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.     

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.     

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.     

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.     

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     

Research of the entry of rare earth elements Eu3+ and La3+ into plant cell

Whether rare earth elements can enter into plant cells remains controversial. This article discusses the ultracellular structural localization of lanthanum (La³⁺) and europium (Eu³⁺) in the intact plant cells fed by rare earth elements Eu³⁺ and La³⁺. Eu-TTA fluorescence analysis of the plasmalemma, cytoplast, and mitochondria showed that Eu³⁺ fluorescence intensities in such structures significantly increased. Eu³⁺ can directly enter or be carried by the artificial ion carrier A23187 into plant cells through the calcium ion (Ca²⁺) channel and then partially resume the synthesis of amaranthin in the Amaranthus caudatus growing in the dark. Locations of rare earth elements La³⁺ and Eu³⁺ in all kinds of components of cytoplasmatic organelles were determined with transmission electron microscope, scanning electron microscope, and energy-dispersive X-ray microanalysis. The results of energy-dispersive X-ray microanalysis indicated that Eu³⁺ and La³⁺ can be absorbed into plant cells and bind to the membranes of protoplasm, chloroplast, mitochondrion, cytoplast, and karyon. These results provide experimental evidence that rare earth elements can be absorbed into plant cells, which would be the basis for interpreting physiological and biochemical effects of rare earth elements on plant cells.     

Immunolocalization of alkaloids and X-ray microanalysis of elements in lupin seeds

Summary Immunolocalization of alkaloids in lupin seeds (Lupinus spp.) has been performed by cryofixation and conventional methods. Alkaloids were localized in the protein bodies of the cotyledon cells. Some immunogold particles in the walls of these cells were also observed. There were no differences in the sites of localization between the two mentioned methods. X-ray microanalysis of elements showed the presence of P, Mg, S, and K in the protein bodies of cotyledon cells in lupin seeds. The role of K⁺ in alkaloids transport is discussed.     

The effects of the adrenoreceptor agonists phenylephrine and isoproterenol on the intracellular ion concentrations of branchial epithelial cells of brown trout (Salmo trutta L.)

Brown trout were fitted with indwelling, intraperitoneal catheters and injected with 4–6 mol · kg^-1 of the -receptor agonist phenylephrine or the -receptor agonist isoproterenol. The intracellular concentrations of sodium, chlorine, potassium and phosphorus in the pavement epithelial cells and the mitochondria-rich cells of the branchial epithelium were measured by X-ray microanalysis 1 h after the injection of the adrenoreceptor agonists. Injection with phenylephrine resulted in a significant increase in intracellular chlorine and potassium in mitochondria-rich cells and a significant but relatively smaller increase in chlorine in pavement epithelial cells. Injection with isoproterenol resulted in a significant increase in sodium and chlorine concentration in pavement epithelial cells and a significant decrease in potassium concentration. The only significant effect of isoproterenol injection on mitochondria-rich cells was a decrease in intracellular chlorine concentration. The results suggest that these adrenoreceptor agonists have a direct effect on the influx of Na⁺ and Cl^- across the branchial epithelium. These effects may be a mechanism for acid-base regulation during the severe stress conditions that elicit catecholamine release in vivo. These results corroborate previous studies using X-ray microanalysis which suggested that pavement epithelial cells are the sites of Na⁺ uptake in freshwater fish whilst Cl^- uptake occurs via mitochondria-rich cells.Abbreviations LTSEM low-temperature scanning electron microscope - MR cells mitochondria-rich cells - PE cells pavement epithelial cells - XRMA X-ray microanalysis     

A new approach for rhizosphere research by X-ray microanalysis of microliter soil solutions

Lolium perenne growing with high root density on a fine nylon mesh (Kuchenbuch and Jungk, 1982) caused the development of element gradients in the rhizosphere below the mesh. Micro-liter soil solutions from 2-mg soil samples were sprayed onto Formvar-coated grids and analyzed by X-ray microanalysis in a transmission electron microscope. The results were comparable to those obtained by flame photometry and atomic absorption spectrometry (AAS) of conventional soil solutions from 1 g soil. X-ray microanalysis of micro-soil solutions allows the application of different extraction procedures to even small amounts of soil usually available from rhizosphere experiments. Information about soil buffering characteristics in the rhizosphere can thus be obtained. Aluminum accumulation in the rhizosphere of small segments of single Picea abies fine roots grown in undisturbed natural forest soil could be detected with this technique.     

