Quantitative and qualitative characterization of aquatic iron oxyhydroxide particles by EF-TEM

Electron energy-loss spectroscopy (EELS) and elemental imaging under the energy-filtered transmission electron microscope are powerful tools for the characterization of iron-rich particles present in natural waters. Features present in EEL spectra (Fe-M_2,3 Fe-L_2,3 and O-K ionization edges) of goethite (α-FeOOH) have been studied with an energy filter operated at 80 keV to determine optimal quantification and elemental imaging of Fe-rich natural aquatic particles in the 30–200 nm range of thickness. For quantitative aims, the Fe-M_2,3 ionization edge cannot be used easily, but the Fe-L_2,3 edge provides more accurate results owing to a better background extrapolation. The partial cross-section of the Fe(III) M shell has been determined for iron oxide. The use of two-windows (jump-ratio) and three-windows (background stripping) imaging methods is discussed in relation to the specimen thickness.     

Elemental composition of pyroantimonate precipitates analysed by electron spectroscopic imaging (ESI) and electron energy-loss spectroscopy (EELS) in vitellogenic ovarian follicles of Drosophila

Ca²⁺ was precipitated with potassium antimonate in vitellogenic follicles of the fruit fly Drosophila melanogaster and the distribution of the precipitates formed was studied by electron microscopy. The microvilli of the oolemma in mid- and late vitellogenic follicles were lined with precipitates. The chemical composition of the precipitates was analysed by electron spectroscopic imaging (ESI). The images produced by inelastically scattered electrons at specific ionization edges were compared, and the nonspecific background signals were subtracted by an image processing system. The presence of Ca²⁺, antimony and oxygen in the precipitates formed could be demonstrated. The elemental composition of the precipitates and of yolk spheres was also analysed by electron energy-loss spectroscopy (EELS). With respect to the precipitates, signals at the calcium L_2,3-edge, the oxygen K-edge and the antimony M_4,5-edge were recorded without deconvolution and background subtraction. The yolk spheres, which were free of precipitates, gave the characteristic signal of the nitrogen K-edge. The applied techniques combine good ultrastructural resolution with the possibility of analysing the elemental composition of histochemical reaction products and cellular structures.     

Localization of Ca2+ -stores and tissue compartments with a Ca2+ -binding capacity in the organ of Corti of the guinea-pig by electron energy-loss spectroscopy

The addition of 10 mM CaCl₂ to glutaraldehyde fixative leads to the formation of small electron-dense deposits in the organ of Corti of the guinea-pig. These precipitates are mainly attached to cell membranes in contact with different extracellular lymphatic fluids. A higher number of precipitates is localized in the acellular parts of tectorial and basilar membrane. Electron energy-loss spectroscopy (EELS) was used to determine the elemental composition of the deposits formed. The spectra showed a prominent signal at the Ca²⁺ L_2,3 ionization edge. Oxygen could also be detected in all the precipitates analysed. EELS analysis of mitochondria of the inner and outer hair cells after conventional fixation (glutaraldehyde followed by post-fixation in OsO₄) revealed a small but significant calcium signal.     

Electron spectroscopic study (ESI,EELS) of Nanoplast-embedded mammalian lung

The potential of Nanoplast melamine resin embedding for the study of mammalian lung parenchyma was examined by means of electron spectroscopic imaging (ESI) and electron energy-loss spectroscopy (EELS). Samples were either fixed with glutaralde-hyde-paraformaldehyde or glutaraldehyde-tannic acid, or were directly transferred to the embedding medium without prior fixation. Organic dehydrants, as well as fixatives containing heavy metals and stains, were omitted. A very high level of ultrastructural detail of chromatin, ribosomes, mitochondria and plasma membranes was achieved by ESI from the Nanoplast-embedded samples. The most prominent gain in ultrastructural detail was achieved when moving from an energy loss just below the L_2,3 edge of phosphorus at 132 eV to an energy loss just beyond this edge. This reflects the prominent P L_2,3 edge observed by EELS of Nanoplast-embedded samples in comparison with conventionally processed samples. Thus, taking into account possible sectioning artefacts, excellent heterochromatin images which rely on the phosphorus distribution can be obtained from Nanoplast-embedded samples by computer-assisted analysis of electron spectroscopic images. In this respect glutaraldehyde-paraformaldehyde fixation is preferable to glutaraldehyde-tannic acid fixation because the presence of silicon, revealed by EELS, in tannic-acid-fixed samples may introduce artefacts in phosphorus distribution images obtained by the three-window method because of the close proximity of the L_2,3 edges of silicon and phosphorus.     

