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

Summary— 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.     

Zinc distribution and excretion in the leaves of the grey mangrove,Avicennia marina (Forsk.) Vierh

Mangroves are important as primary producers in estuarine food chains. Zinc is often a major anthropogenic contaminant in estuarine ecosystems and has potential ecotoxicological consequences for mangrove communities. Accumulation, distribution and excretion of zinc in the leaf tissue of the grey mangrove, Avicennia marina was studied using SEM X-ray microanalysis and Atomic Absorption Spectroscopy. The first leaves of A. marina grown in 500 μg Zn as ZnCl₂ per g of dry soil were found to accumulate 106.3±18.5 μg Zn per g dry tissue, significantly higher than control plants, after a 7-month period. Washings from first leaves contained significantly higher amounts of zinc (0.30±0.14 μg/cm² Zn) than control plants after 1 month, suggesting excretion of zinc from glandular trichomes. SEM X-ray microanalysis revealed salt crystals exuded from glandular tissue on the adaxial surface of first leaves to be composed of alkaline metals and zinc in zinc treated plants. SEM X-ray microanalysis of seedlings dosed with 4 g/l Zn as Zn Cl₂ revealed a decreasing Zn gradient from xylem tissue, through photosynthetic mesophyll, to hypodermal (water) tissue. A subsequent increase in Zn concentration was observed in glandular tissue. Cell wall Zn concentrations were consistently higher than intracellular Zn concentrations.     

Quantitative X-ray microanalysis of calcium in sea urchin eggs after quick-freezing and freeze-substitution

Summary Freeze-substitution was used to study the distribution of calcium in sea urchin eggs, and the validity of the technique was assessed. We followed the fate of both total and exchangeable calcium of sea urchin eggs in two species (Paracentrotus lividus and Arbacia lixula) after the various treatments needed for freeze-substitution and embedding. We compared the calcium content either by X-ray microanalysis of Epon-embedded sections of freeze-substituted eggs (6.2±0.71 mmoles/kg of Epon-embedded tissue) or by flame spectrometry analysis of living eggs (32.3±1.30 nmoles/mg protein). After standardization of units, both values lead to similar total calcium content. We also measured the movements of ⁴⁵Ca from prelabelled eggs. Exchangeable ⁴⁵Ca as well as total calcium appeared unaffected by the preparative treatment for X-ray microanalysis. In conclusion, our preparative technique for X-ray microanalysis can be considered appropriate for our material and allows us to undertake a subcellular quantification of calcium in various organelles.     

Characterization Of Multi-layered Fish Scales (Atractosteus spatula) Using Nanoindentation,X-ray CT,FTIR, and SEM

The hierarchical architecture of protective biological materials such as mineralized fish scales, gastropod shells, ram’s horn, antlers, and turtle shells provides unique design principles with potentials for guiding the design of protective materials and systems in the future. Understanding the structure-property relationships for these material systems at the microscale and nanoscale where failure initiates is essential. Currently, experimental techniques such as nanoindentation, X-ray CT, and SEM provide researchers with a way to correlate the mechanical behavior with hierarchical microstructures of these material systems^1-6. However, a well-defined standard procedure for specimen preparation of mineralized biomaterials is not currently available. In this study, the methods for probing spatially correlated chemical, structural, and mechanical properties of the multilayered scale of A. spatula using nanoindentation, FTIR, SEM, with energy-dispersive X-ray (EDX) microanalysis, and X-ray CT are presented.     

Composition, speciation and distribution of iron minerals in Imperata cylindrica.

A comparative study of the roots, rhizomes and leaves of an iron hyperaccumulator plant, Imperata cylindrica, isolated from the banks of an extreme acidic environment, using complementary techniques: Mösbauer spectroscopy (MS), X-ray diffraction (XRD), scanning electron microscopy (SEM) coupled to energy-dispersive X-ray microanalysis (EDAX) and transmission electron microscopy (TEM), has shown that two main biominerals, jarosite and ferrihydrate-ferritin, accumulate in the different tissues. Jarosite accumulates mainly in roots and rhizomes, while ferritin has been detected in all the structures. A model of iron management in I. cylindrica is presented.     

