W. Jacob

Influence of fixation procedures on the microanalysis of lead-induced intranuclear inclusions in rat kidney.

Using Laser Microprobe Mass Analysis (LAMMA), we studied the chemical composition of lead-induced intranuclear inclusions in rat kidney tissue prepared by three different wet chemical fixation procedures for transmission electron microscopy. Fixation with glutaraldehyde-Na2S gave the same results as fixation with glutaraldehyde only: a high lead concentration could be detected. Therefore, for lead strongly bound to proteins, precipitation procedures are not essential. Post-fixation with osmium tetroxide drastically changed the composition of the inclusions: the lead concentration decreased substantially, while sodium, calcium, and barium were introduced. The osmium tetroxide fixative was found to be the source of the contamination. It also contained aluminum, and we suggest that other proteins (e.g., in neurofibrillary tangles) might be able to take up Al out of solution and that care must be exercised in interpreting the microanalytical results of osmium-fixed material. For the microanalysis of the lead inclusions, fixation with glutaraldehyde only provides a good compromise between preservation of the ultrastructure and maintenance of the element distribution.

Electron energy-loss spectroscopy and electron probe X-ray microanalysis of exhaust aerosols in slurry nebulization inductively coupled plasma atomic spectrometry for ceramic powders

Abstract Exhaust aerosols in inductively coupled plasma (ICP) atomic spectrometry generated from different ceramic powder suspensions (Al 2 O 3 , ZrO 2 and SiC) were investigated by electron energy loss spectroscopy (EELS) and electron probe X-ray micro analysis (EPXMA). Differences in morphology, size and elemental composition of sub-micrometer residues of these refractory powders, collected above ICPs operated with different gasses, were found to depend on the outer gas (N 2 O 2 and Ar), the distance between the impacting stage and the plasma, and on the ceramic powder introduced as a slurry into a suitable nebulizer. Identical exhaust types of particles were observed in the case of Al 2 O 2 and ZrO 2 slurries, also when different outer gases and different operation conditions were used. For SiC, much depends on the outer gas used, as shown by morphological and chemical composition studies. Electron energy loss spectroscopy and element spectroscopic imaging (ESI) were used for the elemental analysis and the localization of the detected elements (Al, Zr, Si, C, O and N) in the individual exhaust particles.

Methodology for spectrum evaluation in quantitative electron energy‐loss spectrometry using the Zeiss CEM902

The spectral energy‐loss intensities acquired with the Zeiss CEM902 do not follow a Poisson law. The intensities are normally distributed and their variances are non‐uniform. When fitting the inverse power law after logarithmic transformation, it is expected that the power law coefficients and their uncertainties will be biased. Due to the particular dependence of the variance on the intensity which is observed in our experiments, it appears that no bias is introduced when using unweighted least‐squares fitting after log‐log transformation. Because the sample variances are estimated with low degrees of freedom, the residual variance of the fit is proposed as a criterion rather than the X2 test to evaluate the optimum width of the fitting window. The optimal width for the integration window can be determined as the window where the signal‐to‐noise ratio is at a maximum. Critical limits for qualitative and quantitative analysis are proposed which are based on the variance of the net intensity. They can be used to test the analytical significance of the signals.

Analytical electron microscopy of silver halide photographic systems

Studies of silver halide (AgX) photographic materials and individual microcomponents by TEM/STEM/SEM/EDX, CL, EFTEM/EELS and digital image analysis techniques are reviewed. Electron-beam-AgX interactions are discussed to clarify relationships between the signals analysed in various operating AEM modes. An optimum strategy of structural and analytical diagnosis of complex silver halide photographic systems by a number of AEM methods is considered, using a number of examples (colour and black-and-white films, AgX microcrystals, and colour coupler dispersions). AEM applications to study of the main stages of the photographic process, i.e., chemical ripening, spectral sensitisation, latent image formation and development are also presented. Current trends and future directions in research of AgX-based photographic systems by AEM techniques are outlined.

ENERGY-FILTERING TEM AND ELECTRON ENERGY-LOSS SPECTROSCOPY OF DOUBLE STRUCTURE TABULAR MICROCRYSTALS OF SILVER HALIDE EMULSIONS

Composite Ag(Br,I) tabular microcrystals of photographic emulsions were studied by the combination of energy‐filtering electron microscopy (EFTEM) and electron energy‐loss spectroscopy (EELS) in conjunction with energy‐dispersive X‐ray (EDX) microanalysis. The contrast tuning under the energy‐filtering in the low‐loss region was used to observe more clearly edge and random dislocations, {11−1} stacking faults in the grain shells parallel to {11−2} edges and bend and edge contours. Electron spectroscopic diffraction patterns revealed numerous extra reflections at commensurate positions in between the Bragg reflections and diffuse honeycomb contours; these were assigned to the number of defects in the shell region parallel to the grain edges and polyhedral clusters of interstitial silver cations, respectively. Inner‐shell excitation bands of silver halide were detected and confirmed by EDX analyses, i.e. the Ag N2,3 edge at 62 eV (probably overlapped with the weak I N4,5 edge at 52 eV and the Br M4,5 edge at 70 eV), the I M4,5 edge at about 620 eV, and the Br L2,3 edge at about 1550 eV energy losses. Energy‐loss near‐edge structure of the Ag M4,5 edge at about 367 eV energy losses and low‐loss fine structure arisen as a result of interband transitions and excitons, possibly superimposed with many electron effects, have been revealed. The crystal thickness was determined by a modified EELS log‐ratio technique in satisfactory agreement with measurements on grain replicas.

