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.     

Annular electron energy-loss spectroscopy in the scanning transmission electron microscope

We study atomic-resolution annular electron energy-loss spectroscopy (AEELS) in scanning transmission electron microscopy (STEM) imaging with experiments and numerical simulations. In this technique the central part of the bright field disk is blocked by a beam stop, forming an annular entry aperture to the spectrometer. The EELS signal thus arises only from electrons scattered inelastically to angles defined by the aperture. It will be shown that this method is more robust than conventional EELS imaging to variations in specimen thickness and can also provide higher spatial resolution. This raises the possibility of lattice resolution imaging of lighter elements or ionization edges previously considered unsuitable for EELS imaging.     

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.     

Electron energy-loss spectroscopy analysis of adriamycin-plasma membrane interaction

Our previous studies on the mechanism of cytotoxic action of the anti-tumour drug adriamycin (ADR) indicated that this anthracyclinic antibiotic strongly modified the molecular architecture of the plasma membrane of human erythrocytes, presumably becoming incorporated within both lipid layers. In order to verify this hypothesis, electron energy-loss spectroscopy (EELS) has been used to compare the P content in control and ADR-treated erythrocyte ghosts. EELS measurements allowed us to reveal a significant reduction in the P/C ratio in erythrocyte ghosts after ADR treatment. This finding seems to reflect a phospholipid ‘dilution’ produced by the incorporation of the drug molecules in the membrane layers. A structural model of the ADR-membrane interaction is proposed.     

Characterization of biological macromolecules by combined mass mapping and electron energy-loss spectroscopy

The combination of scanning transmission electron microscopy (STEM) and parallel-detection energy-loss spectroscopy (EELS) was used to detect specific bound elements within macromolecules and macromolecular assemblies prepared by direct freezing. After cryotransferring and freeze-drying in situ, samples were re-cooled to liquid nitrogen temperature and low-dose (about 10³ e/nm²) digital dark-field images were obtained with single-electron sensitivity using a beam energy of approximately 100 keV and a probe current of approximately 5 pA. These maps provided a means of characterizing the molecular weights of the structures at low dose. The probe current was subsequently increased to about 5 nA in order to perform elemental analysis. The 320 copper atoms in a keyhole limpet haemocyanin molecule (mol.wt = 8 MDa) were detected with a sensitivity of ± 30 atoms in an acquisition time of 200 s. Phosphorus was detected in an approximately 10-nm length of single-stranded RNA contained in a tobacco mosaic virus particle (mol.wt = 130 kDa/nm) with a sensitivity of ± 25 atoms. Near single-atom sensitivity was achieved for the detection of iron in one haemoglobin molecule (mol.wt = 65 kDa, containing four Fe atoms). Such detection limits are only feasible if special processing methods are employed, as is demonstrated by the use of the second-difference acquisition technique and multiple least-squares fitting of reference spectra. Moreover, an extremely high electron dose (about 10¹⁰ e/nm²) is required resulting in mass loss that may be attributable to ‘knock-on’ radiation damage.     

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.     

Extracting physically interpretable data from electron energy-loss spectra

Principal component analysis is routinely applied to analyze data sets in electron energy-loss spectroscopy (EELS). We show how physically meaningful spectra can be obtained from the principal components using a knowledge of the scattering of the probe electron and the geometry of the experiment. This approach is illustrated by application to EELS data for the carbon K edge in graphite obtained using a conventional transmission electron microscope. The effect of scattering of the probe electron is accounted for, yielding spectra which are equivalent to experiments using linearly polarized X-rays. The approach is general and can also be applied to EELS in the context of scanning transmission electron microscopy.     

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.     

