Ultrastructural aluminium detection in amphibian tissues by electron spectroscopic imaging and electron energy-loss spectroscopy

Aluminium causes a variety of toxic effects in living organisms but very little is known about its uptake, pathways and locations of deposition. We have applied electron spectroscopic imaging (ESI) and electron energy-loss spectroscopy (EELS) to locate aluminium at the ultrastructural level in amphibian larvae from acidic ponds. It is found diffusely bound or precipitated in cell organelles. The spatial resolution of aluminium detection is high. The elemental composition of small areas can be demonstrated by EELS. Three different fixation procedures give similar results. The two- and three-window methods at the K- and L-edges are compared.     

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.     

Image-EELS: Simultaneous recording of multiple electron energy-loss spectra from series of electron spectroscopic images

A new approach for element microanalysis with energy-filtering transmission electron microscopy (EFTEM) is presented which was accomplished with the CEM 902 electron microscope (Zeiss, Germany). This method is called Image-EELS, because it is a synthesis of electron energy-loss spectroscopy (EELS) and electron spectroscopic imaging (ESI). Series of energy-filtered images at increasing energy losses are recorded from one area with a TV camera. In a second step the intensity of selected regions in the image stack is measured with an image analysis system and plotted as a function of the energy loss. Thus many spectra from different objects can be calculated from one image series and compared with each other. The spatial resolution of EELS is considerably enhanced, the noise is decreased because many pixels from irregular objects are integrated, and the information from ESI can be analysed as a function of the energy loss.     

Mapping of ELNES on a nanometre scale by electron spectroscopic imaging

The amorphous interfacial layer between Si substrates and diamond films grown by plasma-assisted chemical vapour deposition has been studied by electron spectroscopic imaging. The amorphous layer consists mainly of carbon, which can only be distinguished from the diamond film by analysis of the near-edge structure (ELNES) of the carbon K edge. Series of electron spectroscopic images were acquired across the carbon K edge and were analysed in order to reveal the presence of the π*- and σ*-excitations. After background removal from the corresponding images, phase maps for the distribution of sp² and sp³ hybridized carbon can be obtained. From the whole series of images, electron energy-loss spectra can be extracted for any given area in the images. The results show that the amorphous layer covers large areas along the interface and that regions with only 1–2 nm layer thickness can clearly be analysed. The results obtained with the electron spectroscopic imaging technique will be compared with results obtained on a field emission gun scanning transmission electron microscope.     

Limitations of beam damage in electron spectroscopic tomography of embedded cells

Elemental mapping in the energy filtering transmission electron microscope (EFTEM) can be extended into three dimensions (3D) by acquiring a series of two‐dimensional (2D) core‐edge images from a specimen oriented over a range of tilt angles, and then reconstructing the volume using tomographic methods. EFTEM has been applied to imaging the distribution of biological molecules in 2D, e.g. nucleic acid and protein, in sections of plastic‐embedded cells, but no systematic study has been undertaken to assess the extent to which beam damage limits the available information in 3D. To address this question, 2D elemental maps of phosphorus and nitrogen were acquired from unstained sections of plastic‐embedded isolated mouse thymocytes. The variation in elemental composition, residual specimen mass and changes in the specimen morphology were measured as a function of electron dose. Whereas 40% of the total specimen mass was lost at doses above 10⁶ e^?/nm², no significant loss of phosphorus or nitrogen was observed for doses as high as 10⁸ e^?/nm². The oxygen content decreased from 25 ± 2 to 9 ± 2 atomic percent at an electron dose of 10⁴ e^?/nm², which accounted for a major component of the total mass loss. The specimen thickness decreased by 50% after a dose of 10⁸ e^?/nm², and a lateral shrinkage of 9.5 ± 2.0% occurred from 2 × 10⁴ to 10⁸ e^?/nm². At doses above 10⁷ e^?/nm², damage could be observed in the bright field as well in the core edge images, which is attributed to further loss of oxygen and carbon atoms. Despite these artefacts, electron tomograms obtained from high‐pressure frozen and freeze‐substituted sections of C. elegans showed that it is feasible to obtain useful 3D phosphorus and nitrogen maps, and thus to reveal quantitative information about the subcellular distributions of nucleic acids and proteins.     

