X-ray microanalysis of cell nuclei

The principal component analysis, a multivariate statistical analysis of data, has been used to process X-ray microanalytical data from cell nuclei. Sixty-seven measurements from different areas of chromatin, nucleoli of rat follicular cells, and nucleoli of rat oocyte cells in their antral stage have been studied. The variables are the X-ray characteristic signals for P, S, Al, Fe, Cu, and Zn. This method demonstrates four distinct groups, the chromatin area, which is associated with a higher concentration of P; the compact mass of oocyte nucleolus which possesses the highest content in S, Al, and Zn, and two groups of nucleolar areas. The fibrillar component is richer in S, Al, and Zn than the granular component. The high degree of correlation between these three elements proves the chemical affinity of metals for the proteins (S being the signature for proteins). Cryoembedding in Lowicryl resin at even lower temperatures (213° K in K11M) after quick cryofixation and cryosubstitution in the absence of chemical fixatives gives good ultrastructural preservation and the possibility of simultaneously performing X-ray microanalysis and immuno-cytochemistry.     

X-ray microanalysis of plants containing the platinum group metals

It has been demonstrated that Water hyacinths, grown hydroponically in a solution containing the platinum group metals, accumulate and concentrate some of these metals in their roots. Root samples were examined in a scanning electron microscope, equipped with an energy dispersive spectrometer (SEM-EDS), and platinum was detected on the surface. Further examination in an electron microprobe analyser (EMPA), using both wavelength dispersive spectrometry (WDS) and EDS, show platinum localized in the epidermal region of the root. Ruthenium was detected in root material taken from plants that had accumulated the complexed metal ion from solution.     

生物胺的检测方法评价

生物胺是一类含氮低分子化合物，对动植物和微生物有重要的生理作用。适量的生物胺有助于人体正常的生理功能，过量则会引起不良的生理反应。目前国内外食品中的生物胺的检测主要采用液相色谱，一般仅是组胺检测。实际上许多食品中还含有其它的生物胺（腐胺、尸胺、精胺、亚精胺、色胺、尸胺、苯乙胺、章鱼胺），且其存在会对组胺的毒性有协同作用。因此，多组分生物胺的同时检测对于食品的安全和水产品出口有着重要的意义。本文对国内外比较常见的生物胺检测方法作了叙述和比较。     

Characterization of atmospheric particles by electron probe X-ray microanalysis

Microchemical glass standards were used to validate a quantitation method based on peak-to-background (P/B) ratios from electron probe x-ray microanalysis spectra. This standardless method was applied to the determination of concentrations of individual particles from Malpha or Lalpha lines, as well as from Kalpha lines. The algorithm was tested on particulate glass samples for diameters ranging from 1 to 20 microm. The determined concentrations did not depend on particle size. The certified values for elements were well matched, except for Na, which may migrate under electron bombardment. Finally, classification of qualitative results obtained for aerosol particles was completed by the P/B quantitative method.     

Energy-dispersive X-ray microanalysis of aqueous biologic samples on bulk supports

The present investigation describes a modification of the liquid droplet technique that allows for the quantitative elemental analysis of small volumes (< 100 picoliters) of aqueous biologic samples using a scanning transmission electron microscope (Philips 400 HTG-STEM) equipped with an EDAX energy dispersive detector. Aliquots of samples and standards were micropipetted onto solid beryllium supports under paraffin oil. The oil was washed with organic solvents and the samples frozen and freeze-dried. The samples were excited in a Philips 400-HTG-STEM by scanning a 1-μm, 20-kV electron beam over the surface of the droplets, and the X-ray spectra were collected. Measured X-ray intensities in characteristic peaks were found to be linearly related to the concentration of various elements in the sample. This work demonstrates the feasibility of performing quantitative elemental analysis of minute samples and cells in a scanning transmission electron microscope equipped with an energy dispersive X-ray detector.     

Quantitative X-ray microanalysis of biological specimens

Qualitative X-ray microanalysis of biological specimens requires an approach that is somewhat different from that used in the materials sciences. The first step is deconvolution and background subtraction on the obtained spectrum. The further treatment depends on the type of specimen: thin, thick, or semithick. For thin sections, the continuum method of quantitation is most often used, but it should be combined with an accurate correction for extraneous background. However, alternative methods to determine local mass should also be considered. In the analysis of biological bulk specimens, the ZAF-correction method appears to be less useful, primarily because of the uneven surface of biological specimens. The peak-to-local background model may be a more adequate method for thick specimens that are not mounted on a thick substrate. Quantitative X-ray microanalysis of biological specimens generally requires the use of standards that preferably should resemble the specimen in chemical and physical properties. Special problems in biological microanalysis include low count rates, specimen instability and mass loss, extraneous contributions to the spectrum, and preparative artifacts affecting quantitation. A relatively recent development in X-ray microanalysis of biological specimens is the quantitative determination of local water content.     