Quantitative electron probe microanalysis of nitrogen

Quantitative electron probe microanalysis of nitrogen has been performed on 18 nitride specimens in the voltage range of 4–30 kV. A conventional lead stearate and a new W/Si multilayer analyzer crystal (2d = 5.98 nm) were used simultaneously. The net peak count rates on the latter crystal were approximately 2.8 times higher than on the lead stearate crystal. In addition, significant improvements in the peak to background ratios were achieved, which proved critical when dealing with difficult nitrides such as ZrN, Nb₂N, and Mo₂N. A most conspicuous feature of the new crystal, however, is its effective suppression of higher-order reflections in the wavelength range of N-Kα. As a result, backgrounds are free of interfering lines and spectral artifacts, which facilitates accurate background determination. Two procedures for the analysis of nitrogen in the presence of titanium are also discussed. One procedure is based on the assumption that the area–peak factor (APF, integral k-ratio/peak k-ratio) of the N-Kα peak in Ti–N compounds relative to a nitrogen standard (Cr₂N) has a fixed value which can be used in order to separate the Ti-_ll peak from the N-Kα peak. Extensive tests on a number of compositions in the Ti–N system have shown that it is possible to do quantitative analysis of nitrogen in Ti–N compounds with a relative accuracy of better than 5%. For very low levels of nitrogen (below 15 at%) the APF method was found unsuitable. In such cases, however, the well-known multiple least-squares digital-fitting techniques appeared to do an excellent job. In order to account for the effects of peak shape alterations in the N-Kα peak, all nitrogen spectra were recorded in integral fashion and compared with those of the Cr₂N standard. The shape alterations for the N-Kα peak were much smaller than for B-Kα and C-Kα radiations: The observed values for the area–peak factor relative to Cr₂N differed less than 5% from unity. The present work has resulted in a database containing 144 integral k-ratios for N-Kα and 149 peak k-ratios for the x-ray lines of the metal partners in the nitrides. Our own Gaussian ?(pz) matrix correction procedure “PROZA,” used in conjunction with a set of improved mass absorption coefficients, yielded excellent results for the nitrogen data: An average value of k_calc/K_meas of 1.0051 and a relative root mean square deviation of 3.99%.     

Study of structures at the boundary and defects in organic thin films of perchlorocoronene by high-resolution and analytical transmission electron microscopy

Both the periodic and non-periodic structures of perchlorocoronene (C₂₄Cl₁₂) crystals were characterized by high-resolution transmission electron microscopy (HRTEM), electron diffraction (ED), electron energy-loss spectroscopy (EELS), and energy-filtered transmission electron microscopy (EFTEM). The HRTEM images at the boundary of the C₂₄Cl₁₂ crystals exhibit the flexibility of defect structures, where molecules align to compensate for the discontinuity between two different domains. Emphasized by the filtered images, it was found that the non-periodic regions are created everywhere with a small electron beam irradiation (∼10⁶ electrons nm⁻²) and then spread over the entire regions to completely destroy the periodic structures after a higher electron dose (∼2×10⁶ electrons nm⁻²). The effect of the electron beam irradiation was monitored by ED, EELS, and EFTEM, where periodic structures and content elements are well preserved up to 10⁶ electrons nm⁻², but chlorine atoms decreased with a much higher electron dose. This is explained by the breakage of the C–Cl bond to detach chlorine atoms, confirmed by energy-loss near the edge structures (ELNES) of carbon π? peaks and chlorine loss at the edge of the specimen, as well as by theoretical simulation. The detachment of chlorine is localized at the peripheral edge around a hole confirmed by core-loss EFTEM imaging.     

Improving the analysis of small precipitates in HSLA steels using a plasma cleaner and ELNES

The change from producing high strength low alloy (HSLA) steel sheet by conventional thick slab casting to producing it by direct charged thin slab casting causes a major change in the evolution of the precipitation. A key area of interest is the composition of the sub-10nm precipitates used to produce dispersion hardening. Carbon extraction replicas are frequently used to study precipitates in steels and other metals. When used with annular dark field imaging, this technique gives high contrast images of the precipitates while the thin carbon film adds little background or additional characteristic signals to either electron energy loss spectra or energy dispersive X-ray spectra. The method has the additional major advantage of removing the ferromagnetic matrix when studying HSLA steels. However, when the precipitates contain carbon, the C K-edge is dominated by the contribution from the amorphous carbon film. A plasma cleaner can be used to thin this carbon film to approximately 0.5 nm or less and then the contribution from the carbon in the precipitate can be separated from that in the carbon film using the electron energy loss near edge structure. A similar approach can be taken to separate the oxygen content of the precipitate from that of oxides formed from low-level impurities in the amorphous carbon during the plasma thinning process. In most cases, the precipitate studied here contained little or no oxygen even for the smallest sizes examined (approximately 4 nm). The precipitates contain mainly nitrogen with little carbon. For some compositions, the precipitates are clearly sub-stoichiometric.     