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.     

Application of Synchrotron Radiation X-Ray Fluorescence (μ-SRXF) and X-Ray Microanalysis (SEM/EDX) for the Quantitative and Qualitative Evaluation of Trace Element Accumulation in Woody Plants

Different analytical techniques were applied to describe the localization of lead and chromium in the tissues of walnut (Juglans regia) and maple (Acer saccharinum) plants exposed to soils that had been artificially contaminated with heavy metals. Two X-ray-based techniques, synchrotron radiation X-ray fluorescence (μ-SRXF) and X-ray microanalysis (SEM/EDX), were utilized in association with induced coupled plasma optical emission spectrometry (ICP-OES). These techniques allowed the definition of maps showing a preferential accumulation of lead in the root periderm of both plants and a limited translocation of the metal to the stems. Accordingly, estimation of the lead concentration with ICP-OES showed that roots contained, on a dry weight basis, 20 to 40 times more metal than stems. Chromium, supplied to the plants as Cr³⁺ or Cr⁶⁺, was taken up only in the latter case and in limited amounts. In general, walnut was more efficient than maple in the uptake of lead and chromium. The combination of X-ray-based techniques and ICP-OES proved to be a powerful quantitative tool in mapping, with high definition, the sites of metal storage in plant tissues.     

Investigations on the cuticle of the polychaete elytra using energy dispersive X-ray analysis

Energy dispersive X-ray analysis of elements with Z>11 in a SEM (scanning electron microscope) was used to investigate the elytra ofLagisca extenuata, Lepidonotus clava, andHarmothoe areolata from Naples (Italy) and Banyuls (France). High concentrations of halogens and a few other elements were found in certain papillae in samples from both locations. Additional TEM-examinations and X-ray analysis of thin sections revealed that the halogen concentration is inversely related to the collagen content of the matrix. The halogens are presumably bound to tyrosines, which occur in these structures. In addition, accumulation of Mn²⁺ and possibly Fe³⁺ in the papillae might depend on environmental conditions. The results show that valuable information about the chemical composition of biological structures can be obtained by energy dispersive X-ray analysis. Moreover, the results indicate that this method may be useful for environmental investigations.This work is part of a doctoral thesis, written at the Zoological Institute of the University at Hamburg.     

X-ray microanalysis of ion distribution within root cortical cells of the halophyte Suaeda maritima (L.) Dum.

The ion content of compartments within cortical cells of mature roots of the halophyte Suaeda maritima grown at 200 mol·m^-3 NaCl has been studied by X-ray microanalysis of freeze-substituted thin sections. Sodium and Cl were found in the vacuoles at about four-times the concentration in the cytoplasm or cell walls, whereas K was more concentrated in the cell walls and cytoplasm than in vacuoles. The vacuolar Na concentration was 12- to 13-times higher than that of K. The Na concentration of cell walls of cortical cells was about 95 mol·m^-3 of analysed volume. The cytoplasmic K concentration within the mature cortical cells was estimated to be 55 mol·m^-3 of analysed volume.     

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.     

Application of X-ray microanalysis to cell suspensions of oil palm (Elaeis guineensis Jacq.)

X-ray microanalysis has been used to determine the elemental composition of oil-palm (Elaeis guineesis) cell suspensions without the use of cryoprotectants. Results based on individual cells were gathered over a typical growth cycle of 14 d. During the log phase (5–7 d) there is an increase in the number of cells containing high concentrations of both K (400 mmol kg^-1 dry weight) and P (400 mmol kg^-1 dry weight). Morphologically these cells had thin cell walls and were frequently joined to other cells (two to five cells per clump).     

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.     