Improvement of quantitation of biological X-ray microanalysis

Absolute measurements of elemental concentrations within thin biological samples are often made by reference to a series of standards which resemble the samples in chemical and physical properties and the linear relationship between (p-b)/c and concentration. This principle requires that the chemical and physical properties of the matrix remain constant throughout a series of standards with different elemental contents and throughout different regions of the samples. Some of the changes undergone by specimens during X-ray microanalysis, e.g. loss of elements or organic mass loss, are also influenced by the composition of the matrix. A simple empirical modification to the linear (p-b)/c versus concentration relationship is presented to account for some of these effects and therefore improve quantitation of analyses.     

X-ray microanalysis of freeze-dried and frozen-hydrated cryosections

The elemental composition and the ultrastructure of biological cells were studied by scanning transmission electron microscopy (STEM) combined with energy dispersive X-ray microanalysis. The preparation technique involves cryofixation, cryoultramicrotomy, cryotransfer, and freeze-drying of samples. Freeze-dried cryosections 100-nm thick appeared to be appropriate for measuring the distribution of diffusible elements and water in different compartments of the cells. The lateral analytical resolution was less than 50 nm, depending on ice crystal damage and section thickness. The detection limit was in the range of 10 mmol/kg dry weight for all elements with an atomic number higher than 12; for sodium and magnesium the detection limits were about 30 and 20 mmol/kg dry weight, respectively. The darkfield intensity in STEM is linearly related to the mass thickness. Thus, it becomes possible to measure the water content in intracellular compartments by using the darkfield signal of the dry mass remaining after freeze-drying. By combining the X-ray microanalytical data expressed as dry weight concentrations with the measurements of the water content, physiologically more meaningful wet weight concentrations of elements were determined. In comparison to freeze-dried cryosections frozen-hydrated sections showed poor contrast and were very sensitive against radiation damage, resulting in mass loss. The high electron exposure required for recording X-ray spectra made reproducible microanalysis of ultrathin (about 100-nm thick) frozen-hydrated sections impossible. The mass loss could be reduced by carbon coating; however, the improvement achieved thus far is still insufficient for applications in X-ray microanalysis. Therefore, at present only bulk specimens or at least 1-μm thick sections can be used for X-ray microanalysis of frozen-hydrated biological samples.     

Quantification for the X-ray microanalysis of cryosections

Some problems of the quantitative analysis of diffusible elements in cryosections are reviewed. The two prevalent methods for obtaining concentrations from X-ray data, one based on characteristic radiation alone and the other on continuum-normalization, are recapitulated. Both methods seem suitable at cellular level while the latter seems preferable at finer spatial resolution. Recourse to both methods together is desirable in the analysis of frozen-hydrated sections especially when there is no peripheral standard. Selective local contamination is a particular hazard in the analysis of chlorine. In the case of sodium, physical parameters set restrictive limits to the minimum concentration measurable by ‘energy-dispersive’ X-ray spectrometry (about 20 mm kg^?1) and to the spatial resolution attainable by diffractive X-ray spectrometry (～0·2 μm). One obvious danger to meaningful quantitative analysis is inadvertent redistribution of diffusible elements during the moments preceding the freeze-quenching of a tiny piece of tissue. Data are presented to show that concentration changes due to simple evaporation are a real hazard prior to the quenching of sub-millimetre size samples.     

Biological applications of electron probe microanalysis in france

The contribution of French science to the field of biological X-ray microanalysis is reviewed. The main analytical microscopy centers are listed, and their methods and main results are summarized.     

Cytochemical investigation of the digestive gland of two strombidae species (Strombus gigas and Strombus pugilis) in relation to the nutrition

Strombus gigas and Strombus pugilis are threatened species and aquaculture represents a good alternative solution to the fishing. In this study, we highlighted the intracellular digestion process in the digestive gland of two Strombidae species, S. gigas and Strombuspugilis, by the cytochemical characterization of two lysosomal enzymes: acid phosphatase and arylsulfatase. In order to check the efficiency of artificial food digestion, we conducted the characterization on freshly collected, starved and artificially fed individuals of S. pugilis. TEM observations of digestive gland sections from freshly collected individuals of both species revealed the presence of acid phosphatase and arylsulfatase activity mostly located in the apical third of digestive cells. Both enzymes were also detected in artificially fed individuals. In response to the starvation, acid phosphatase is not produced anymore by digestive cells, while arylsulfatase is still present. To our knowledge, this is the first cytochemical validation of intracellular digestion of artificial food in Strombidae. This study highlights the intracellular digestion of artificial food developed for Strombidae aquaculture. Moreover, we have shown that the lysosomal activity could be used as a feed index. Microsc. Res. Tech. 2012. © 2012 Wiley Periodicals, Inc.     