Electron spectroscopic imaging of antigens by reaction with boronated antibodies

Two small homogeneous markers for electron spectroscopic imaging (ESI) containing eight dodecaborane cages linked to a poly-α,ε- l -lysine dendrimer were synthesized; one of these was made water soluble by the attachment of a polyether. The markers were coupled to the sulfhydryl group of (monovalent) antibody fragments (Fab') by a homobifunctional cross-linker. While the coupling ratios of the poorly water-soluble compound did not exceed 20%, the polyether-containing variant reacted quantitatively. Its suitability for immunolabelling was tested in a study of the mechanism of the transcellular transport of an administered heterologous protein (bovine serum albumin, BSA) through ileal enterocytes of newborn piglets by endocytotic vesicles in comparison to conventional immunogold reagents. The post-embedding technique was employed. The boronated Fab' gave rise to considerably higher tagging frequencies than seen with immunogold, as could be expected from its form- and size-related physical advantages and the dense packing of BSA in the vesicles. The new probe, carrying the antigen-combining cleft at one end and the boron clusters at the opposite end of the oval-shaped conjugate, add to the potential of ESI-based immunocytochemistry.     

Quantitative electron spectroscopic imaging studies of microelectronic metallization layers

The combination of focused ion beam (FIB) sample preparation and quantitative electron spectroscopic imaging is an ideal tool for the investigation of layered structures used in microelectronic metallization schemes. In the present work, Si₃N₄/Cu/Si₃N₄/SiO₂/Si and Al/TiN/Ti/SiO₂/Si metallization layers produced by physical vapour deposition are investigated. We apply series of energy filtered images in the low loss region for a mapping of the sample thickness which makes it possible to refine the parameters of the FIB process. We also show how series of energy filtered images in the core loss region can be used to obtain elemental distribution images and chemical bonding information on these samples on a nanometre scale. For materials with a small grain size and/or a strong variation in Bragg orientation, the intensity distribution of the elemental map is strongly influenced by the superimposed Bragg contrast. This detrimental effect can be reduced greatly by using hollow cone illumination, as is demonstrated for polycrystalline Cu. One striking feature observed in Cu layers prepared with FIB is strong, regularly arranged contrast variations caused by subsurface defects in the Cu grains. We suppose that these defects are a consequence of a strong interaction of Ga atoms from the FIB with Cu.     

Experimental ionization cross-sections of phosphorus and calcium by electron spectroscopic imaging

The absolute partial electron scattering cross-section for the phosphorus L_2,3-shell ionization was measured by electron spectroscopic imaging using poliovirus as a primary standard. The equivalent calcium cross-section was obtained in relation to phosphorus using the stoichiometric ratio for these two elements in hydroxyapatite, Ca₁₀(PO₄)₆(OH)₂. At 80kcV, the partial cross-section of phosphorus was 2.26 times 10^?20 and 2.68 × 10^?20cm²/atom for poliovirus and hydroxyapatite, respectively, at 150eV loss for a 15-cV energy window and an acceptance angle of 15 mrad. Under the same conditions the calcium cross-section was 0.49 × 10^?20 cm²/atom at 360 eV loss. The experimental values are slightly higher than the theoretical cross-sections calculated either by hydrogenic or Hartree—Slater approaches.     

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.     

Energy-filtered transmission electron microscopy of multilayers in semiconductors

Quantitative analysis of novel semiconductors with wide or ultrathin multilayers of atomic dimensions is very important in order to control electronic and optical properties, but rather difficult due to the limited resolution in most techniques. In this paper we attempt to assess how effectively the total As dopant concentration in ultrathin As doped layers in InP and the Ti atomic fraction in a Ti _x Al_{1− x} N multilayer can be analysed quantitatively using energy-filtered imaging. These two materials have characteristic edges located at widely different energy losses, with the L edge of As being above 1000 eV, while that of Ti is around 450 eV. We have quantified the As concentration using the three-window technique and theoretical cross-sections and we find that the resolution limit is dominated by the signal-to-noise ratio in this delta-doped specimen. However, the accuracy of the Ti atomic fraction in Ti _x Al_{1− x} N can be as good as 10 at% for specimens of uniform thickness made by focused ion beam milling. We will compare our results with measurements of the composition made using Fresnel contrast, high resolution imaging and high angle annular dark field techniques.     

