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.     

Localization of lanthanum tracer in oral epithelium using transmission electron microscopy and the electron microprobe

Although water soluble tracers have been used to localize intercellular permeability barriers with the transmission electron microscope, there is the possibility of translocation or loss of the tracer during processing. This study compares the localization of lanthanum tracer in keratinized oral epithelium after routine processing with lanthanum seen after using freeze drying to avoid aqueous fixation and dehydration. The electron probe was used to identify the lanthanum tracer in the tissue and to distinguish it from other electron-dense material. After incubating small biopsies of rat palate mucosa in 1% lanthanum nitrate, specimens were either routinely processed for electron microscopy or quick frozen, dehydrated, fixed in osmium vapour and infiltrated with epoxy resin. Examination in the transmission electron microscope indicated that preservation of the freeze dried tissue did not compare favourably with that of normally processed tissue, but the distribution of the electron-dense tracer in the intercellular spaces and the extent of the penetration through the epithelia was similar in the two types of preparations. Confirmation of the tracer as lanthanum was obtained by wavelength dispersive X-ray analysis with the electron probe. The results indicate that no appreciable shift in the localization of the tracer is introduced by routine aqueous fixation and dehydration for electron microscopic examination.     

Energy Dispersive X-ray Tomography for 3D Elemental Mapping of Individual Nanoparticles

Energy dispersive X-ray spectroscopy within the scanning transmission electron microscope (STEM) provides accurate elemental analysis with high spatial resolution, and is even capable of providing atomically resolved elemental maps. In this technique, a highly focused electron beam is incident upon a thin sample and the energy of emitted X-rays is measured in order to determine the atomic species of material within the beam path. This elementally sensitive spectroscopy technique can be extended to three dimensional tomographic imaging by acquiring multiple spectrum images with the sample tilted along an axis perpendicular to the electron beam direction.Elemental distributions within single nanoparticles are often important for determining their optical, catalytic and magnetic properties. Techniques such as X-ray tomography and slice and view energy dispersive X-ray mapping in the scanning electron microscope provide elementally sensitive three dimensional imaging but are typically limited to spatial resolutions of > 20 nm. Atom probe tomography provides near atomic resolution but preparing nanoparticle samples for atom probe analysis is often challenging. Thus, elementally sensitive techniques applied within the scanning transmission electron microscope are uniquely placed to study elemental distributions within nanoparticles of dimensions 10-100 nm.Here, energy dispersive X-ray (EDX) spectroscopy within the STEM is applied to investigate the distribution of elements in single AgAu nanoparticles. The surface segregation of both Ag and Au, at different nanoparticle compositions, has been observed.     

The kinetics and mechanism of lead (II) biosorptionby powderized Rhizopus oligosporus

The kinetics and mechanism of lead biosorption by powderized Rhizopus oligosporus were studied using shake flask experiment. The optimum biomass concentration and initial solution pH for lead sorption at initial lead concentrations ranging from 50–200 mg/l was obtained at 0.5 g/l and pH5, respectively. In term of the ratio of initial lead concentration to biomass concentration ratio, the highest lead adsorption was obtained at 750 mg/g which gave the maximum lead uptake capacity of 126 mg/g. The experimental data of lead sorption by R.oligosporus fitted well to the Langmuir sorption isotherm model, indicating that the sorption was similar to that for an ion-exchange resin. This means that the sorption is a single layer metal adsorption that occurred as a molecular surface coverage. This assumption was confirmed by the examination of lead sorption using transmission electron microscope and energy dispersive X-ray analysis, which showed that during sorption most of the lead was adsorbed on the surface of cell.     

