreas. By means of cytochemistry and X-ray microanalysis it was shown that membrane-limited vesicles are important sites of deposition for lead, copper, zinc and probably smaller amounts of cadmium. The vesicles also contained phosphorus. They are identical with the reported cuprosomes and belong to the lysosomal system. In addition, considerable amounts of lead, copper, and cadmium were found in small structures outside of membrane-limited organelles.Abbreviations EDX energy-dispersive X-ray - P. scaber Porcellio scaber - STEM scanning transmission electron microscope - TEM transmission electron microscope - v volume - wt weight     

The slug foot as a site of uptake of copper molluscicide

Copper has been demonstrated in the feet of slugs exposed to the molluscicide copper sulfate. Using the scanning electron microscope coupled with X-ray microanalysis, copper was localized to the epithelium of the foot. Under the transmission electron microscope the subcellular distribution of copper was obtained by precipitating the molluscicide with potassium ferrocyanide. The reaction product was both electron opaque and insoluble in the preparative media. The precipitate was localized in the zonula adhaerens and septate junctions between adjacent epithelial cells. Reaction product was also present in some of the pinocytotic vesicles of the epithelial cells. The identity of the reaction product was confirmed using X-ray microanalysis.     

Use of a Technovit 7200 VLC to Facilitate Integrated Determination of Aluminum by Light and Electron Microscopy

Technovit 7200 VLC is an excellent embedding medium for both inorganic histochemistry by light microscopy and X-ray microanalysis by scanning and transmission electron microscopy. Liver samples from rats after intraperitoneal treatment with aluminum chloride were fixed in glutaraldehyde and embedded in the resin. Thick sections were easily cut on an ultramicrotome and stained with aluminon for aluminum (Al). An intense positive reaction with aluminon was observed in the Kupffer cells by light microscopy. The surface structures of the same resin block cut for light microscopy were observed under a scanning electron microscope fitted with an energy dispersive X-ray spectrometer. The Kupffer cells appeared white in the backscattered mode. Localization of Al in the Kupffer cells was confirmed by an X-ray distribution map in the scanning electron microscope. Subcellular localization of Al in the Kupffer cells was performed on the same semithin sections using a transmission electron microscope equipped with an energy dispersive X-ray spectrometer. Most Al was found in lysosomes of the Kupffer cells. The resin was stable in the electron beam and chlorine-free.     

The element distribution in ultrathin cryosections of cultivated fibroblast cells

A preparation method for measurements of the intracellular distribution of elements in tissue culture cells is described which is based on cryofixation, cryoultramicrotomy, cryotransfer and X-ray microanalysis in a scanning transmission electron microscope. Dry weight concentrations of phosphorus, sulfur, chlorine and potassium in the nucleus, the cytoplasm and the mitochondria of L929 fibroblast cells of the mouse are reported. The preparation and quantitation procedures are discussed with respect to present limitations and possible improvements of the method.     

A combined scanning and transmission electron microscopic study and electron probe microanalysis of human pineal acervuli

Summary Untreated, decalcified and trypsinized acervuli from human pineal bodies were studied with the scanning and transmission electron microscope as well as by electron probe microanalysis. The mulberry-like acervuli are composed of a various number of spherical lobes (135–800 m) between which clustered groups of globuli (4–14 urn in diameter) are observed. The acervular lobes are very probably formed by an aggregation of these globuli. Small round particles 125–500 Å in diameter are observed on the surface of the pineal concretions. These are not influenced by either decalcification or trypsin treatment. The acervular mineral corresponds morphologically to hydroxyapatite. The electron probe microanalysis reveals the existence of calcium and phosphorus as main components of the acervuli. Small quantities of magnesium and strontium were also detected.Dedicated to Professor Berta Scharrer on the occasion of her 70th birthdayWith the technical assistance of Mr. P.A. MilliquetThe author wishes to thank Mr. Bauer and Mr. Fryder (Nestec SA, La Tour de Peilz) for the use of the Cambridge Stereoscan electron microscope and Dr. T. Jalanti (C.M.E., Lausanne) for his help with the use of the X-ray microanalyser     

CELL WALL STRUCTURE AND IRON DISTRIBUTION IN THE GREEN ALGA STAURASTRUM LUETKEMUELLERI (DESMIDIACEAE)1

The cell wall of Staurastrum luetkemuelleri Donnat & Ruttner was examined with scanning electron microscope (SEM) using whole cells, in thin sections with transmission electron microscope (TEM), and in air dried whole cells and unstained thin sections with X-ray microanalysis in the scanning-transmission electron microscope (STEM). The cell wall was ornamented with spines and wartlike structures. Spines were solid structures, consisting of deposits of cell wall material between two main cell wall layers. The wart-like structures were pore organs extending through the cell wall and the mucilaginous layer outside the cell wall. The pore cylinder was surrounded by deposits of cell wall material similar to the ones in the spines. X-ray microanalysis of selected areas on whole cells from a natural population showed iron accumulation in discrete locations on the cell extensions of S. luetkemuelleri. In the unstained thin sections iron was found only in the cell wall deposits in the spines. Cells grown in laboratory cultures failed to show iron accumulation regardless of readdition of iron-EDTA (Fe-EDTA) to the culture medium.     