Image-spectroscopy--II. The removal of plural scattering from extended energy-filtered series by Fourier deconvolution.

The increased spectral information obtained by acquiring an EFTEM image-series over several hundred eV allows plural scattering to be removed from loss images using standard deconvolution techniques developed for the quantification of EEL spectra. In this work, both Fourier-log and Fourier-ratio deconvolution techniques have been applied successfully to such image-series. Application of the Fourier-log technique over an energy-loss range of several hundred eV has been achieved by implementation of a novel method that extends the effective dynamic range of EFTEM image-series acquisition by over four orders of magnitude. Experimental results show that the removal of plural scattering from EFTEM image-series gives a significant improvement in quantification for thicker specimen regions. Further, the recovery of the single-scattering distribution using the Fourier-log technique over an extended energy-loss range is shown to result in an increase in both the ionisation-edge jump-ratio and the signal-to-noise ratio.     

Element specific imaging with high lateral resolution: an experimental study on layer structures

Planar defects and individual layers in ceramic material are chemically imaged by high resolution energy-filtering TEM using a post-column imaging electron energy filter. Objects are barium layers in the cuprate superconductor NdBa₂Cu₃O_7−δ (isostructual to YBa₂Cu₃O_7−δ) as well as planar defects and precipitates of β-WB in tungsten- and chromium-doped TiB₂. The barium layers with a spacing of 0.42 nm in the cuprate are resolved in jump-ratio images using the Ba_N edge. In the boride system the β-WB precipitates with thickness of 0.8 nm can be chemically imaged in elemental maps of B_K, Ti_L ,Cr_L and W_M. The B as well as the Ti map show a decrease in intensity at the precipitates, whereas in the W map an increase in intensity is observed. The boron-deficient layers with a spacing of 0.38 nm in the β-WB precipitate can be resolved in boron jump-ratio images. Additionally, defects containing single boron-deficient layers are chemically imaged. Hence structures in the dimension of interatomic distances can be imaged with respect to their elemental constituents. Although high resolution electron spectroscopic images contain strong interference contrast from elastic scattering, after normalization or background subtraction the element specific images are dominated by chemical contrast.     

New examples for near-edge fine structures in electron energy loss spectroscopy

The electron energy loss near-edge structures of the K and L edges of elements occurring in sulfides and oxides of copper and zinc have been investigated. Furthermore, the K edge of carbon in some carbonates and the L edges of sulfur and phosphorus in sulfates and phosphates have been measured and the near-edge fine structures of these edges have been found to be characteristic for the complex ions CO^2-₃, SO^2-₄. Thus, near-edge fine structures of EELS edges can be very useful as a fingerprint for rapid identification of chemical compounds such as carbonates, sulfates, phosphates and some oxides in the TEM.     

Demonstration of aluminium in polyphosphate of Laccaria amethystea (Bolt. ex Hooker) Murr. by means of electron energy-loss spectroscopy

Toxic aluminium species in acidified soil damage the rootlets of trees. The symbiotic association with ectomycorrhizal fungi may, however, protect the rootlets by sequestration of aluminium in polyphosphates. Electron energy-loss imaging and spectroscopy was used to identify the relative amount of aluminium in the polyphosphate of the mycelium of the ectomycorrhizal fungus Laccaria amethystea. A three-window method at the L_2,3 edge yielded results which were in agreement with the spectra obtained at the aluminium K edge. The aluminium net distribution images were recorded at 85 eV, the backgrounds at 70 eV and 60 eV, setting the energy selecting slit to 5 eV and the beam current to 15 μA. The method can be used to study the sequestration of aluminium in mycelia of ectomycorrhizal fungi under different stress situations.     