Comparing the Detection of Iron-Based Pottery Pigment on a Carbon-Coated Sherd by SEM-EDS and by Micro-XRF-SEM

The same sherd was analyzed using a scanning electron microscope with energy dispersive spectroscopy (SEM-EDS) and a micro X-ray fluorescence tube attached to a scanning electron microscope (Micro-XRF-SEM) to compare the effectiveness of elemental detection of iron-based pigment. To enhance SEM-EDS mapping, the sherd was carbon coated. The carbon coating was not required to produce Micro-XRF-SEM maps but was applied to maintain an unbiased comparison between the systems. The Micro-XRF-SEM analysis was capable of lower limits of detection than that of the SEM-EDS system, and therefore the Micro-XRF-SEM system could produce elemental maps of elements not easily detected by SEM-EDS mapping systems. Because SEM-EDS and Micro-XRF-SEM have been used for imaging and chemical analysis of biological samples, this comparison of the detection systems should be useful to biologists, especially those involved in bone or tooth (hard tissue) analysis.     

Subcellular distribution of chromium in accumulating plant Leersia hexandra Swartz

The subcellular distribution of chromium in Leersia hexandra Swartz, a Cr-accumulating plant found in China, was studied by differential centrifugation, transmission electron microscope and energy dispersive analysis of X-ray. Subcellular fractionation of Cr-containing tissues showed that most of the accumulated Cr was isolated to the cell walls in roots and the vacuoles in leaves. When the plant was grown in a nutrient solution containing 60 mg L^?1 Cr, 83.2% of the root Cr was localized in the cell wall fraction, while 57.5% of leaf Cr was localized in the vacuole and cytoplasm fraction. Transmission electron microscopic analysis revealed that those cell compartments contained especially electron dense areas. Energy dispersive X-ray spectra showed the electron dense areas contained high Cr. However, the dark electron precipitates were never observed in the plant cells without Cr treatment. In all treatment groups (5, 30 and 60 mg L^?1), the fraction containing the lowest level of Cr was the organelle fraction in roots as well as leaves. These results indicated that Cr accumulated in the L. hexandra was preferentially stored in the cell walls of roots and the vacuoles of leaves. This phenomenon diverted Cr ions from metabolically active compartment (chloroplast, mitochondria), resulting in a reduction of Cr toxicity in the plant cell.     

Combined structural and chemical analysis of the anammoxosome: a membrane-bounded intracytoplasmic compartment in anammox bacteria

Anammox bacteria have unique intracellular membranes that divide their cytoplasm into three separate compartments. The largest and innermost cytoplasmic compartment, the anammoxosome, is hypothesized to be the locus of all catabolic reactions in the anammox metabolism. Electron tomography showed that the anammoxosome and its membrane were highly folded. This finding was confirmed by a transmission electron microscopy study using different sample preparation methods. Further, in this study electron-dense particles were observed and electron tomography showed that they were confined to the anammoxosome compartment. Energy dispersive X-ray analysis revealed that these particles contained iron. The functional significance of a highly folded anammoxosome membrane and intracellular iron storage particles are discussed in relation to their possible function in energy generation.     

The X-ray microanalysis method for investigation of chemical composition of normal and pathologic human peripheral blood lymphocytes

The X-ray microanalysis method for investigation of chemical composition, mainly trace elements, of normal and pathologic human peripheral blood lymphocytes has been presented. The method in question consists of two stages. During the first stage the scanning electron microscope with the energy dispersive X-ray spectrometer has been used. The second stage of the method consisted of employing the above microscope with the microwave dispersive spectrometer. The method enables the short-term determination of the various elements content within the lymphocytes with particular insight into the content of elements in a single cell. The preliminary investigations showed significant differences in the content of chlorine, sulfur and lead in normal human lymphocytes and that from patients with cancer of the larynx.     

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.     

Ultrastructural localization of silver deposits in the receptacle cells of carnation flowers

Carnations were treated with a silver thiosulphate complex to prevent wilting of the flowers. The ultrastructural localization of silver and sulphur in the receptacle tissue was investigated by electron microscopy. Electron-dense deposits were present in the receptacle tissue. Coarse-grained deposts (diam. 60–100 nm) were predominantly observed at the inner side of the cell wall, whereas fine-grained deposits (diam. 20–60 nm) were predominantly present inside the cell-wall region and in the intercellular spaces. These particles were analyzed for chemical elements by X-ray analytical electron microscopy (Philips EM 400 plus Edax energy dispersive analyzer, type 711). In both types of deposits, the presence of silver and sulphur was verified. Point analysis revealed that in both precipitates the S/Ag ratio was of the same order.Abbreviations CTEM conventional transmission electron microscope - STEM scanning transmission electron microscope The AEM unit is a joint unit of the Erasmus University of Rotterdam, the University of Leyden, and the Health Organization TNO. The analytical microscope was purchased with a grant from the Dutch Organization for Pure Scientific Research (ZWO) through BION     

