Parallel recording of electron energy loss spectra

Two silicon photo diode array devices were tested as parallel recording detectors for electron energy loss spectrometry (EELS). The direct bombardment of a Reticon photodiode array detector with high energy electrons (80 keV) causes an irreversible increase in diode dark current. The dark current saturates the detector amplifier after a dose of 10^?6 C/diode making it unsuitable for EELS. A scintillator coupled SIT vidicon is sensitive enough to count two high energy electrons with a spatial resolution of 100 μm, corresponding to 5 eV energy resolution with the electron optical system described. The large pixel-to-pixel gain variation inherent in the scintillator and vidicon can be reduced by averaging the spectrum over a large area of the target perpendicular to the dispersion direction. The L-edge of calcium for a 4 × 10^?3 weight fraction concentration biological specimen is observable in a 40 s parallel recorded spectrum. The minimum detectable concentration of calcium is estimated tobe ten times better for EELS than EDS X-ray analysis.     

Thickness measurements with electron energy loss spectroscopy

Measurements of thickness using electron energy loss spectroscopy (EELS) are revised. Absolute thickness values can be quickly and accurately determined with the Kramers-Kronig sum method. The EELS data analysis is even much easier with the log-ratio method, however, absolute calibration of this method requires knowledge of the mean free path of inelastic electron scattering lambda. The latter has been measured here in a wide range of solids and a scaling law lambda approximately rho(-0.3) versus mass density rho has been revealed. EELS measurements critically depend on the excitation and collection angles. This dependence has been studied experimentally and theoretically and an efficient model has been formulated.     

Quantification and thickness correction of EFTEM phosphorus maps

We describe a method for correcting plural inelastic scattering effects in elemental maps that are acquired in the energy filtering transmission electron microscope (EFTEM) using just two energy windows, one above and one below a core edge in the electron energy loss spectrum (EELS). The technique is demonstrated for mapping low concentrations of phosphorus in biological samples. First, the single-scattering EELS distributions are obtained from specimens of pure carbon and plastic embedding material. Then, spectra are calculated for different specimen thicknesses t, expressed in units of the inelastic mean free path lambda. In this way, standard curves are generated for the ratio k0 of post-edge to pre-edge intensities at the phosphorus L2,3 excitation energy, as a function of relative specimen thickness t/lambda. Thickness effects in a two-window phosphorus map are corrected by successive acquisition of zero-loss and unfiltered images, from which it is possible to determine a t/lambda image and hence a background k0-ratio image. Knowledge of the thickness-dependent k0-ratio at each pixel thus enables a more accurate determination of the phosphorus distribution in the specimen. Systematic and statistical errors are calculated as a function of specimen thickness, and elemental maps are quantified in terms of the number of phosphorus atoms per pixel. Further analysis of the k0-curve shows that the EFTEM can be used to obtain reliable two-window phosphorus maps from specimens that are considerably thicker than previously possible.     

Parallel electron energy-loss spectroscopy free from gain variation

A new method is presented for removing the effect of the gain variation of parallel detectors used for the quantitation of trace elements with electron energy loss spectroscopy (EELS). Use of the ratio of two first-difference spectra eliminates the effect of gain variation of the detector, therefore eliminating the need for gain normalization and dark current subtraction. This method is particularly suitable for revealing small signals superimposed on a large background, a typical scenario for trace element quantitation of both biological and inorganic materials. This method has been tested on a system with a cooled CCD camera as the parallel detector and illustrated by the analysis of low concentration Ca in an organic matrix. The method is expected to be generally applicable to spectral analysis affected by gain variations of parallel detectors.     

Quantitative electron energy loss spectroscopy in biology

The potential for applying electron energy loss spectroscopy (EELS) in biology is assessed. Some recent developments in instrumentation, spectrometer design, parallel detection and elemental mapping are discussed. Quantitation is demonstrated by means of the spectrum from DNA which gives an elemental ratio for N:P close to the expected value. A range of biologically important elements that can be usefully analyzed by EELS is tabulated and some possible applications for each are indicated. Detection limits and the effects of radiation damage are illustrated by spectra from the protein, insulin, and from the fluorinated amino-acid, histidine. Calcium detectability under optimum conditions may be as low as 1 mmol/kg dry weight. The application of EELS to analysis of cryosectioned adrenomedullary (chromaffin) cells is described in order to help determine the composition of the secretory granule. Water content can be determined from the amount of inelastic scattering as measured by the low-loss spectrum. The nitrogen/phosphorus ratio can be measured to provide information about the relative concentrations of ATP, chromogranin, and catecholamines. Quantitative EELS elemental maps are obtained in the STEM mode from chromaffin cells in order to measure the distribution of light elements.     