Effect of titanium carbonitride (Ti(C,N)) decomposition on failure mechanisms in inconel 617 alloy

Titanium Carbonitride (Ti(C,N)) decomposition in Inconel 617 alloy creep‐exposed at 650°C for 574 hours is reported using analytical electron microscopy techniques. Cr‐enriched M₂₃C₆‐type carbides enveloped in fine gamma prime particles thought to be precipitated from the decomposition reaction are observed in the alloy. The morphology of the M₂₃C₆ carbides is irregular and blocky and the particle size up to 5μm, whereas the morphology of gamma prime particles is mostly spherical and up to 30 nm in size. Intergranular carbides are mostly secondary precipitates of the M₂₃C_c type (M predominantly Cr) and these respond to solution heat treatment and precipitate on the grain boundaries as a result of ageing. The ability of intragranular MX to decompose is sensitive to the N content, high N resists decomposition. Decomposed intragranular MX provides an excess source of C which can react locally with Cr to form heat treatable intragranular fine Cr₂₃C₆ precipitates. M₆C can segregate in interdendritic locations during melting which may be the reason for high content of Mo in M₂₃C₆. These precipitates are generally very small and contribute to an additional hardening effect and are the reason for the onset of voiding and cracking along the grain boundaries that ultimately lead to a reduced creep rupture life. Microsc. Res. Tech. 78:336–342, 2015. © 2015 Wiley Periodicals, Inc.     

TEM and HREM study of Al3Zr precipitates in an Al-Mg-Si-Zr alloy

The structure of Al₃Zr precipitates in Al‐1.0Mg‐0.6Si‐0.5Zr (in wt.%) alloy was investigated using conventional transmission electron microscopy (TEM) and high‐resolution TEM (HREM). After annealing of the alloy in the temperature range 450–540 °C, spherical precipitates of metastable L1₂‐Al₃Zr phase appeared nearly homogeneously within the matrix, and elongated particles were found at grain boundaries. L1₂‐structured Al₃Zr were about 20–30 nm in diameter and coherent with the matrix. Inside some of them, planar faults parallel to {100} planes were revealed by use of HREM. Most probably, these faults are an indication of the transition stage of transformation to the stable D0₂₃‐type Al₃Zr phase. The elongated precipitates (about 100 nm) were identified as D0₂₂‐type Al₃Zr. Energy‐dispersive X‐ray analysis showed that they contain, apart from Al, mainly Zr with small amounts of Si. The substitution of Al by Si increased the stability of the D0₂₂‐Al₃Zr as compared with D0₂₃‐Al₃Zr.     

Point-projection imaging of unstained ferritin clusters

A point-projection microscope is described which has been used to obtain images of isolated, unstained ferritin clusters on a tungsten substrate. The microscope images the contour of the collection of protein shells associated with each cluster, rather than the iron core of each ferritin molecule accessible to TEM imaging Quasi, three-dimensional images of cluster morphology are obtained by controlled field-desorption of an immobile, condensed benzene multilayer which initially surrounds and covers each cluster. The imaging process appears to be nondestructive provided a critical electric field strength, F_c, is not exceeded For large, weakly bound ferritin clusters F_c≈4V/nm, a value well above that required for imaging.     

Effects of laser pulsing on analysis of steels by atom probe tomography

When performing atom probe tomography analysis, laser pulsing was found very helpful for some types of steels, which are prone to premature sample failure under voltage pulsing. Rather accurate chemical compositions for both matrix and precipitates were obtained by voltage- and laser-pulsed mode. Some special issues on the effects of laser are discussed. A simple correction based on the ¹³Cⁿ⁺ and ¹⁰Bⁿ⁺ peak was used to quantify C in the carbide (M₂₃C₆, M=Fe, Cr, Mo) and B in the boride (M₃B₂, M=Mo, Fe, Cr, V). The mass resolution in laser mode is sufficient to resolve ⁵⁶Fe²⁺ and ¹⁴N₂¹⁺ at 28 Da. Small peak shifts were found in the spectrum due to reflectron aberrations.     

Extended energy-loss fine structure spectroscopy of structural modifications in Nd2CuO4−δ at the oxygen K edge