Successful application of spatial difference technique to electron energy-loss spectroscopy studies of Mo/SrTiO3 interfaces

The electron energy‐loss near‐edge structure (ELNES) of Mo/SrTiO₃ interfaces has been studied using high spatial resolution electron energy‐loss spectroscopy (EELS) in a dedicated scanning transmission electron microscope. Thin films of Mo with a thickness of 50 nm were grown on (001)‐orientated SrTiO₃ surfaces by molecular beam epitaxy at 600 °C. High‐resolution transmission electron microscopy revealed that the interfaces were atomically abrupt with the (110)_Mo plane parallel to the substrate surface. Ti‐L_2,3 (～460 eV), O‐K (～530 eV), Sr‐L_2,3 (～1950 eV) and Mo‐L_2,3 (～2500 eV) absorption edges were acquired by using the Gatan Enfina parallel EELS system with a CCD detector. The interface‐specific components of the ELNES were extracted by employing the spatial difference method. The interfacial Ti‐L_2,3 edge shifted to lower energy values and the splitting due to crystal field became less pronounced compared to bulk SrTiO₃, which indicated that the Ti atoms at the interface were in a reduced oxidation state and that the symmetry of the TiO₆ octahedra was disturbed. No interfacial Sr‐L_2,3 edge was observed, which may demonstrate that Sr atoms do not participate in the interfacial bonding. An evident interface‐specific O‐K edge was found, which differs from that of the bulk in both position (0.8 ± 0.2 eV positive shift) and shape. In addition, a positive shift (0.9 ± 0.3 eV) occurred for the interfacial Mo‐L_2,3, revealing an oxidized state of Mo at the interface. Our results indicated that at the interface SrTiO₃ was terminated with TiO₂. The validity of the spatial difference technique is discussed and examined by introducing subchannel drift intentionally.     

Water mapping in hydrated soft materials

We present a method based on spatially resolved electron energy-loss spectroscopy in the cryo-STEM to map the spatial distribution of water in frozen-hydrated polymers. The spatial resolution is limited by the dose constraints imposed by radiation damage, and to stay within these constraints, the use of fine electron-probe sizes comes at the cost of reduced counts in the energy-loss spectra. Thus, at the resolution limit, the detection of isolated water-rich pixels or the identification of minor variations in water content across the specimen is complicated because one must distinguish significant fluctuations from noise. Here we develop a criterion with which to guide such a distinction. We characterize the intrinsic noise associated with spectral measurements under given illumination and acquisition conditions. We then use that noise in combination with scatter diagrams to threshold spectrum images and objectively identify statistically significant compositional fluctuations. We illustrate these ideas using a simulated spectrum dataset for a hypothetical blend of hydrophilic and hydrophobic homopolymers. We show that while a direct inspection of the water map may not allow any meaningful conclusions to be drawn, after applying the thresholding approach we can clearly identify the regions of the specimen that are rich in water. We also experimentally study a model blend system comprised of hydrophilic poly(vinyl pyrrolidone) (PVP) dispersed in a hydrophobic matrix of poly(styrene) (PS). By MLS fitting using damaged and undamaged PVP reference spectra, we determine that the critical dose characteristic of dry PVP is approximately 8000 e/nm2 using 200 keV incident electrons. Irradiating frozen-hydrated PVP gives rise to noticeable hydrogen evolution at doses of approximately 1500 e/nm2. To stay within this constraint we use doses of 400 e/nm2 and a pixel spacing in the spectrum imaging of 100 nm. In order to quantitatively map the water, PVP, and PS compositions, we measure their total inelastic scattering cross-sections. Direct inspection of the composition maps reveals the presence of large water-rich domains of the order of approximately 1 microm and the scatter-diagram thresholding approach identifies small water-rich domains one pixel in size.     

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.     

Highly spatially resolved electron energy-loss spectroscopy in the bandgap regime of GaN

The possibilities of obtaining information about interband scattering processes in the bandgap regime of GaN from electron energy-loss spectra, taken in a dedicated scanning transmission electron microscope (STEM), are investigated. With the help of precise simulations of the zero-loss peak it is feasable to process, extract and analyse data in the extreme low-loss regime of a few electronvolts. The accuracy of the results is restricted predominantly by instrumental broadening functions. By modelling these accurately, it is possible to eliminate the effects of the tail of the zero-loss peak and to extract the low-loss spectrum together with the correct value for the bandgap of GaN. Furthermore, differences in the shapes of the low-loss spectra can be revealed, depending on the microstructural features, probed at different beam locations.     