Egg-shell calcium and the hatching of Globodera rostochiensis

The Ca²⁺ content of Globodera rostochiensis egg-shells was investigated by X-ray microanalysis. When intact eggs were treated with 5 mM sodium 1, 2-di (2-aminoethoxy) ethane-N,N,N',N',-tetra-acetate (EGTA) only part of the egg-shell Ca²⁺ was removed. Similarly treated opened egg-shells lost almost all their Ca²⁺. We think that Ca²⁺ of intact egg-shells which is accessible to EGTA is in the outer part of the shell and that the inaccessible Ca²⁺ is in the inner lipoprotein layer. Much Ca²⁺ was removed from opened egg-shells by the hatching agents Zn²⁺, La³⁺ and decationised potato-root exudate, but none by dilute acetic acid or Mg²⁺. Hatching agents, by binding to or replacing Ca²⁺, may change lipoprotein membrane structure. Eggshells treated with potato-root exudate contained about 3 times as much Ca²⁺ as untreated shells, because the treatment makes additional binding sites available. Our results suggest that three types of Ca²⁺ -binding sites occur in the egg-shell.     

Localization of Nickel in Tissues of Streptanthus polygaloides Gray (Cruciferae) and Endemic Nickel Hyperaccumulators from California

The genus Streptanthus Nutt. is one of the most important indicators of ultramafic floras in western North America. This genus contains taxa that are endemic or tolerant of ultramafic soils. Streptanthus polygaloides is an annual nickel hyperaccumulator strictly confined to ultramafic soils throughout the Californian Sierra Nevada foothills. Nickel concentration in S. polygaloides populations was evaluated by elemental microanalysis using inductively coupled plasma mass spectrometry (ICP-MS). Representative samples of S. polygaloides roots, stems, leaves, flowers, and fruits were studied by scanning electron microscopy (SEM) coupled to an energy-dispersive X-ray probe (SEM-EDX). Results show Ni accumulation values between 0.09 and 1.18 %, and a distribution pattern similar to that observed in other Ni hyperaccumulator taxa, with the leaf epidermis accumulating the largest concentrations.     

An X-ray microanalytical study of the ferricyanide reaction for the electron cytochemical demonstration of succinate dehydrogenase activity in isolated mitochondria

Synopsis The cytochemical technique for the demonstration of succinate dehydrogenase activity by ferricyanide reduction and simultaneous coupling with Cu²⁺ has been investigated by the combined techniques of transmission electron microscopy and X-ray microanalysis. The localization of the final reaction product of succinate dehydrogenase activity within isolated mouse liver mitochondria is described, and variations in the composition of the final reaction product are shown to occur, both at various sites within the mitochondrion, as well as after different reaction times.     

Elemental analysis using differential absorption techniques

X-ray differential absorption microanalysis is presented as a technique for trace element analysis of hydrated biological specimens of about 0.1–5 μm thickness. For the study of the light elements (Z?20), the absorption technique minimizes the radiation dose and, thus, damage to such specimens when compared with X-ray fluorescence. A Scanning Transmission X-ray Microscope (SXTM) is described, which has been used to map the concentration of calcium in bone with better than 300 nm spatial resolution and a sensitivity to 5% calcium by weight. Future plans are briefly discussed that offer the hope of achieving 0.1% trace element sensitivity and 75 nm spatial resolution.     

Ion compartmentation in Porphyra umbilicalis determined by electron-probe X-ray microanalysis

The ion composition of cell compartments in the intertidal red alga Porphyra umbilicalis adapted for two weeks in 3.5 x artificial seawater was determined by X-ray microanalysis of unfixed, frozen, bulk specimens. A procedure is described for the calculation of ion concentrations in the main cell compartment, cytoplasm, vacuoles and plastid. The results indicate high K⁺ and low Na⁺ concentrations in cytoplasm and plastid. Sodium ions are preferentially localized in vacuoles. Both, vacuoles and plastid contain high Cl^- concentrations.     

Role of intestinal mucus in crystal biogenesis: an electron-microscopical,diffraction and X-ray microanalytical study

Summary In the posterior intestine of the sea-water eel, mucus plays an important role in biocrystallization of calcium ions. By means of transmission and scanning electron microscopy associated with X-ray microanalysis and X-ray diffraction it has been possible to determine the role of mucous fibers as nucleation sites. Biocrystallization occurs in 2 steps: (1) Calcification of mucus. As soon as mucus is excreted in the intestinal lumen, it is loaded with calcium, as shown by lanthanum affinity and X-ray microanalysis on freeze-dried tissues. (2) Genesis of crystals. Needleshaped crystallites build up in coalescent spherites in the intestinal lumen near the microvilli. Genesis occurs as follows: (a) crystallite mineralization by nucleation in an organic matrix composed of glycoproteinaceous mucous fibers, followed by the appearance of spherites; (b) coalescence in spherites and association of spherites in rhombohedra; (c) extrusion of organic material during the final step of crystallization.     