Quantitative electron spectroscopic imaging in bio-medicine: evaluation and application

Electron spectroscopic imaging (ESI) with the energy-filtering transmission electron microscope enables the investigation of chemical elements in ultrathin biological sections. An analysis technique has been developed to calculate elemental maps and quantitative distributions from ESI sequences. Extensive experience has been obtained with a practical implementation of this technique. A procedure for more robust element detection has been investigated and optimized. With the use of Fe-loaded Chelex beads, the measurement system has been evaluated with respect to the linearity of the element concentration scale, the reproducibility of the measurements and the visual usage of image results. In liver specimens of a patient with an iron storage disease the detectability of iron was tested and we tried to characterize iron-containing components.   The concentration measurement scale is approximately linear up to a relative section thickness of ≈ 0.5. Monitoring of this parameter is therefore considered to be important. The reproducibility was measured in an experiment with Fe-Chelex. The iron concentration differed by 6.4% between two serial measurements. Element distributions are in many applications interpreted visually. For this purpose the frequently used net-intensity distributions are regarded as unsuitable. For the quantification and visual interpretation of concentration differences mass thickness correction has to be performed. By contrast, for the detection of elements the signal-to-noise ratio is the appropriate criterion.   Application of ESI analysis demonstrated the quantitative chemical capabilities of this technique in the investigation of iron storage diseases. Based on an assumed ferritin iron loading in vivo , different iron components can be discerned in liver parenchymal cells of an iron-overloaded patient.     

Electron spectroscopic imaging of the morphology of in situ polyethylene ‘shish kebabs’

The simultaneous imaging of the crystalline and rigid amorphous areas of semi-crystalline polyethylene is made possible by electron spectroscopic imaging (ESI) in an energy-filtering transmission electron microscope (EFTEM). Conventional bright-and dark-field imaging modes mainly provide information about the non-crystalline areas which react chemically with classical staining agents such as ruthenium tetroxide. The ability of ESI to image structures which are inaccessible to the staining agent is shown in TEM micrographs of nascent polyethylene ‘shish kebabs’ embedded in a Lowicryl HM20 matrix.     

Toward quantitative core-loss EFTEM tomography

Core-loss EFTEM tomography provides three-dimensional structural and chemical information. Multiple inelastic scattering occurring in thick specimens as well as orientation-dependent diffraction contrast due to multiple elastic scattering, however, often limit its applications. After demonstrating the capability of core-loss EFTEM tomography to reconstruct just a few monolayers thin carbon layer covering a Fe catalyst particle we discuss its application to thicker samples. We propose an approximate multiple-scattering correction method based on the use of zero-loss images and apply it successfully to copper whiskers, providing a significant improvement of the reconstructed 3D elemental distribution. We conclude this paper by a general discussion on experimental parameters affecting the accuracy of EFTEM 3D elemental mapping.     

Electron spectroscopy imaging to study ELNES at a nanoscale

Series of energy‐filtered TEM images have been acquired with very narrow energy slit using a post‐column energy filter. This allowed us to reconstruct spectra with an energy resolution estimated to 2 eV, and a spatial resolution in the order of 0.5 nm. In that way, fine structures of the N‐K edge in AlN/GaN heterostructures have been investigated and compared to EELS spectra. The fine structure in the two nitrides is very sensitive to the local environment. Very good agreement between ESI and EELS spectra was found. Moreover, this technique allowed analysis of the AlN/GaN interface at a nanoscale. The second example is an application of the technique to construct bonding maps. In this case, maps differentiating AlN nanoprecipitates with either the cubic or the hexagonal phase were created.     

Demonstration of calcium in dermal melanocytes of Xenopus laevis and Poecilia reticulata with electron energy-loss spectroscopy and electron spectroscopic imaging

The subcellular distribution of calcium in dermal melanocytes of Xenopus laevis and Poecilia reticulata has been analysed. Using two cytochemical methods, phosphate precipitation and a combined oxalate-pyroantimonate technique, electron energy-loss spectroscopy and electron spectroscopic imaging have been applied for elemental analysis. Both precipitation techniques revealed a high calcium content in the melanosomes of both species. Calcium was also located in the vicinity of collagen fibrils and in the plasma membrane.     

