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.     

Electron energy-loss spectroscopy (EELS) of iodine in thyroxine: parameters of radiation-induced loss of iodine

The loss of iodine during irradiation with electrons was measured in order to determine whether it can be detected by electron spectroscopic imaging (ESI). Since iodine can be bound to organic molecules, it could be a candidate for tracer studies by ESI in biomedical research. A solution of thyroxine spread over a carbon film was used as a test specimen. Thyroxine contains four iodine atoms covalently bound to aromatic rings. For ESI, higher doses have to be used than in conventional electron microscopy. The iodine content was therefore measured after irradiation with doses of up to 3 × 10⁷ el nm^?2. The measurements were carried out for different specimen thicknesses, temperatures and dose rates, and it was found that iodine can be detected with ESI if moderate dose rates are used at a temperature of ?160 °C.     

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.     

Fourier-ratio deconvolution techniques for electron energy-loss spectroscopy (EELS)

We discuss several ways of using Fourier-ratio deconvolution to process low-loss spectra. They include removal of the tail arising from the zero-loss peak, extraction of the spectrum of a particle from data recorded from the particle on a substrate, separation of the bulk and surface components in spectra recorded from samples of the same composition but different thickness, and investigation of interface energy-loss modes. We also demonstrate the use of a Bayesian-equivalent procedure based on the Richardson–Lucy algorithm.     

Quantitative water mapping of cryosectioned cells by electron energy-loss spectroscopy

A direct technique based on electron energy-loss spectroscopy (EELS) in the scanning transmission electron microscope (STEM) has been developed to map subcellular distributions of water in frozen-hydrated biological cryosections. Previously, methods for water determination have been indirect in that they have required the cryosections to be dehydrated first. The new approach makes use of spectrum-imaging, where EELS data are collected in parallel at each pixel. Several operations are required to process the spectra including: subtraction of the detector dark current, deconvolution by the detector point-spread function, removal of plural inelastic scattering and correction for the support film. The resulting single scattering distributions are fitted to standard reference spectra at each pixel, and water content can be determined from the fitting coefficients. Although the darkfield or brightfield image from a hydrated cryosection shows minimal structure, the processed EELS image reveals strong contrast due to variations in water content. Reference spectra have been recorded from the major biomolecules (protein, lipid, carbohydrate, nucleic acid) as well as from vitrified water and crystalline ice. It has been found that quantitative results can be obtained for the majority of subcellular compartments by fitting only water and protein reference spectra, and the accuracy of the method for these compartments has been estimated as ± 3·5%. With the present instrumentation the maximum allowed dose of 2 × 10³ e/nm² limits the useful spatial resolution to around 80 nm for ± 5% precision at a single pixel. By averaging pixel intensities a value of 56·8% with a precision of ± 2·0% has been determined for the water content of liver mitochondria. The water mapping technique may prove useful for applications to cell physiology and pathophysiology.     

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.     

The application of electron spectroscopic imaging for quantification of the area fractions of calcium-containing precipitates in nervous tissue

Energy-filtering transmission electron microscopy has been applied to the quantification of area fractions of calcium-containing cytochemical reaction products in central nervous tissue and the retina of fish. The method of electron spectroscopic imaging using electrons with an energy loss of 250 eV produces images with a very high, structure-sensitive contrast. This is a suitable imaging condition for the reliable detection of reaction products and structural details in unstained ultrathin sections. The images were recorded with a sensitive TV camera and evaluated with the integrated digital image-analysis system of the Zeiss CEM 902 energy-filtering electron microscope. An empirical procedure was developed which objectively detects reaction products and calculates characteristic values, taking into account different staining intensities. This new and sensitive method enabled an assessment to be made of the influence of temperature and light adaptation on cytochemically detectable calcium in nervous tissue of fish. Higher amounts of calcium-containing reaction product were detected in synaptic clefts of the optic tectum in warm-adapted fish than in cold-adapted fish. In synaptic vesicles of photoreceptor cells in the fish retina, higher amounts of reaction product were found in dark-adapted fish than in light-adapted fish.     