Design of Magnetic Polymeric Particles as a Stimulus-Responsive System for Gastric Antimicrobial Therapy

The treatment of peptic ulcers induced by H. pylori remains challenging due to the deep mucous layer location of bacteria preventing antimicrobial drug access. The present work aimed to design and evaluate in vitro dual responsive (both pH and magnetic field-sensitive) polymeric magnetic particles loaded with amoxicillin as a smart drug carrier for deep mucous layer penetration and in situ drug release. Magnetite particles were produced by the co-precipitation method and subsequently coated with the Eudragit^®S100 and amoxicillin by using the spray-drying technique. The physicochemical characterization of the obtained particles was carried out by optical and scanning electron microscopy, X-ray powder diffraction, Fourier transform infrared spectroscopy, nitrogen adsorption/desorption isotherms, and vibrating sample magnetometry. Additionally, drug release tests and antibacterial activity tests were evaluated in vitro. Microparticles presented 17.2?±?0.4 μm in size and their final composition was 4.3?±?1.5% of amoxicillin, 87.0?±?2.3% of Eudragit, and 9.0?±?0.3% of magnetite. They were both pH and magnetic field responsive while presenting antimicrobial activity. On one side, magnetic field responsiveness of particles is expected to prompt them to reach bacterium niche in deep mucous layer by means of magnetic forces. On the other side, pH responsiveness is expected to enable drug release in the neutral pH of the deep mucous layer, preventing undesired delivery in the acidic gastric lumen. Smart microparticles were designed presenting both pH and magnetic field responsiveness as well as antimicrobial activity. These may be promising assets for peptic ulcer treatment.     

SIMS Investigation of the Distribution of K+, Na+, Mg2+ and Ca2+ Cations in Birch Pollen

Two microanalytical techniques were used to investigate the inorganic cation content and distributions in birch (Betula verrucosa Ehrh.) pollen. With intact pollen grains. X-ray microanalysis (EDX) could only give a mean ionic composition. Secondary Ion Microscopy and Spectrometry (SIMS) appeared to be a more suitable technique to image ion distributions in the different pollen structures. This was carried out with samples prepared using a new vapour phase technique designed to improve ion retention. Transmission electron microscopy (TEM)showed good structural preservation of the samples. Monovalent ion (K⁺, Na⁺) distribution showed features different from those of the divalent cations (Ca²⁺, Mg²⁺). In the vegetative cell, the alkaline cations were mainly distributed in the most internal part of the cytoplasm and they were probably associated with starch grains or concentrated in dry vacuoles. Calcium distribution correlated well with the areas in the cytoplasm of the vegetative cell containing a dense network of mitochondria and endoplasmic reticulum. Within the pollen grain, the sperm cell appeared to contain the most calcium. Calcium was also abundant in the sporoderm. These results reveal the potential of SIMS for pollen studies that include germination, the monitoring of air pollutants and the allergens-ion interactions.     

Preparation techniques for electron probe microanalysis of cells

Abstract

Electron probe X-ray microanalysis can be used to determine the elemental composition of cells and tissues in defined functional states. However, reliable quantitative data can be expected only after appropriate cryopreparation techniques. The most versatile preparation and analysis technique can be outlined by four succeeding steps: 1. Cryofixation, 2. Cryoultramicrotomy, 3. Cryotransfer including freeze-drying, 4. Energy dispersive X-ray microanalysis.     

Polyphosphate polymers during early embryogenesis of Periplaneta americana

Inorganic polyphosphates (PolyP) are linear polymers of phosphate (Pi) residues linked by high-energy phosphoanhydride bonds. Despite a wide distribution, their role during insect embryogenesis has not been examined so far. In this study, we show the mobilization of PolyP polymers during the embryogenesis of the cockroach Periplaneta americana. PolyP was detected by enzymatic and fluorimetric assays and found to accumulate in two main sizes by agarose gel electrophoresis. Confocal microscopy showed their presence in small vesicles. In addition, X-ray microanalysis of small vesicles showed considerable amounts of calcium, sodium and magnesium, suggesting an association of PolyP with these elements. Variations of the free Ca⁺², Pi and PolyP levels were observed during the first days of embryogenesis. Our results are consistent with the hypothesis that phosphate ions modulate PolyP variation and that PolyP hydrolysis result in increasing free Ca⁺² levels. This is the first investigation of PolyP metabolism during embryogenesis of an insect and might shed light on the mechanisms involving Pi storage and homeostasis during this period. We suggest that PolyP, mainly stored in small vesicles, might be involved in the functional control of Ca⁺² and Pi homeostasis during early embryogenesis of P. Americana.     