Some of the properties of flame retardant medium density fiberboard made from rubberwood and recycled containers containing aluminum trihydroxide

The flame retardancy of medium density fiberboard (MDF) made from mixture of rubberwood fibers and recycled old corrugated containers was studied. Aluminum trihydroxide (ATH) was used as a fire retardant additive and mixed with the fibers to manufacture experimental MDF panels using wet process. Phenol formaldehyde (PF) resin in liquid, 2% based on oven dry weight of fibers, was used along with 0%, 10%, 15% and 20% of ATH. The flame retardant test was done using the limiting oxygen index (LOI) test. The other properties investigated include internal bond strength, thickness swelling and water absorption. The results showed that ATH loading increased as the LOI of MDF increased. This demonstrated that ATH could improved the fire retardant property of MDF at sufficient loading. An increase in concentration of ATH showed an increase in the IB values of MDF made without resin. MDF panels made without resin showed a progressive increase in internal bond as the composition of recycled old corrugated containers fiber increased. Addition of resin improved internal bond strength and reduced thickness swelling, and water absorption. Thickness swelling of panel increased as the composition of recycled old corrugated containers fiber increased. Scanning electron microscopy (SEM) and energy dispersive X-ray analysis (EDAX) showed that there is indication of ATH and resin filling the void space in between fibers.     

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     

Mitigation of the corrosion-causing Desulfovibrio desulfuricans biofilm using an organic silicon quaternary ammonium salt in alkaline media simulated concrete pore solutions

Abstract

Organic silicon quaternary ammonium salt (OSA), an environmentally friendly naturally occurring chemical, was used as a bacteriostatic agent against sulphate-reducing bacteria (SRB) on a 20SiMn steel surface in simulated concrete pore solutions (SCP). Four different media were used: No SRB (NSRB), No SRB and OSA (NSRB?+?OSA), With SRB (WSRB), With SRB and OSA (WSRB?+?OSA). After biofilm growth for 28 days, optimized sessile SRB cells survived at the high pH of 11.35 and as a result these cells caused the breakdown of the passive film due to the metabolic activities of the SRB. Corrosion prevention results showed that the OSA was effective in mitigating the growth of the sessile SRB cells and reduced corrosion in the SCP. These results were further confirmed by scanning electron microscope images, energy dispersive X-ray analysis, confocal-laser scanning microscopy, X-ray photoelectron spectroscopy and corrosion testing using electrochemical analysis.     

Manganese in the shell of Centropyxis (Rhizopodea: Protozoa)

Summary The proteinaceous shells of Centropyxis hirsuta contain a relatively high concentration of manganese in an amorphous state. The concentrations of manganese fill the alveoli which are characteristic of the shell structure. Observations based on cultured animals and subsequently examined by X-ray diffraction, a solid state energy dispersive X-ray analyser, and an analytical electron microscope, lead to the conclusions that manganese is selectively absorbed by Centropyxis hirsuta and deposited in the shell.     