The discovery of the superconducting electron-doped compound Nd₁₈₅Ce₀₁₅CuO_4?δ has stimulated great interest in its micro- and crystal structure, since the superconducting properties depend on parameters such as nonstoichiometry, phase composition, heat treatment and microstructure. The work presented herein is focused on the determination of the oxygen environment in the undoped parent compound Nd₂CuO₄ and in the structural modification Nd₂CuO₃₅ The analysis of the oxygen K (O 1s) edge extended electron energy-loss fine structure (EXELFS) of the tetragonal parent compound Nd₂CuO₄ and of the orthorhombic modification Nd₂CuO₃₅ is reported by using electron energy-loss spectroscopy in combination with transmission electron microscopy. Nd₂CuO₃₅ is produced by in situ heating and reduction of Nd₂CuO₄ in the transmission electron microscope. The EXELFS of the O 1s electron energy-loss edges is analysed with the classical extended X-ray absorption fine structure (EXAFS) treatment and compared with ab initio multiple scattering EXAFS calculations for both structural modifications. Highly accurate information on the local atomic environment of the oxygen atoms in Nd₂CuO₃₅ is obtained from EXELFS analysis using Nd₂CuO₄ as a standard. The results are in accordance with the structural data gained from X-ray diffraction analysis. This applies especially to the more complicated structure of Nd₂CuO₃₅ determined recently.     

Resolution Of M23C6/Austenite Phase Boundary Defect Structures By Weak Beam Microscopy

The weak beam dark field imaging technique has been successfully applied to the problem of imaging misfit dislocations at the interface between M₂₃C₆ precipitates and an austenite matrix. The technique eliminates Moiré fringe contrast which otherwise obliterates the contrast from interfacial dislocations. Three sets of parallel dislocations were observed with spacings and line vectors consistent with them being edge dislocations with Burgers vectors of a/3<111>.     

Spatially resolved electron energy-loss studies of metal–ceramic interfaces in transition metal/alumina cermets

Composites consisting of an alumina matrix and 20 vol.% transition metal (Ni or Fe) particles, prepared by hot pressing powder blends, have been studied using spatially resolved transmission electron energy-loss spectroscopy (EELS), and, to a lesser extent, by high-resolution electron microscopy (HREM). Particular attention was paid to the elucidation of the chemical bonding mechanisms at the metal-ceramic interface; EELS spectra from interfacial regions being obtained via a spatial difference technique. From both qualitative and quantitative interpretation of EELS near-edge structures, as well as observed HREM images, the data appear to be consistent with the presence of an Al-terminated alumina at the interface and the formation of direct transition metal – aluminium bonds in Al(O₃M) (M = Ni or Fe) tetrahedral units, possibly as a result of the dissolution and interfacial reprecipitation of Al during processing. These results correlate well with similar model studies on diffusion-bonded Nb/Al₂O₃ interfaces and may, in the light of recent theoretical electronic structure calculations, have implications for the resultant interfacial bond strength in such materials.     

Improving energy resolution of EELS spectra: an alternative to the monochromator solution

In this paper, we propose a numerical method which can routinely improve the energy resolution down to 0.2-0.3eV of electron energy-loss spectra acquired in a transmission electron microscope. The method involves measurement of the point-spread function (PSF) corresponding to the spectrometer aberration and to the incident energy spread, and then an inversion of this PSF so as to restore the spectrum. The chosen algorithm is based on an iterative calculation of the maximum likelihood solution known to be very robust against small errors in the PSF used. Restorations have been performed on diamond and graphite C-K edges acquired with an initial energy resolution of around 1eV. After reconstruction, the sharp core exciton lines become clearly visible for both compounds and the final energy resolution is estimated to be about 200-300meV. In the case of graphite, restorations involving both energy resolution and angular resolution have been successfully conducted. Finally, restorations of Fe L(2,3) and O-K edges measured for various iron oxides will be shown.     

Quantitative energy-filtered image analysis in cytochemistry. II. Morphometric analysis of element-distribution images

A combination of energy-filtered electron microscopy (EFEM) and an image-analysis system (IBAS/2000) is used for a morphometric analysis of chemical reaction products in cells. Electron energy-loss spectroscopic element-distribution images are acquired from cytochemical reaction products in a variety of cellular objects: (1) colloidal thorium particles in extra-cellular coat material, (2) iron-containing ferritin particles in liver parenchymal cells, (3) barium-containing reaction products in endoplasmic reticulum stacks, (4) elements present in lysosomal cerium- and barium-containing precipitates connected with acid phosphatase (AcPase) or aryl sulphatase (AS) enzyme activity. Areas or area fractions are determined from such element-distribution images by application of an objective image segmentation method. By superposition of two or more element-distribution images, mutual element relations are qualitatively established in lysosomal cerium- and barium-containing precipitates connected with acid phosphatase (AcPase) or aryl sulphatase (AS) enzyme activity. By comparing electron spectroscopic images (ESI) with element-distribution images, the mutual contrast per element relations are quantitatively investigated. The obtained gain in resolution in such electron energy-loss spectroscopic element-distribution images will be explained and discussed.