Biosorption of Copper by a Bacterial Biofilm on a Flexible Polyvinyl Chloride Conduit

Inexpensive technologies with less-than-optimal efficiencies as a strategy for countering economic restraints to pollution control have been evaluated by using a laboratory-scale biotreatment process for copper-containing effluent. Economizing measures include the use of polyvinyl chloride (PVC) cylinders fashioned from commercially available flexible PVC conduit to support a biofilm that was cultured in an inexpensive medium prepared in wastewater. The biofilm was challenged by aqueous copper solution in a bioreactor and subsequently analyzed under a scanning electron microscope with energy-dispersive X-ray microanalysis.     

Biosorption of copper by a bacterial biofilm on a flexible polyvinyl chloride conduit

Inexpensive technologies with less-than-optimal efficiencies as a strategy for countering economic restraints to pollution control have been evaluated by using a laboratory-scale biotreatment process for copper-containing effluent. Economizing measures include the use of polyvinyl chloride (PVC) cylinders fashioned from commercially available flexible PVC conduit to support a biofilm that was cultured in an inexpensive medium prepared in wastewater. The biofilm was challenged by aqueous copper solution in a bioreactor and subsequently analyzed under a scanning electron microscope with energy-dispersive X-ray microanalysis.     

Silicon in rat liver organelles: Electron probe microanalysis

Summary Electron probe microanalysis of unfixed freeze-substituted rat liver tissue embedded in Spurr's low viscosity epoxy resin demonstrated the occurrence of Si as well as P, S, and Cl in the nucleus, nucleolus, mitochondria, and rough endoplasmic reticulum. Chemical analysis confirmed that the Si in the organelles did not originate from instrumental contaminants. This suggests that Si may be involved in the biochemistry of these subcellular organelles.Supported by Grant GM-08229-12-13 from the National Institutes of Health, USPHS.We are grateful to the Kevex Corporation and Mr. Glenn W. Meyer, Sales Engineer, for the use of the Kevex X-ray spectrometer; we wish to thank as well the Perkin-Elmer Corporation and their Western Branch Manager, Mr. Michael E. Mullen and Senior Microscopist, Mr. Minoru Shinorhara, for use of and assistance with the Hitachi HU 12 A transmission electron microscope. We also wish to acknowledge Mary Louise Chiappino for her technical assistance in preparing the thin sections, the final micrographs and the X-ray photographs, and Darlene Lum for technical assistance in the laboratory.     

Electron probe X-ray microanalysis of cultured myogenic C2C12 cells with scanning and scanning transmission electron microscopy

Heterogeneity of the elemental content of myogenic C2C12 cultured cells was studied by electron probe X-ray microanalysis (EPXMA) with scanning (SEM EPXMA) and scanning transmission electron microscopy (STEM EPXMA). The best plastic substrate for growing cells was Thermanox. For STEM EPXMA, a Formvar film coated with carbon was found to be suitable substrate. The cells examined by scanning transmission electron microscopy showed great heterogeneity in their elemental content in comparison with the cells examined in the scanning electron microscope despite of an almost identical preparation procedure for EPXMA. Nevertheless the K/Na ratios obtained from both methods of EPXMA were very close (4.1 and 4.3). We conclude that the observed discrepancy in the elemental content obtained by the two methods may be due to differences in instrumentation and this must be taken into account when planning a comparative study.     

The element distribution in ultrathin cryosections of cultivated fibroblast cells

Summary A preparation method for measurements of the intracellular distribution of elements in tissue culture cells is described which is based on cryofixation, cryoultramicrotomy, cryotransfer and X-ray microanalysis in a scanning transmission electron microscope. Dry weight concentrations of phosphorus, sulfur, chlorine and potassium in the nucleus, the cytoplasm and the mitochondria of L929 fibroblast cells of the mouse are reported. The preparation and quantitation procedures are discussed with respect to present limitations and possible improvements of the method.The paper was presented partly as a poster at the joint meeting on clectron microscopy of the Deutsche Gesellschaft für Elektronenmikroskopie and the Belgische Vereniging voor Elektronenmikroskopie in Antwerpen, 11.–16. 09. 1983     

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.     