Thickness dependence of signal/background ratio of inner-shell electron excitation loss in eels

The influence of the specimen thickness on the core-edge loss intensity has been studied using the relative specimen thickness (t_R) as the standard rule for the specimen thickness in EELS. The core-edge loss intensity monotonously increased with the increasing relative specimen thickness, 0 < t_R ? 1, and monotonously decreased with increasing relative specimen thickness, t_R ? 1, behind the maximum of core-edge intensity at t_R ? 1. “Optimum specimen thickness” to get the highest core-edge intensity is suggested for the thickness of t_R = 1. The thickness-dependent factor T(t_R) should be considered in a practical measurement in EELS.     

Electron energy loss analysis of near-trace-element concentrations of calcium

The quantitation of near-trace-element concentrations of calcium (25 ppm atomic fraction) with electron energy loss spectroscopy (EELS) is demonstrated. The data collection, with an energy-stabilized parallel recording spectrometer, subsequent signal processing, and quantitation procedures are described. The quantitative results obtained with EELS, in the biologically relevant range of 1 to 100 mmol/kg, are directly compared with simultaneously collected and previously validated energy-dispersive X-ray spectroscopy (EPMA). The experimentally determined sensitivity of EELS for Ca detection is five-fold better than for EPMA, and the theoretically attainable sensitivity of EELS is ten-fold better than for EPMA. However, the attainment of this sensitivity with EELS is technically more difficult and limited by specimen thickness. The sensitivity of EELS experimentally demonstrated in this study permits the detection of three calcium atoms in a 10 nm diameter spot of an organic matrix, with a field-emission-gun-equipped scanning transmission electron microscope.     

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.     

Calculation of the electronic structure of carbon films using electron energy loss spectroscopy.

The detailed understanding of the electronic properties of carbon-based materials requires the determination of their electronic structure and more precisely the calculation of their joint density of states (JDOS) and dielectric constant. Low electron energy loss spectroscopy (EELS) provides a continuous spectrum which represents all the excitations of the electrons within the material with energies ranging between zero and about 100 eV. Therefore, EELS is potentially more powerful than conventional optical spectroscopy which has an intrinsic upper information limit of about 6 eV due to absorption of light from the optical components of the system or the ambient. However, when analysing EELS data, the extraction of the single scattered data needed for Kramers Kronig calculations is subject to the deconvolution of the zero loss peak from the raw data. This procedure is particularly critical when attempting to study the near-bandgap region of materials with a bandgap below 1.5 eV. In this paper, we have calculated the electronic properties of three widely studied carbon materials; namely amorphous carbon (a-C), tetrahedral amorphous carbon (ta-C) and C60 fullerite crystal. The JDOS curve starts from zero for energy values below the bandgap and then starts to rise with a rate depending on whether the material has a direct or an indirect bandgap. Extrapolating a fit to the data immediately above the bandgap in the stronger energy loss region was used to get an accurate value for the bandgap energy and to determine whether the bandgap is direct or indirect in character. Particular problems relating to the extraction of the single scattered data for these materials are also addressed. The ta-C and C60 fullerite materials are found to be direct bandgap-like semiconductors having a bandgaps of 2.63 and 1.59eV, respectively. On the other hand, the electronic structure of a-C was unobtainable because it had such a small bandgap that most of the information is contained in the first 1.2 eV of the spectrum, which is a region removed during the zero loss deconvolution.     

Bandgap measurement of thin dielectric films using monochromated STEM-EELS

High-resolution electron energy-loss spectroscopy (HR-EELS), achieved by attaching electron monochromators to transmission electron microscopes (TEM), has proved to be a powerful tool for measuring bandgaps. However, the method itself is still uncertain, due to Cerenkov loss and surface effects that can potentially influence the quality of EELS data. In the present study, we achieved an energy resolution of about 0.13 eV at 0.1 s, with a spatial resolution of a few nanometers, using a monochromated STEM-EELS technique. We also assessed various methods of bandgap measurement for a-SiNx and SiO₂ thin dielectric films. It was found that the linear fit method was more reliable than the onset reading method in avoiding the effects of Cerenkov loss and specimen thickness. The bandgap of the SiO₂ was estimated to be 8.95 eV, and those of a-SiNx with N/Si ratios of 1.46, 1.20 and 0.92 were measured as 5.3, 4.1 and 2.9 eV, respectively. These bandgap-measurement results using monochromated STEM-EELS were compared with those using Auger electron spectroscopy (AES)-reflective EELS (REELS).     