Mistakes encountered during automatic peak identification in low beam energy X-ray microanalysis

Automated peak identification in electron beam excited X-ray microanalysis with energy dispersive X-ray spectrometry (EDS) is subject to occasional mistakes even on well-separated, high-intensity peaks arising from major constituents. The problem is exacerbated when analysis conditions are restricted to operation in the "low beam energy scanning electron microscopy" (i.e. "low voltage scanning electron microscopy" or LVSEM) regime where the incident beam energy is 5 keV or less. These low beam energy microanalysis conditions force the analyst to use low fluorescence yield L-shell and M-shell peaks rather than higher yield K-shell and L-shell peaks typically selected for elements of intermediate and high atomic number under conventional high beam energy (>10 keV) conditions. Misidentifications can arise in automated peak identification procedures when only a single energy channel is used to characterize an EDS peak. The effect of the EDS measurement process is to convolve the closely spaced Lalpha-Lbeta and Malpha-Mbeta peaks into a single peak with a peak channel shift of 20 eV or more from the Lalpha or Malpha value, which is typically sought in an X-ray database. An extensive list of problem situations encountered in low beam energy microanalysis is presented based upon observed peak identification mistakes as well as likely troublesome situations based upon proximity in peak energy. Robust automatic peak identification requires implementation of peak fitting that utilizes the full peak shape.     

Cryopreparation of mammalian tissue for X-ray microanalysis in STEM

A cryopreparation technique for studies of ultrastructure and distribution of diffusible elements in biological tissue is described. Electron microscopical contrast and characteristic X-ray spectra are found to be poor in completely frozen-hydrated ultrathin cryosections of fresh chemically untreated tissue. Both STEM contrast and detection of characteristic X-rays are enhanced by careful freeze-drying in the microscope. Although the ultrastructure is affected by ice crystals, intracellular compartments can be identified by STEM without staining and studied by X-ray microanalysis.     

Quantitative X-ray microanalysis of hydrogen peroxide within plant cells

Using quantitative X-ray microanalysis in combination with CeCl3-based cytochemical staining of hydrogen peroxide (H2O2) we have developed a new solution for quantification of H2O2 at the subcellular level. Quantitative X-ray microanalysis of plastic-embedded leaves of Populus euphratica Oliv. showed that the obtained cerium precipitates by CeCl3 staining were the mixture of cerium perhydroxides and cerium phosphate, in which the fractions of CePO4 were: (1) 52-74% in cell walls of fresh leaf segments, and (2) 34-70% in the cytoplasm in 10 mM H2O2-treated leaf segments that were previously freeze-dried. Taking the concentration of cerium phosphate as staining background, we reached the cellular concentration of cerium perhydroxides and the corresponding concentration of H2O2. Results showed that H2O2 was present in the cytoplasm of rehydrated leaf segments (29-58 mM), but in fresh leaves, H2O2 was observed in the walls of all measured cell types (17-74 mM).     

Electron spectroscopic imaging and X-ray microanalysis of acrylic fibres

Acrylic fibres are synthetic fibres produced by extruding viscous solutions of acrylonitrile co-polymers. A spin finish is applied during the fibre-forming process. In this work the structure of acrylic fibres has been correlated with spin finish distribution by a combined application of transmission electron microscopy (TEM), electron spectroscopic imaging (ESI) and X-ray microanalysis (XRMA). Many irregularly shaped microcavities at the periphery of the fibres were detected by TEM. XRMA revealed that potassium and phosphorus are the elements most often found inside the spin finish material. ESI revealed that phosphorus is constantly present in all the microcavities. Therefore it seems likely that a preferential distribution of the spin finish is found inside the microcavities, just at the periphery of the fibre. This work is also an example of how both ESI and XRMA are powerful tools in morphological studies.     