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.     

Stoichiometry and valence measurements of niobium oxides using electron energy-loss spectroscopy

Qualitative and quantitative electron energy‐loss spectroscopy analyses have been performed on niobium and stable niobium oxides (NbO, NbO₂ and Nb₂O₅). At integration windows (Δ) greater than 75 eV, k‐factor analysis can be used to distinguish between the stoichiometry of the three oxides within 5.7% error. As seen in other metal oxides, with increasing oxidation state the metal ionization edges shift to higher energies relative to the O‐K edge. Normalized M_2,3 white‐line intensities show a strong correlation with 4d occupancy for each compound. The data are in correspondence with that observed in the literature for 4d transition metals using normalized L_2,3 white lines. Lastly, a distinctive energy‐loss near‐edge, structure of the O‐K edge was observed for each oxide, which could be used as a fingerprint for analysis of unknowns.     

Analysis of the calcium distribution in predentine by EELS and of the early crystal formation in dentine by ESI and ESD

Predentine is a collagen-rich extracellular matrix between the odontoblasts and the dentine with a width of about 15–20 μm. Electron energy-loss spectroscopy of rat incisors shows a significantly higher calcium content in the predentine at the predentine-dentine border than in the middle region of the predentine. At the predentine-dentine border in the dentine, the calcium and the phosphate groups combine to form apatite crystallites. Electron spectroscopic diffraction with zero-loss filtering revealed that the earliest crystallites contain only Debye-Scherrer rings of apatite, which are fewer in number and more diffuse than the diffraction rings from the mature crystallites. We therefore conclude that the early crystallites still contain lattice defects, which are annealed out to some degree with crystal growth. Electron spectroscopic imaging with zero-loss filtering also showed that the earliest crystallites are chains of dots (or small islands); they build up strands composed of islands, which rapidly acquire a needle-like character and coalesce laterally to form ribbon- or plate-like crystallites. The parallel strands sometimes appear to reinforce the macroperiod of the collagen microfibrils (67 nm) by tiny holes without any crystal-substance lined up perpendicular to the parallel strands of the crystallites.     

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.     

Detecting single atoms of calcium and iron in biological structures by electron energy-loss spectrum-imaging

As techniques for electron energy‐loss spectroscopy (EELS) reach a higher degree of optimization, experimental detection limits for analysing biological structures are approaching values predicted by the physics of the electron scattering. Theory indicates that it should be possible to detect a single atom of certain elements like calcium and iron contained in a macromolecular assembly using a finely focused probe in the scanning transmission electron microscope (STEM). To test this prediction, EELS elemental maps have been recorded with the spectrum‐imaging technique in a VG Microscopes HB501 STEM coupled to a Gatan Enfina spectrometer, which is equipped with an efficient charge‐coupled device (CCD) array detector. By recording spectrum‐images of haemoglobin adsorbed onto a thin carbon film, it is shown that the four heme groups in a single molecule can be detected with a signal‐to‐noise ratio of ～10 : 1. Other measurements demonstrate that calcium adsorbed onto a thin carbon film can be imaged at single atom sensitivity with a signal‐to‐noise ratio of ～5 : 1. Despite radiation damage due to the necessarily high electron dose, it is anticipated that mapping single atoms of metals and other bound elements will find useful applications in characterizing large protein assemblies.     

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.     

Investigation and use of plasmon losses in energy-filtering transmission electron microscopy

The electron spectroscopic imaging (ESI), diffraction (ESD) and different types of electron energy-loss spectroscopy (EELS) modes in an energy-filtering transmission electron microscope can all be used for the investigation and analytical use of plasmon losses. Shifts of plasmon losses caused by differences in composition can be detected with an accuracy of 0.1 eV by parallel-recorded EELS (PEELS). The dispersion of plasmon losses and the cut-off angle θ_c can be observed by angle-dispersive EELS and by recording spectra at different scattering angles θ. ESD patterns with a selected energy window width of 1 eV enable the dispersion and its anisotropy to be imaged by characteristic intensity distributions between the primary beam and the first Bragg diffracted beams. The ESI mode can be used for the selective imaging of precipitates and for the investigation of the excitation volume of plasmons in small particles.     