A method for the characterization of surface-modified asbestos fibres by electron energy-loss spectroscopy and electron spectroscopic imaging

A method for the characterization of surface‐treated asbestos fibres with electron microscopy is presented. Electron spectroscopic imaging (ESI) of organosilane‐treated chrysotile asbestos fibres has been carried out. Initially, the region below the carbon edge was inspected in ESI mode for its effectiveness as a background correction. Elemental mapping was performed on standard untreated fibres to take into account non‐characteristic signals from extrapolation errors and camera artefacts. The highest resulting pixel value that results from non‐characteristic signals was used as a threshold for further background correction in the net images. Samples for electron energy‐loss spectroscopy were prepared in two different ways, either by gluing on grids, or by using perforated carbon foils. The results show that the use of a conducting carbon film is necessary for the analysis of such electrically insulating asbestos fibres. Focusing of the electron beam on the individual fibres results in a thermal effect promoting the evaporation of the organosilane reaction products.

Phosphorus, calcium and lead distribution in collagen in lead induced soft tissue calcification : an ultrastructural and X-ray microanalytical study

Repeated intraperitoneal injections of lead acetate in rats caused a calcification of the skin of the abdomen near the site of the injections. In the lead-induced calcifications, electron dense collagen bundles could be observed. On the surface of the collagen fibrils, needle-like crystals were visible. With energy-dispersive X-ray analysis, phosphorus, calcium and lead were detected in the electron dense collagen bundles. X-ray maps of the P-K alpha, Ca-K alpha, and Pb-L alpha plus Pb-L beta lines showed an equivalent distribution along the collagen fibrils for phosphorus and calcium. The occurrence of the most electron dense areas in the STEM-image was comparable to the lead distribution. A good correlation existed between the structural and the elemental images of the same area. Although the medicinal use of preparations containing lead is no longer recommended, some are still prescribed. From our results we can conclude that they should not be applied to injured or inflamed skin.

A simplified ‘sandwich’ technique for in situ embedding and perpendicular sectioning of monolayer cultures of human skin fibroblasts

In the processing of cell cultures, grown as a monolayer in tissue culture dishes for electron microscopy, the sectioning of the monolayer is an essential step. The monolayer can be sectioned either parallel or perpendicular to the plane of growth. Several methods for the perpendicular way of sectioning have already been described. We propose a simplified method in which the monolayer is sandwiched between two layers of resin, one of which is a pre‐polymerized block, the other being a layer of resin, applied at a second stage. Sectioning of this ‘flat embedded’ specimen yields thin sections perpendicular to the plane of growth of the monolayer without elaborate orientating procedures. The advantage of this procedure is that it can be done using only routine embedding techniques, avoiding special materials or complex manipulations. This sandwich technique provides an excellent mechanical fixation of the monolayer and protects it against external damage.

The use of low-cost frame grabber boards on PC-AT computers for ESI: possibilities and limitations

A Zeiss EM902 microscope was connected to a PC‐AT computer, equipped with a Data Translation DT2861 frame grabber board. Using this system, the three‐window background extrapolation algorithm was implemented to perform elemental mapping in the electron spectroscopic imaging mode of an energy filtering transmission electron microscope.

Cryo-analytical electron microscopy: new insight into the understanding of crystalline and electronic structure of silver halide

Recent results of structural and analytical characterization of AgX (X equals Br, I) microcrystals of photographic emulsions by a number of cryo-analytical electron microscopy (AEM) and image analysis techniques are presented. Monte Carlo simulations of electron beam-AgX interactions have been made for a better understanding of relationships between various signals which can be analyzed in the corresponding AEM modes. Combined cryo-ESI/EDX multielement mapping was performed on AgX grain thin sections and on tabular AgX microcrystals. The contrast imaging under filtering with selected energy windows has been applied to image the morphology, crystal and defect structures of composite tabular AgX microcrystals. Extra reflections at commensurate positions in between the main Bragg reflections and weak diffuse intensity honeycomb contours caused by twins and/or {111} stacking faults in the shell region parallel to {112} grain edges and polyhedral clusters of Ag+ interstitials, respectively, were found in selected-area electron spectroscopic diffraction patterns. Low-loss collective excitations due to plasmons and excitons possibly superimposed with interband transitions and many- electron effects were revealed by cryo-EFTEM/EELS. The real and imaginary parts of dielectric permittivity were determined by means of a Kramers-Kronig analysis. Elemental inner shell edges of Ag and halides above 50 eV energy loss have been detected by EELS in accordance with cryo-STEM/EDX- analyses. The local crystal thickness was determined by a modified EELS log-ratio technique in agreement with measurements on grain replicas.