Separation of bulk and surface-losses in low-loss EELS measurements in STEM

To identify major features in low electron energy loss spectra, the different excitations (bulk plasmons, interband transitions, surface plasmons, Cherenkov and surface guided modes) must be delineated from each other. In this paper, this process is achieved by noting the linear thickness dependence of bulk processes contrasted with the constant thickness behavior of surface excitations. An alternative approach of analyzing bulk plasmon-loss is also introduced. Using a new algorithm, the parameters of plasma generation-plasmon energy E(P,0), a damping parameter DeltaE(P) and the coefficient of the dispersion relation gamma were obtained from a single curve fitting on the example of Si. The ability to separate surface-losses from the rest of the data permitted identification of the fine structure of the surface-losses. The strong peak at 8.2 eV characteristic of non-radiative surface plasmon excitations was measured for Si. Analysis of surface excitations indicates that a 10ASiO2 surface coating layer is still present despite careful cleaning the specimen. Dielectric functions deduced from the EELS data prove to be considerably affected by the presence of the surface-losses for samples as thick as 800A.     

Estimation of section thickness and quantification of iron standards with EELS

The possibilities of using electron energy-loss spectroscopy (EELS) for quantification of elemental concentrations in ultrathin sections are examined. Dynabeads, which are polystyrene beads with a known iron content, are proposed as internal iron standards. The quantity of an element present depends on the thickness of the specimen. A prerequisite for estimation of absolute section thicknesses with EELS is the knowledge of the mean free path λ of electrons in the specimen. This factor is determined for the embedding resins Epon and Nanoplast by comparing EELS data with directly observed thicknesses in re-embedded sections. Dynabeads were found to include iron in a homogeneous distribution and to be stable in the electron beam.     

Some effects of electron channeling on electron energy loss spectroscopy

As an electron beam (of order 100 keV) travels through a crystalline solid it can be channeled down a zone axis of the crystal to form a channeling peak centered on the atomic columns. The channeling peak can be similar in size to the outer atomic orbitals. Electron energy loss spectroscopy (EELS) measures the losses that the electron experiences as it passes through the solid yielding information about the unoccupied density of states in the solid. The interaction matrix element for this process typically produces dipole selection rules for small angle scattering. In this paper, a theoretical calculation of the EELS cross section in the presence of strong channeling is performed for the silicon L23 edge. The presence of channeling is found to alter both the intensity and selection rules for this EELS signal as a function of depth in the solid. At some depths in the specimen small but significant non-dipole transition components can be produced, which may influence measurements of the density of states in solids.     

Quantitative EFTEM mapping of near physiological calcium concentrations in biological specimens

Although electron energy-loss spectroscopy (EELS) in the scanning transmission electron microscope (STEM) provides high sensitivity for measuring the important element, calcium, in biological specimens, the technique has been difficult to apply routinely, because of long acquisition times required. Here we describe a refinement of the complementary analytical technique of energy-filtered transmission electron microscopy (EFTEM), which enables rapid imaging of large cellular regions and measurement of calcium concentrations approaching physiological levels. Extraction of precise quantitative information is possible by averaging large numbers of pixels that are contained in organelles of interest. We employ a modified two-window approach in which the behavior of the background signal in the EELS spectrum can be modeled as a function of specimen thickness t expressed in terms of the inelastic mean free path λ. By acquiring pairs of images, one above and one below the Ca L_2,3 edge, together with zero-loss and unfiltered images, which are used to determine a relative thickness (t/λ) map, it is possible to correct the Ca L_2,3 signal for plural scattering. We have evaluated the detection limits of this technique by considering several sources of systematic errors and applied this method to determine mitochondrial total calcium concentrations in freeze-dried cryosections of rapidly frozen stimulated neurons. By analyzing 0.1 μm² areas of specimen regions that do not contain calcium, it was found that the standard deviation in the measurement of Ca concentrations was about 20 mmol/kg dry weight, corresponding to a Ca:C atomic fraction of approximately 2×10⁻⁴. Calcium concentrations in peripheral mitochondria of recently depolarized, and therefore stimulated and Ca loaded, frog sympathetic neurons were in reasonable agreement with previous data.     

