Scanning electron probe x-ray microanalysis of elemental distributions in freeze-dried cryosections of Bacillus coagulans spores.

High-resolution scanning electron probe X-ray microanalysis had been employed to examine elemental distributions in freeze-dried cryosections of Bacillus coagulans spores. Calcium, manganese, and phosphorus were concentrated in the protoplast and the coat. Iron was found in the coat but not in the protoplast, whereas the silicon seen on the coat of other spore species was absent. Sulfur was present in the coat, but was distributed over a broader area than the other elements, which suggested that phosphorus and the metal ions were located in the outer coat layer.     

Microincineration and elemental X-ray microanalysis of single Bacillus cereus T spores

Single whole spores of bacillus cereus T were analyzed by scanning electron microscopy and electron microprobe X-ray microanalysis before and after high-temperature (600 degrees C) ashing in air. High-temperature ashing consisted of a centripetal oxidation of the spore surface combined with pyrolysis of the spore's interior. Ashing of single spores produced a compact central ash particle, mimicking the much larger unashed spore body in outline but containing craterlike microregions, and a peripheral thin ash film. Ashing mostly eliminated the spore's organic matrix; however, ash residues still gave residual carbon-characteristic X-ray counts. Ashing of single spores produced a two-, five-, and six-fold increase of potassium, magnesium, and calcium X-ray intensities, respectively. Iron, although low in actual counts, became detectable after ashing. Phosphorus characteristic X-rays were decreased by 41% after ashing, while volatilization lowered sodium and manganese X-ray intensities by over 80%. High-temperature ashing enhanced element-characteristic X-ray intensities of the non-volatilizable mineral(ized) elements of spores by compacting them into ash residues, more so than by simply abolishing their organic matrix. Microincineration appears a generally useful preconcentration technique for elemental detection and localization in X-ray microanalysis.     

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.     

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.     

Location of Calcium Within Bacillus Spores by Electron Probe X-Ray Microanalysis

Spectroscopic microanalysis of the element-characteristic X rays produced by a scanning electron microprobe was employed to detect calcium and carbon in both intact and thin-sectioned spores of Bacillus cereus T and B. megaterium QM B1551. Linear scan profiles and multilinear scan images of the X-ray emissions for calcium (Ca(Kalpha)) were compared with those for carbon (C(Kalpha)) as an index of mass. Location was accomplished by stereological comparisons with secondary electron images and conventional transmission electron micrographs. Although the elements could be detected at the attogram level theoretically, spatial resolution was limited to approximately 500 to 1,000 nm in an intact spore, e.g., by the primary electron beam diameter, the electron-excited spore microvolume, and the type of specimen support. The resolution was improved to approximately 100 to 200 nm by use of thin-sectioned spores, with precautions to prevent calcium leakage from the specimen during preparations. In both intact and sectioned spores, calcium was distributed throughout the spore, similarly to carbon, and concentrated mainly in a central region corresponding to the spore protoplast.     

Disturbances of mineral metabolism in teeth of rats receiving corticosteroids for 3 generations.

Distribution of calcium and phosphorus was investigated with quantitative and qualitative methods in teeth of rats chronically treated with low doses of corticosteroids for 3 generations. In animals from 2nd and 3rd generation, scanning electron microscopy revealed focal lesions in enamel and dentin. The disturbances of the mineral metabolism in teeth of corticosteroid-treated rats were also reflected by abnormal calcium and phosphorus content in enamel and dentin, as assessed by X-ray microanalysis.     

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     

Mineralized dermal layer of the Brazilian tree-frog Corythomantis greeningi

Some species of anuran amphibians possess a calcified dermal layer (the Eberth-Kastschenko layer) located between the "stratum spongiosum" and the "stratum compactum." This layer consists of calcium phosphate deposits, proteoglycans, and glycosaminoglycans. Although regarded as a protective layer against desiccation, a calcium reservoir, or possibly a remnant of a dermal skeleton present in anuran ancestors, very little is known about its origin, structure, and function. Thus, we studied the structure and composition of the mineralized dermal layer of Corythomantis greeningi, a peculiar hylid from the Brazilian semiarid region (caatinga), using conventional and cryosubstitution methods combined with transmission, scanning, and analytical electron microscopy. Results show that the dermal layer consists of dense, closely juxtaposed, globular structures. Although the electron opacity of the globules was variable, depending on the type of preparation, crystal-like inclusions were present in all of them, as confirmed by dark field microscopy. Electron probe X-ray microanalysis showed calcium, phosphorus, and oxygen, and electron diffraction revealed a crystalline structure comparable to that of a hydroxyapatite.     