A post-embedding avidin-biotin peroxidase system to demonstrate the light and electron microscopic localization of lectin binding sites in rat kidney tubules

Summary A post-embedding method for the light and electron microscopic demonstration of lectin binding sites in rat kidney tubules is described. The use of biotinylated lectins, followed by treatment with avidin peroxidase and the DAB—H₂O₂ sequence, produced intense staining of acrylic sections at the electron microscope level: brush borders and associated structures, cytoplasmic granules, basal infoldings and basement membrane—plasmalemmal interfaces of proximal tubules bound erythrophytohaemagglutinin, while distal tubules were mainly unstained. At the light microscope level, epoxy resin sections showed a similar staining pattern after etching, as did acrylic resin sections after intensification of the final reaction product. The binding of wheatgerm agglutinin to cytoplasmic granules and brush border structures in the proximal tubules was abolished, at both the light and electron microscope levels, by the competing sugar tri-N—acetylchitotriose. Epoxy resin ultrathin sections required etching before staining was achieved in the electron microscope, and results were far inferior to those obtained with acrylic resin. This method allows rapid and inexpensive screening of large numbers of lectins, if required, at both the light and electron microscope levels, using reagents that are stable for long periods of time.     

Qualitative electron probe analysis of secretory ameloblasts and odontoblasts in the rat incisor

Summary Rapidly frozen growing rat incisors were freeze fractured and freeze dried in preparation for energy dispersive X-ray emission microanalysis in a scanning electron microscope. Ca levels were found to be elevated in the distal cell body of odontoblasts, whereas Ca was uniformly low over all parts of the cell body of secretory ameloblasts. The results suggest fundamental differences in the mechanisms by which these two cell types process Ca, and that Ca possibly diffuses through the secretory ameloblast layer on its way to the enamel.     

Novel technique for internal structure and elemental distribution analyses of granular sludge from reactors for wastewater treatment

A novel technique for internal structure and elemental distribution analyses of granular sludge is presented. Sludge samples were freeze-dried and embedded in epoxy resin to form a module, which were then ground and polished to obtain sequential cross-sections. The cross-sections were analyzed by scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy (EDX). SEM observations showed that one granule was formed having several cores with different inorganic minerals, rather than a single core. EDX results indicate that the main elements of the granules are O, Ca, Mg, and P. In addition, the distribution areas of calcium and magnesium in the granule do not coincide.     

Biosorption of Cd(II) from Aqueous Solution by Pseudomonas plecoglossicida: Kinetics and Mechanism

In our present study, we investigated the mechanism of Cd(II) biosorption from aqueous solution by Pseudomonas plecoglossicida using different instrumental techniques. The adsorption kinetics fitted well with the pseudo second-order model, suggesting that the Cd(II) adsorption by P. plecoglossicida consisted of a chemisorption and a physisorption process. Compared with the dead P. plecoglossicida cells, the live cells demonstrated the same adsorption capacity of Cd(II). Scanning electron microscope with energy dispersive X-ray spectroscopy analysis revealed that the main mechanism of adsorption was the combination of Cd(II) with the organic functional groups in the cell wall of P. plecoglossicida. Furthermore, Fourier transform infrared spectroscopic analysis of the metal-loaded biosorbent confirmed the participation of -NH, -OH, -CH, and -CONH groups in the uptake of Cd(II). Moreover, cation transport test revealed that ionic exchange interactions were involved in the Cd(II) adsorption. However, it only played a minor role in the Cd(II) biosorption process.     

Concentration of elements in mitotic chromatin as measured by x-ray microanalysis

Unfixed frozen-dried and uncoated tissue sections of the mouse duodenum were placed on carbon planchets and analyzed in a scanning electron microscope fitted with energy dispersive X-ray equipment. Computer analysis of the X-ray spectra allowed elemental microanalysis of the nucleus, cytoplasm, and mitotic chromatin regions in the cryptal and villus enterocytes. The peak to continuum ratio of S, Cl, K, and Ca were higher in mitotic chromatin than any of the other sites measured. The redistribution of Ca at mitosis is postulated to help explain both chromosome condensation and assembly of the mitotic spindle apparatus.     

A review of sources of spurious silicon peaks in electron microprobe X-ray spectra of biological specimens.

The presence of Si peaks in X-ray spectra of biological specimens can lead to misinter-pretation of data if one is unaware of potential sourees of artifactual Si. Silicontcontamination may be acquired from oils or greases during the course of specimen preparation or from the vacuum system of the electron microscope. In energy dispersive X-ray detection, the level of the Si peak cannot be reduced below that of the internal fluorescence peak obtained from the dead layer of the detector.