Ultrastructural and electron spectroscopic analyses of cyanobacteria and bacteria

The extracellular sheath material and some intracellular cell components of cyanobacteria and phosphate-accumulating sewage bacteria were analysed by electron spectroscopic imaging (ESI) and electron energy-loss spectroscopy (EELS). The specimens were embedded in water-soluble Nanoplast resin without any previous fixation and ultrathin sections were examined in a Zeiss CEM 902 microscope. A high sulphur content was detected in the inner sheath of the cyanobacterium Gloeothece. The elemental composition of some cell components and inclusion bodies, such as carboxysomes and cyanophycin, was determined by ESI and EELS. In addition, the phosphate content in specific granules of phosphate-accumulating sewage bacteria was estimated by EELS and nuclear magnetic resonance spectroscopy.     

The glycosomes of trypanosomes: number and distribution as revealed by electron spectroscopic imaging and 3-D reconstruction

Computer-aided 3-D reconstruction of typanosomes from 0·35-μm-thick sections imaged on the Zeiss 902 electron microscope are being used to study the dynamics of cell organization. Segregation of glycolytic enzymes into glycosomes raises questions concerning the distribution and biogenesis of these organelles. Direct counts of glycosomes from Trypanosoma evansi indicate 30–40 per cell and for the closely related T. brucei, 65 per cell. These figures contrast with the estimates of others who have used model-based morphometric methods to obtain a value of 230 per cell.     

Development,quantitative performance and applications of a parallel electron energy-loss spectrum imaging system

The development of a parallel electron energy-loss spectrum imaging system is presented. The analytical performance of the imaging technique was investigated and the system applied to materials science problems. The system, which allows acquisition and storage of a parallel electron energy-loss spectrum at each pixel of an image, was developed by interfacing a multichannel analyser and a microscope to a computer workstation. In the experimental conditions used for imaging, detection limits and quantification errors were large and varied as a function of spatial resolution and the range of chemical elements of interest in the image. Applications of this imaging technique in materials science showed that quantitative chemical information is provided by the system and that the use of relative thickness maps and detailed statistical analysis of the spectrum-image allowed an unbiased interpretation of the images. As energy-loss spectra are available after processing, spectroscopic information about the analysed material can be used to provide supplementary information.     

Reflection electron imaging and spectroscopy studies of aluminium metal reduction at α-aluminium oxide (0,1,2) surfaces

Cleaved α-aluminium oxide (0,1,2) surfaces are studied using combined techniques of scanning reflection electron microscopy and reflection electron energy-loss spectroscopy. An α-aluminium oxide (0,1,2) surface can terminate with a layer of either oxygen or Al atoms depending on where the bonds break. Aluminium metal can be reduced only from the domains initially terminated with oxygen by an intensive electron beam. The interpretation of this damage effect is based on the desorption process of oxygen atoms after Auger decay. The resistance to beam damage of the domains initially terminated with Al is found to be due to the protection of the surface adsorbed amorphous oxygen layer.     

Subcellular localization of boron in cultured melanoma cells by electron energy-loss spectroscopy of freeze-dried cryosections

Boron neutron capture therapy (BNCT) is based on the ability of the non‐radioactive isotope ¹⁰B to capture thermal neutrons and to disintegrate instantaneously. This reaction opens a way to selectively destroy tumour cells after specific uptake of ¹⁰B. In this paper, a method based on electron energy‐loss spectroscopy is presented for detecting and quantifying boron in freeze‐dried cryosections of human melanoma cells. A practical detection limit of around 6 mmol kg^?1 in 0.1‐µm² areas is estimated using specimens prepared from standard boron solutions. Preliminary results of boron mapping in the spectrum‐imaging acquisition mode reveal boron penetration and probably spot‐like accumulation within melanoma cells when exposed to culture medium containing sodium borocaptate.     

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.