Ultrastructural evidence for iron accumulation within the tube of Vestimentiferan Ridgeia piscesae

This study reports on the accumulation of iron within the tube wall of the deep sea vent macro invertebrate Vestimentiferan Ridgeia piscesae collected from Juan de Fuca ridge. Combining an array of approaches including environmental scanning electron microscope (ESEM), electron probe micro-analysis (EPMA), X-ray microanalysis (EDS) and transmission electron microscope (TEM), we provide evidences for the influence of prokaryotic organisms on the accumulation of metals on and within the tube wall. Two types of iron-rich minerals such as iron oxides and framboidal pyrites are identified within or on the tube wall. Our results reveal the presence of prokaryotic organism is apparently responsible for the early accumulation of iron-rich minerals in the tube wall. The implications of the biomineralisation of iron in tube wall at hydrothermal vents are discussed.     

Electron microscopical localization of the lead in peripheral blood neutrophils of the rat with timm sulphide silver method and X-ray probe microanalysis.

The distribution of lead ions in rat peripheral neutrophils was investigated using Timm sulphide silver method. In the neutrophils of adult male rats the Pb-precipitates were electron microscopically localized at 1.5, 3.0 and 6.0 hours after a single intraperitoneal injection of lead at dose 150 mgPb kg b. w. After increased lead ions input to cell matrix a formation of cytoplasmic metal deposits as well as an aggregation of Pb-complexes within deep invaginations of nuclear membrane was detectable. Furthermore, the neutrophils were prepared to examination by scanning electron microscope according to Domaga?a et al. (1979). In neither case there was a clear difference of neutrophil surface morphology between exposed and control populations. The same preparations were then used to X-ray probe microanalysis, and correlation between the presence of Timm reaction product and lead indication was obtained.     

Autofluorescence and Ultrastructure in the Myxomycete Diachea leucopodia (Physarales)

Autofluorescence is reported for the first time in Myxomycete fruiting bodies. Ultrastructure of stalked sporangia of Diachea leucopodia (Didymiaceae, Physarales) was studied using scanning and transmission electron microscopy, energy-dispersive X-ray microanalysis, and fluorescence microscopy. External and internal properties of the peridium that surround the spores and capillitium exhibit autofluorescence. The stalk is composed of calcareous granules and energy-dispersive X-ray microanalysis demonstrates that the elemental composition of the peridium, capillitium, and stalk has varying concentrations of calcium.     

Specific Features of the Formation of a Halo during Pathogenesis as a Response of Cereal Epidermal Cells to Penetration of Powdery Mildew Causative Agents

A cytophysiological study was carried out of the functional status of a halo as a response of the host plant to contact with a powdery mildew pathogen. Interactions of the powdery mildew causative agents with barley, wheat, wheat–wheat-grass hybrids, wheat-aegilops lines, and aegilops with different genotypic resistance lead to the expression of haloes during pathogens, which are induced by infection pegs of the primary growth tubes, appressoria, and hyphal lobes. Haloes are visualized using cytochemical reactions to proteins and scanning electron microscopy. The observed differences in the size of haloes and intensity of their staining (uniform or zonal) are related, to a great extent, to individual reactions of the plant cell at the penetration site and, to a lesser extent, to the level of genotypic resistance. An analysis of electron microscopy and cytochemistry studies suggests that the halo as a physiologically active zone is localized at the level of the plant cell plasmalemma. Active taxis of the cell organelles to the site of infection during the formation of a halo suggests that some kind of informational signals to changes in the cell metabolism are spread from the halo zone, which lead to compatible or incompatible interactions.     

X-ray microanalysis of freeze-dried digestive mucus in Anguilla anguilla L: modifications of ion content during the early steps of secretion

Digestive mucus of sea-water adapted eels has been observed and analyzed by the scanning electron microscope (SEM) after rapid freezing at liquid nitrogen temperature followed by freeze-drying. No chemical procedures were used in this technique. This allowed the maintenance of the mucous coating.Preliminary X-ray microanalysis carried out on freeze-fractured and freeze-dried samples of the oesophagus showed a decrease of K⁺ and an increase of Ca²⁺ and Cl⁻ from the basal part of the mucous cell towards its the apical part. This technique has proven to be satisfactory for it prevents translocation and loss of diffusible elements in situ and allows X-ray microanalysis in the SEM.