Thermolysis preparation of zinc(II) oxide nanoparticles from a new micro-rods one-dimensional zinc(II) coordination polymer synthesized by ultrasonic method

The reaction of the zinc(II) acetate and 1,4-bis(3-pyridyl)-2,3-diaza-1,3-butadiene (3-bpdb) in presence of perchlorate anions produces a new one-dimensional coordination polymer, {[Zn(μ-3-bpdb)(3-bpdb)₂(H₂O)₂](ClO₄)₂·3-bpdb}_n (1). The compound 1 has been characterized by IR, ¹HNMR and ¹³CNMR spectroscopies. The single crystal X-ray data shows an infinite one-dimensional structure that grows in two- and three-dimensions by hydrogen bonding and π-π stacking. The compound 1 also has been synthesized at micro-size by sonochemical processes and characterized by IR and X-ray powder diffraction (XRD). The scanning electron microscopy (SEM) shows compound 1 has been grown as micro-rod morphology. The thermal stabilities at bulk and micro-size scale were studied by thermal gravimetric (TG) and differential thermal analysis (DTA). The ZnO nanoparticles were obtained by direct calcination at 400 °C under air atmosphere and by thermolysis in oleic acid at 200 °C. The obtained zinc(II) oxide nanoparticles were characterized by X-ray diffraction (XRD) and scanning electron microscopy (SEM).     

Crystalline calcium in littorinid mucus trails

Previous work has shown that the feet of terrestrial and freshwater snails are important in calcium regulation, often secreting granules of CaCO₃. This phenomenon has not, until now, been observed in marine snails. Here we report the presence of CaCO₃ granules in the trail mucus of Littorina littorea (L.), L. saxatilis (Olivi) and L. obtusata (L.) Fixed mucus trails on plastic coverslips were examined by X-ray microanalysis under the SEM. Of the single-metal granules observed in the mucus trails the most abundant were of calcium (means: L. littorea, 440 mm⁻²; L. saxatilis, 401 mm⁻²; L. obtusata, 348 mm⁻²) followed for each species by silicon (maximum mean density: L. saxatilis, 120 mm⁻²) and iron (maximum mean density: L. saxatilis, 65 mm⁻²) granules. Single-metal granules of Al, Ti, Mg and P were also found but only in the mucus trails of L. obtusata, perhaps reflecting its different collection site from the other two species. The mean size of the calcium granules showed significant interspecific variation (L. littorea, 1.32 μm diameter±0 08 μm, n = 143; L. saxatilis, 1.80 μm±0.12, n = 113; L. obtusata, 2.14 μm±0.09, n = 167). Most calcium granules (L. littorea, 80%, n = 35; L. saxatilis, 57%, n = 113; L. obtusata, 69%, n = 167) were attached to, or embedded within, microthreads of mucus which tended to run parallel to the direction of locomotion. The significance of this is unknown although it may imply that the CaCO₃ granules are secreted with the mucus. It is concluded that calcium losses via this route are too small for pedal mucus to function significantly in ionoregulation of calcium. The calcium in the trail may therefore perform other functions, for example indicating trail polarity.     

The use of X-ray microanalysis to demonstrate the uptake of the molluscicide copper sulphate by slug eggs

Summary Copper has been localized in copper sulphate treated eggs of the slug Agriolimax reticulatus (Mü). This has been accomplished using a freeze-fracture technique, the freeze-dried halves of the fractured eggs being analysed in the scanning electron microscope, using both energy dispersive and wavelength dispersive X-ray microanalysis systems. The distribution of copper obtained using these methods has been compared with that achieved using a standard histochemical technique. Both techniques revealed that the copper is initially retained in the perivitelline membranes. The application of X-ray microanalysis to such studies is discussed.This research was supported by the Agricultural Research Council (G.B.), Grant No. AG 72/13     

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.     

Interspecific variations in the surface ultrastructure of the gills of freshwater mullets

SEM studies were made on the gills of freshwater mullets,Rhinomugil corsula andSicamugil cascasia, to correlate surface ultrastructure of various gill units with their probable functions. Two types of lamellated gill rakers of the former fish are suited for plankton feeding and the short, stumpy and transversely beaded gill rakers of the latter reflect the varied food and feeding habit of the fish.R. corsula has numerous mucous glands on the epithelium covering the gill arch and gill filaments,S. cascasia has fewer. In accordance with the differences in the density and distribution of the mucous glands, the microridged epithelial cells also show variations in their architectural plan. In both species the epithelium of the secondary lamellae is smooth, probably an adaptation for better gaseous exchange.