Quantitative electron spectroscopic imaging in bio-medicine: Methods for image acquisition,correction and analysis

Many questions about the metabolism of specific elements in the human body might be answered if elemental concentrations could be measured in situ in cells. With electron energy-loss spectroscopic imaging (ESI), concentrations can potentially be determined with high spatial resolution. The theory of the quantification procedure has already been derived. Many practical instrument-related problems, however, have to be solved. In the current research an energy-filtering TEM is used and the image-acquisition chain is examined in detail. Quantification requires images to be recorded over a large dynamic range. To solve this problem, the use of optical attenuation filters has been introduced. The use of the combination of a scintillator screen and a TV-camera as a detection system has consequences for the processing of the data. Corrections for the camera photometric sensitivity and, to some extent, for shading are necessary. Further consequences of such a detection system for the correction of the element a-specific spectral background and element detection are discussed. The derived methodology is tested in several ways and finally applied for the quantitative analysis of iron in liver parenchymal cells of a porphyria cutanea tarda patient.     

The electron energy-loss spectroscopic analysis of inhaled smoke particles

This paper details the use of electron spectroscopic imaging in the elemental analysis of smoke particles inhaled by smoke-death victims. The results show that these particles have a varied structure and composition. Because of this, these particles may play a far more significant role in smoke inhalation injuries than has previous been recognized.     

The in situ architecture of Escherichia coli ribosomal RNA derived by electron spectroscopic imaging and three-dimensional reconstruction

The structures of the large and small ribosomal subunits of Escherichia coli were reconstructed using spectroscopic electron microscopy and quaternion-assisted angular reconstitution to resolutions of better than 4 nm. In addition, the distributions of phosphorus within these complexes were reconstructed. The three-dimensional reconstruction of the distribution of this atomic element is an extension of microanalysis (in two dimensions) for phosphorus identification and mapping, as a signature of the arrangement of the phosphate backbones of the constituent ribosomal RNAs. The results on both the phosphorus reconstructions and the total reconstructions (protein and ribosomal RNA) reveal several passageways through both subunits. The structures correspond favourably with other independent reconstructions of the whole E. coli ribosome from cryoelectron micrographs and their accompanying models of translation (Frank et al ., Nature , 376, 441–444, 1995; Stark et al ., Structure , 3, 815–821, 1995). The overall reconstructions in conjunction with the phosphorus (rRNA) distributions are the first to be achieved synchronously for this nucleoprotein complex.     

ESI contrast analysis: a new approach for element analysis with energy-filtering transmission electron microscopy (EFTEM)

Element microanalysis with energy-filtering transmission electron microscopy can be performed in different ways. Electron energy-loss spectroscopy (EELS) records the intensity as a function of the energy loss from selected regions. Elemental mapping with electron spectroscopic imaging (ESI) uses energy-filtered images at an element-specific energy loss from which a background image has to be subtracted. A combination of these two approaches is Image-EELS, which records a series of energy-filtered images, each at a different energy loss and measures the intensity of selected regions as a function of the energy loss. As an additional procedure ESI contrast analysis is introduced; with this we can investigate the image contrast as a function of the energy loss. The contrast can be measured for the total image or for selected regions as the standard deviation of the grey levels divided by the mean grey level. This procedure is added as a new feature to the existing Image-EELS program, so that EELS intensity spectra and the contrast can be directly compared. Alternatively, the contrast can be calculated step by step from individually recorded electron spectroscopic images, so that only simple equipment for image analysis is sufficient to realize this new method. Inner shell ionizations produce characteristic, but often weak element edges in energy-loss spectra which can be detected more sensitively and reliably as a rapid increase in contrast. Regional analysis of the contrast as a function of the energy loss, combination of data from different regions and the possibility to increase the intensity during recording of the images expand the application range of this new analytical method which bridges the gap between ESI and EELS analysis. With ESI contrast analysis it is possible to discriminate between faint element signals and artefacts in elemental mapping. This new approach for element detection is especially advantageous for biological objects which usually contain very small amounts of the interesting elements in heterogeneous and complex objects. As an example, nervous tissue of a fish was analysed after cytochemical precipitation of endogenous calcium.