A differentially pumped low-energy ion beam system for an ultrahigh-vacuum atom-probe field-ion microscope

An ultrahigh-vacuum (UHV) differentially pumped low-energy (50-3000 eV) ion beam system for the in situ irradiation of specimens in a UHV atom-probe field-ion microscope (FIM) was designed and constructed. The ion beam system consisted of a Finkelstein-type ion source, an Einzel lens, and a magnetic mass analyzer. The ion source was connected to the analyzer chamber by small apertures which resulted in differential pumping between the ion source and the analyzer chamber; during a typical in situ irradiation of a specimen in the atom-probe FIM the total pressure was maintained at approximately 10(-7) Torr. In the case of helium ion irradiation the optimum ion-current density was approximately 0.5 microA cm(-2) for 300-eV He+ ions at the atom-probe FIM specimen. After the completion of a helium ion irradiation the pumpdown time from 5 x 10(-7) to approximately 3 x 10(-10) Torr in the atom-probe FIM chamber was 0.5 h.     

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.     

A system for acquiring simultaneous electron energy-loss and X-ray spectrum-images

A compositional imaging system based on simultaneous scanning electron energy‐loss spectroscopy (EELS) and energy‐dispersive X‐ray spectroscopy (EDS) was developed. This system utilizes the combined power of EELS and EDS for quantitative compositional imaging at nanometre resolution. The system is particularly suitable for, but not limited to, biological research, as it simultaneously provides sensitive maps of an element such as Ca or P from EELS and of many other elements from EDS. Degradation of resolution by specimen drift is prevented by correcting for drift during data acquisition, using image cross‐correlation. Several advanced features are implemented for real‐time and/or off‐line quantitative analysis, and the performance of the system is illustrated with practical applications to compositional imaging of cardiac muscle.     

An electron energy loss spectral library

Although the general characteristics of an energy loss spectrum are reasonably well known, tables of atomic energy levels are not always sufficient to uniquely identify all features in a given spectrum. Systematic variation on an element-to-element basis across the periodic table can be substantial and has not yet been documented in a single reference source. A wide range of specimens has been studied using EELS, resulting in a fairly extensive data base, which is now being prepared for general distribution to the scientific community in the form of a spectral library.     

Energy-filtering TEM and electron energy-loss spectroscopy of double structure tabular microcrystals of silver halide emulsions

Composite Ag(Br,I) tabular microcrystals of photographic emulsions were studied by the combination of energy-filtering electron microscopy (EFTEM) and electron energy-loss spectroscopy (EELS) in conjunction with energy-dispersive X-ray (EDX) microanalysis. The contrast tuning under the energy-filtering in the low-loss region was used to observe more clearly edge and random dislocations, {11⁻1} stacking faults in the grain shells parallel to {11⁻2} edges and bend and edge contours. Electron spectroscopic diffraction patterns revealed numerous extra reflections at commensurate positions in between the Bragg reflections and diffuse honeycomb contours; these were assigned to the number of defects in the shell region parallel to the grain edges and polyhedral clusters of interstitial silver cations, respectively. Inner-shell excitation bands of silver halide were detected and confirmed by EDX analyses, i.e. the Ag N_2,3 edge at 62 eV (probably overlapped with the weak I N_4,5 edge at 52 eV and the Br M_4,5 edge at 70 eV), the I M_4,5 edge at about 620 eV, and the Br L_2,3 edge at about 1550 eV energy losses. Energy-loss near-edge structure of the Ag M_4,5 edge at about 367 eV energy losses and low-loss fine structure arisen as a result of interband transitions and excitons, possibly superimposed with many electron effects, have been revealed. The crystal thickness was determined by a modified EELS log-ratio technique in satisfactory agreement with measurements on grain replicas.     

A new analytical method for characterising the bonding environment at rough interfaces in high-k gate stacks using electron energy loss spectroscopy

Determining the bonding environment at a rough interface, using for example the near-edge fine structure in electron energy loss spectroscopy (EELS), is problematic since the measurement contains information from the interface and surrounding matrix phase. Here we present a novel analytical method for determining the interfacial EELS difference spectrum (with respect to the matrix phase) from a rough interface of unknown geometry, which, unlike multiple linear least squares (MLLS) fitting, does not require the use of reference spectra from suitable standards. The method is based on analysing a series of EELS spectra with variable interface to matrix volume fraction and, as an example, is applied to a TiN/poly-Si interface containing oxygen in a HfO₂-based, high-k dielectric gate stack. A silicon oxynitride layer was detected at the interface consistent with previous results based on MLLS fitting.