Determination of chloride efflux by X-ray microanalysis versus MQAE-fluorescence

The importance of chloride channels for the cell is demonstrated by a number of serious human diseases that are due to mutations in chloride channels. The most well-known of these diseases is cystic fibrosis. Investigations into the mechanisms of the disease and possible treatments require the study of chloride fluxes at the level of individual cells. The present study compares two methods for studies of chloride transport: X-ray microanalysis and MQAE fluorescence with image analysis. As an experimental system, the cAMP-activated chloride channel in cultured respiratory epithelial cells was chosen. Both methods showed that stimulation with the cAMP-elevating agents forskolin and IBMX decreased the chloride content of the cells by about 20-27%. Inducing a driving force for chloride by replacing extracellular chloride by nitrate resulted in a chloride efflux that was significantly increased in the presence of forskolin and IBMX. This study shows that X-ray microanalysis and MQAE fluorescence are adequate and comparable methods for measuring cAMP-dependent chloride transport in individual cells.     

Evaluation of quantitative procedures for X-ray microanalysis of environmental particles

Scanning electron microscopy combined with energy-dispersive X-ray spectrometry is particularly suited to characterizing morphology and elemental composition of individual microparticles. Although not straightforward, quantitative X-ray microanalysis of low-Z-containing particles is achievable using atmospheric thin-window X-ray detectors. A critical aspect of light element analysis is the choice of substrate material. In this work, particles were deposited on specially developed boron substrates. Three case studies were investigated successively in the order of increasing difficulty. Firstly, hundreds of calcium carbonate (CaCO(3)) particles ranging in size from 0.3 to 10 microm were analyzed. Three quantitative procedures were tested: the "k-ratio" method, conventional ZAF correction, and Monte Carlo simulations. Average relative errors obtained by the reverse Monte Carlo quantitative program named CASINO were better than 2.5 wt %, carbon included. Secondly, further evaluation was carried out on a finely crushed biotite mineral, containing more than nine elements. Finally, airborne particulate matter, consisting of a complex heterogeneous mixture of particles, was investigated. By applying the Monte Carlo quantitative procedure, the observed particles were easily classified into particle types. Pure compounds (e.g., CaSO(4).2H(2)O, SiO(2), CaCO(3), etc) were directly assigned according to stoichiometry. In some cases (marine-derived particles), a partial reactivity of atmospheric particles was demonstrated by quantitative analysis.     

Personal-computer-based system for electron beam X-ray microanalysis of biological samples

A system based on a personal computer has been developed which provides a relatively inexpensive way to equip an electron microscopy laboratory for quantitative elemental analyses of cryosectioned biological samples. This system demonstrates the feasibility of making an X-ray analyser from a personal computer, together with commercially available hardware and software components. Hardware and software have been assembled to drive the beam in a scanning electron microscope, collect and analyse X-ray spectra, and save, retrieve, and analyse data. Our software provides a menu-controlled user interface to direct spectra acquisition and analysis. Spot analyses, video images, and quantitative elemental images may be obtained and results transferred in ASCII format to other computers. Wet weight, as well as dry weight, concentrations are calculated, if measurements were made of areas of the hydrated sample before it was freeze-dried. Grey-level copies of video and quantitative elemental images may be made on a laser printer.     

Quantitative study of spurious X-ray production in thin film microanalysis

When X-ray microanalysis is performed in a TEM on a thin area of a specimen, some a priori indistinguishable spurious photons produced in other zones of this specimen are always recorded. Several mechanisms contribute to this production. For instance, some Bremsstrahlung and characteristic photons are generated by secondary and Auger electrons; a conservative upper bound to this particular contribution is calculated for several materials, and the present approach is compared to the Monte Carlo simulation. It is then shown that, in special test-specimens, the total extraneous contribution of the thick parts of the specimen to the spectra recorded in a thin zone can be measured; different instruments may now be compared in this respect. In the HB5 STEM, this total contribution remains low; its main cause is beam scattering in the specimen, not before it. Finally, an experimental procedure for estimating this bulk contribution in any specimen of interest is proposed. Calculations and experiments are illustrated for the case of gallium arsenide.     

Agar standards for quantitative X-ray microanalysis of resin-embedded plant tissues

Calibration standards for quantitative X-ray microanalysis of resin-embedded plant tissue were prepared by adding 6600 mM KC1 to 5% agar. Agar blocks with an edge length of 1–2 mm were rapidly frozen, freeze-dried and embedded in styrene-methacrylate. Dry sections 1 μm thick were mounted on adhesive-coated grids. Apart from fine-scale inhomogeneities caused by ice crystal formation, the KC1 is evenly distributed in the agar blocks. The peak-to-continuum values of K and Cl were highly linearly correlated to the K and Cl contents over the whole concentration range.