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.     

Characterization of nanophase Al-oxide/Al powders by electron energy-loss spectroscopy

Al nanoparticles were prepared by the inert gas condensation method. After passivation with oxygen and air exposure we obtained a powdered sample of an Al-oxide/Al nanocomposite material. In the present paper we describe the use of the electron energy-loss spectroscopy (EELS) technique in a transmission electron microscope to characterize such nanostructured powders compared with a microcrystalline commercial aluminium foil. Energy-filtered images showed the presence of an alumina overlayer of ≈ 4 nm covering the aluminium nanoparticles (23 nm in diameter). EELS analysis enabled us to determine the total amount of Al₂O₃ and metallic Al and the structure of the alumina passivation overlayer in the sample. In particular, the extended energy-loss fine structure analysis of the data showed a major presence of Al tetrahedrally coordinated with oxygen in the alumina passivation layer of Al nanoparticles instead of the octahedral coordination found for a conventional Al foil. This surprising effect has been attributed to the nanoscopic character of the grains. The analysis of the electron-loss near-edge structure also determines the presence of a certain degree of aggregation in this kind of powdered sample as result of the coalescence of the nanocrystalline grains. The procedure presented here may have the potential to solve other problems during characterization of nanostructured materials.     

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.     

Development of a high energy resolution electron energy-loss spectroscopy microscope

We have developed a high energy resolution electron energy-loss spectroscopy (EELS) microscope, which can take spectra from specified small specimen areas and specified small reciprocal space areas to investigate detailed electronic structures. The EELS microscope is equipped with retarding Wien filters as the monochromator and the analyser. The filters are designed to achieve a stigmatic focus. The energy resolutions are 12 meV and 25 meV for cases without and with a specimen, respectively. Spatial and momentum resolutions are 30–110 nm in diameter and 1.1 nm⁻¹ in angular diameter, respectively. EELS spectra are presented to show the performance of this instrument.     

In situ analysis of gas composition by electron energy-loss spectroscopy for environmental transmission electron microscopy

We have developed methods for using in situ electron energy-loss spectroscopy (EELS) to perform quantitative analysis of gas in an environmental transmission electron microscope. Inner-shell EELS was able to successfully determine the composition of gas mixtures with an accuracy of about 15% or better provided that some precautions are taken during the acquisition to account for the extended gas path lengths associated with the reaction cell. The unique valence-loss spectrum associated with many gases allowed simple methodologies to be developed to determine gas composition from the low-loss region of the spectrum from a gas mixture. The advantage of the valence loss approach is that it allows hydrogen to be detected and quantified. EELS allows real-time analysis of the volume of gas inside the reaction cell and can be performed rapidly with typical acquisition times of a few seconds or less. This in situ gas analysis can also be useful for revealing mass transport issues associated with the differential gas diffusion through the system.     

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.     

EELS log-ratio technique for specimen-thickness measurement in the TEM

We discuss measurement of the local thickness t of a transmission microscope specimen from the log-ratio formula t = λ In (I_t/I₀) where I_t and I₀ are the total and zero-loss areas under the electron-energy loss spectrum. We have measured the total inelastic mean free path λ in 11 materials of varying atomic number Z and have parameterized the results in the form λ = 106F (E₀/E_m)/ln (2βE₀/E_m) where F = (1 + E₀/1,022)/(1 + E₀/511)², the incident energy E₀ is in keV, the spectrum collection semiangle β is in mrad, and E_m = 7.6Z^0.36. This formulation should allow absolute thickness to be determined to an accuracy of ±20% in most inorganic specimens.