Parazoanthus axinellae Extract Incorporated Hybrid Nanostructure and Its Potential Antimicrobial Activity**

This study, it was aimed to examine the change in the antimicrobial effect of sea anemone Parazoanthus axinellae extract by forming its nanoflowers. A scanning electron microscope (SEM) and energy dispersive X-ray spectroscopy (EDX) were expended to observe the morphologies of the Cu NFs that had been produced. Fourier transform infrared spectroscopy (FT-IR) and X-ray diffraction (XRD) techniques were expended to analyze the managing assemblies in P. axinellae extract, which perform an effective part in the synthesis routine, as well as the crystal assembly of NFs. P. axinellae extract mediated the HNFs (Hybrid nanoflowers) are at high, pure crystalline nature, flower shape with a crystallographic system at the nanoscale with mean crystallite size 21.9 nm using XRD, and average particle size ~10 nm by SEM. The broad absorption band at 2981–2915 cm⁻¹ in the FT-IR spectra of anemone extract and Cu-anemone NFs represents the unique peak of hydroxy groups. In addition, Cu NFs were tested for their antibacterial properties. Cu NFs have been discovered to exhibit antibacterial properties. It is suggested that P. axinellae extract and various inorganic components be used to synthesize a variety of NFs and assess their suitability for usage in biomedical fields.     

Chloride and sulphur concentrations in chloroplasts of spinach

Chloride concentrations within individual chloroplasts, the adjacent cytoplasm and nearby vacuoles of spinach mesophyll cells (Spinacea oleracea L. cv. Hybrid 102) were determined by means of electron probe X-ray microanalysis in the cleavage plane of quench frozen tissue, which was maintained at liquid nitrogen temperatures. The accuracy of quantitative data obtained with this technique is greatly improved by the adoption of a peak to background ratio method and use of carbon slurry standards, which mimic the quench frozen tissue and its X-ray fluorescence. Chloroplasts were incapable of maintaining relatively high levels of Cl^? under conditions of low Cl^? availability (zero Cl^? or 20 μM Cl^? in nutrient solution), and under conditions of Cl^? stress (100 or 200 mM Cl^?) chloroplasts had only a limited capacity to maintain a Cl^? concentration at a level below that of the cytoplasm and vacuole. However, under conditions of Cl^? stress the concentration of Cl^? in cytoplasm immediately adjacent to chloroplasts was substantially higher than in the chloroplasts or more distant cytoplasm. Thus, Cl^? levels in chloroplasts are apparently not as tightly regulated as was suggested by estimates of Cl^? concentration based on aqueous isolation of chloroplasts. Levels of S in chloroplasts were relatively high (equivalent to 40–60 mM SO₄^2? in S standards) and constant for all treatments, with the possible exception of lower S levels in chloroplasts of leaves approaching premature senescence as a result of salt stress. It is implied that the stability of the S-content results largely from its presence in macromolecular components of chloroplasts (sulfolipids and proteins).     

Applications of Environmental Scanning Electron Microscopy (ESEM) in botanical research

Abstract

Conventional Scanning Electron Microscopy (SEM) is limited by artefacts from sample preparation, while Environmental Scanning Electron Microscopy (ESEM) permits observations of hydrated, non-conductive samples without any preparation. In this short review, the two systems are described and some examples given. In addition, a study of trace element localization by X-ray ESEM microanalysis in Azolla caroliniana cultured in the presence of trace elements is presented. The highest concentration occurred in roots and stem. Leaves showed lower accumulation, with concentrations decreasing from the base to the apex of the shoot, and sharp differences between ventral and dorsal lobes of single leaves, the former accumulating more than the latter. The epidermal cells in the ventral lobes of basal leaves were largely lost in treated plants. The differential localisation of trace elements in the plant protected the dorsal lobes, which are the main photosynthetic part of the plant, the nitrogen-fixing cyanobacterial colonies and the apical meristems from potentially adverse effects of trace elements.     

TRACKING POLLEN FLOW OF SOLANUM ROSTRATUM (SOLANACEAE) USING BACKSCATTER SCANNING ELECTRON MICROSCOPY AND X-RAY MICROANALYSIS

A technique using micronized metal powders was developed for both general labeling of pollen and marking of individual pollen grains. After labeling, pollen flow is analyzed by the use of backscatter scanning electron microscopy and X-ray microanalysis. To test the efficiency and efficacy of the technique, we assessed differences in pollen distribution in Solanum rostratum, an enantiostylous species with dimorphic anthers which are putatively feeding and pollinating anthers. Pollen from each set of anthers was labeled using different micronized metal powders. We could not confirm the differentiation of functional anthers in S. rostratum. This technique provides an efficient and convenient method for tracking pollen movement within and between flowers, and anthers within a single blossom can be differentially marked.