Quantitative and localized element analysis in cross-sections of spruce [Picea abies (L.) Karst.] needles with different degrees of damage

Summary Proton-induced X-ray microanalysis (micro-PIXE) permits the simultaneous determination of the content and the distribution of elements with atomic numbers higher than Z = 13 in biological samples. This method was used to investigate element content and localization in cross-sections of 6-month-old spruce needles. It was possible to detect the elements silicon, phosphorus, sulphur, chlorine, potassium, calcium, manganese, iron and zinc in semithin (10 m) sections of the needles. The localization of the cationic elements like potassium, calcium and manganese was determined in the one-dimensional line scan mode and in the two-dimensional raster scan mode. To demonstrate the usefulness of this method for forest decline research, element content and localization were compared in needles from two trees, which differed in their degree of damage. We were able to detect differences in the amount of cations and in their distribution inside the needles.     

Functional morphology of phagocytosing alveolar macrophages. Long-term electron microscopic and X-ray microanalytical investigations on the rat model

A single instillation of 1 ml iron dextran (containing 191.3 mg iron(III)hydroxide and 200 mg dextran) was administered under anaesthesia by a polyvinyl catheter into the lower lobe of the right lung in one hundred 4-week-old wistar rats. The animals were killed at intervals ranging between 1 min and 4 weeks. The lower lobe of the right lung was examined by light and electron (transmission and scanning) microscopy. In addition, X-ray microanalyses were performed on tissue sections in the transmission and scanning electron microscopes. The process of phagocytosis of iron dextran by alveolar macrophages can be subdivided into three stages, which we have termed the "phase of attachment" (from 1 to 5 min), followed by the "phase of phagocytosis" (from 5 to 20 min) and finally the "resident macrophage stage" (from 1 to 24 h). X-ray microanalysis shows a high phosphorus content even if iron dextran is concentrated on the surface of macrophages. Phagocytosis of particles between 15 and 40 A in size occurs within minutes, the particles being engulfed in phagosomes, which form as double-layered invaginations of the cell membrane into the interior of the cell. The fusion of phagosomes with lysosomes produces phagolysosomes (type 2 lysosomes) in which iron dextran is broken down into lamellar residual bodies. In these lamellar bodies X-ray microanalysis shows that in addition to abundant iron, there is a high phosphorus content, which may indicate the involvement of surfactant. Only 1 h after instillation, free particles of iron dextran can no longer be demonstrated in the alveoli, although a proportion of the iron dextran remains in resident macrophages (pulmonary tissue macrophages) and some is also found in splenic macrophages.     

Intranuclear silicon detection in a subcutaneous connective tissue cell by energy-dispersive x-ray miscroanalysis using fresh air-dried spread.

Silicon was detected by energy-dispersive x-ray microanalysis in the nucleus of a subcutaneous connective tissue cell of mice fed normally. To eliminate contamination, pieces of connective tissue were spread on copper grids and examined without any treatment by an energy-dispersive spectrometer with a scanning transmission apparatus attached to an electron microscope. Scanning transmission electron microscopy of the spread has demonstrated a well-preserved ultrastructure. Fibrous structures, nuclei and nucleoli of cells and mitochondrial granules were recognized. Electron probe analysis showed peaks for silicon at three spots on the nucleus of a cell in addition to those for phosphorus, sulfur, chlorine, potassium and calcium, whereas no peak of silicon could be detected at the spots on nuclei of other cells, mitochondrial granules and electron-lucent area on the same grid as the above. Silicon appears to play a significant role in the nucleus. Applicability of the technique to know the distribution of easily contaminating elements and diffusible substances is shown.     

Acidocalcisomes of trypanosomatids have species-specific elemental composition

The elemental composition and stoichiometric profile of elements present in acidocalcisomes of different genera of the Trypanosomatidae family (insect, plant, and mammalian parasites) submitted to parallel cultivation conditions were studied. X-ray microanalysis using transmission electron microscopy in conjunction with a morphometric approach was used to investigate the elemental content, number, distribution, and volumetric density of acidocalcisomes of different species. Microanalytical data showed that the different parasites possess the same elemental composition (oxygen, sodium, magnesium, phosphorus, calcium, iron, and zinc) in their acidocalcisomes. However, the relative concentrations of the elements varied among species, but not within acidocalcisomes of individual species. Iron was detected in acidocalcisomes of all species analyzed, characterizing this element as a constituent of these organelles. Taken together, the results strongly indicate a species-specific composition of acidocalcisomes in trypanosomatid parasites.     

Measurement of Total Calcium in Neurons by Electron Probe X-ray Microanalysis

In this article the tools, techniques, and instruments appropriate for quantitative measurements of intracellular elemental content using the technique known as electron probe microanalysis (EPMA) are described. Intramitochondrial calcium is a particular focus because of the critical role that mitochondrial calcium overload plays in neurodegenerative diseases. The method is based on the analysis of X-rays generated in an electron microscope (EM) by interaction of an electron beam with the specimen. In order to maintain the native distribution of diffusible elements in electron microscopy specimens, EPMA requires "cryofixation" of tissue followed by the preparation of ultrathin cryosections. Rapid freezing of cultured cells or organotypic slice cultures is carried out by plunge freezing in liquid ethane or by slam freezing against a cold metal block, respectively. Cryosections nominally 80 nm thick are cut dry with a diamond knife at ca. -160 °C, mounted on carbon/pioloform-coated copper grids, and cryotransferred into a cryo-EM using a specialized cryospecimen holder. After visual survey and location mapping at ≤-160 °C and low electron dose, frozen-hydrated cryosections are freeze-dried at -100 °C for ~30 min. Organelle-level images of dried cryosections are recorded, also at low dose, by means of a slow-scan CCD camera and subcellular regions of interest selected for analysis. X-rays emitted from ROIs by a stationary, focused, high-intensity electron probe are collected by an energy-dispersive X-ray (EDX) spectrometer, processed by associated electronics, and presented as an X-ray spectrum, that is, a plot of X-ray intensity vs. energy. Additional software facilitates: 1) identification of elemental components by their "characteristic" peak energies and fingerprint; and 2) quantitative analysis by extraction of peak areas/background. This paper concludes with two examples that illustrate typical EPMA applications, one in which mitochondrial calcium analysis provided critical insight into mechanisms of excitotoxic injury and another that revealed the basis of ischemia resistance.     

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     

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.     

X-ray microanalytical studies on cryofixed blood cells of the ascidian Phallusia mammillata

Summary Cryofixed blood morula cells of Phallusia mammillata (Cuvier), which are considered to be vanadium-accumulating cells, were examined by X-ray microanalysis using STEM (scanning transmission electron microscopy) and SEM (scanning electron microscopy). It is thought that cryopreparation preserves the native distribution of diffusible elements such as sodium, chlorine, and potassium, and prevents the displacement of vanadium, all of which may occur during conventional preparation. The results show that morula cell globules contain a large amount of sulphur and chlorine, and some sodium, magnesium, bromium and potassium, but very little or no vanadium.     

The characteristics of osteoclast localization in the bones of the extremities of the crested salamander Pleurodeles waltlii]

Localization of polynuclear osteoclasts in limb bones of ribbed newt were analyzed in consideration of calcium content in different bone structures. With using the methods of light and scanning electron microscopy and X-ray microanalysis it was shown that a critical level of cartilage calcification is need to activate osteoclasts. Osseous tissue can bind more calcium salt without resorption.     

Energy dispersive X-ray microanalysis of neurosecretory granules of mouse pituitary on fresh air-dried tissue spreads

Electron microscopy of fresh air dried spreads of unstained posterior lobe tissue from mouse pituitary disclosed neurosecretory granules. Each granule showed a seemingly homogeneous dense core surrounded by a halo and a bounding membrane. The area between granules in the cytoplasm was relatively well preserved. The energy dispersive X-ray microanalysis revealed peaks for sulfur, chlorine and potassium in two granules. The third granule displayed peaks for phosphorus and chlorine. These elements probably contribute to the high electron density of the granules. There was no peak for calcium, in contrast to the dense bodies of human blood platelets.     

The chemoreceptor surface of the taste disc in the frog,Rana esculenta. An ultrastructural study with lanthanum nitrate

Summary The distribution of lanthanum on the taste disc of the frog,Rana esculenta, afteren bloc staining of the tongue with lanthanum nitrate was studied at the ultrastructural level by means of scanning electron microscopy (in the secondary electron mode or in the back scattered electron mode), energy-dispersive X-ray microanalysis, transmission electron microscopy and electron spectroscopic imaging. It was consistently found that lanthanum distribution on the surface of the taste disc is not homogeneous and that the surface of putative receptor cells is in contact with strongly lanthanum-positive material. Calcium co-localizes with lanthanum at that level. These results suggest that different microenvironments exist at the surface of the taste disc and that this could be relevant to the receptor function.     

Specialized calciferous cells in the marine algaRhodogorgon carriebowensis and their implications for models of red algal calcification

Summary Calcification inRhodogorgon carriebowensis J. Norris et Bucher was associated with a particular cell type in the cortex. Calciferous cells were 4–6 times the length of cortical assimilatory cells. The distal two-thirds of the calcifying cell was invested with a thick wall that stained with periodic acid Schiff. Thick fibrils formed a reticulum and surrounded grains of calcium carbonate that ranged in shape from rhombohedral to subspherical and were up to 200 nm in greatest dimension. The proximal third of the cell was a tapering uncalcified stalk. The narrow base of the cell was attached to the subtending cell of the fascicle by a normal septum with a pit plug. The cell within the calcified wall matrix was usually flattened and had a very small volume. Cellular contents were dense; even when organelles could be discerned, they could not be identified. X-ray microanalysis revealed that other elements commonly found mixed with calcium carbonate are virtually absent from mineral deposits inR. carriebowensis, but electron diffraction study showed d-spacings that varied from those of pure calcite. Current models of red algal calcification are discussed in light of the findings on this alga.Abbreviations CaCO³ calcium carbonate - DIG differential interference contrast - PAS periodic acid Schiff - SEM scanning electron microscopy - TEM transmission electron microscopy