A generalization of atom location by channelling enhanced microanalysis

The technique of atom location by channelling enhanced microanalysis (ALCHEMI), has limitations in practical application. A generalization of this technique, with the underlying idea that additional relationships can be generated by performing these experiments at an appropriate number of orientations depending upon the stoichiometry of the original compound has been developed. The precise number of orientations required to solve the distributions in a whole class of related compounds is expressed in terms of the stoichiometry of the original compound along with the alloying additions.     

Quantification for the X-ray microanalysis of cryosections

Some problems of the quantitative analysis of diffusible elements in cryosections are reviewed. The two prevalent methods for obtaining concentrations from X-ray data, one based on characteristic radiation alone and the other on continuum-normalization, are recapitulated. Both methods seem suitable at cellular level while the latter seems preferable at finer spatial resolution. Recourse to both methods together is desirable in the analysis of frozen-hydrated sections especially when there is no peripheral standard. Selective local contamination is a particular hazard in the analysis of chlorine. In the case of sodium, physical parameters set restrictive limits to the minimum concentration measurable by ‘energy-dispersive’ X-ray spectrometry (about 20 mm kg^?1) and to the spatial resolution attainable by diffractive X-ray spectrometry (～0·2 μm). One obvious danger to meaningful quantitative analysis is inadvertent redistribution of diffusible elements during the moments preceding the freeze-quenching of a tiny piece of tissue. Data are presented to show that concentration changes due to simple evaporation are a real hazard prior to the quenching of sub-millimetre size samples.     

A comparison of two models for the characteristic X-ray fluorescence correction in thin foil analysis

It is shown that Philibert & Tixier's calculation for the contribution of characteristic fluorescence in thin foil X-ray microanalysis is in error. The calculation of Nockolds et al. for this contribution is shown to be correct.     

Real-time quantitative elemental analysis and mapping: microchemical imaging in cell physiology

Recent advances in widely available microcomputers have made the acquisition and processing of digital quantitative X-ray maps of one to several cells readily feasible. Here we describe a system which uses a graphics-based microcomputer to acquire spectrally filtered X-ray elemental image maps that are fitted to standards, to display the image in real time, and to correct the post-acquisition image map with regard to specimen drift. Both high-resolution quantitative energy-dispersive X-ray images of freeze-dried cyrosections and low-dose quantitative bright-field images of frozen-hydrated sections can be acquired to obtain element and water content from the same intracellular regions. The software programs developed, together with the associated hardware, also allow static probe acquisition of data from selected cell regions with spectral processing and quantification performed on-line in real time. In addition, the unified design of the software program provides for off-line processing and analysing by several investigators at microcomputers remote from the microscope. The overall experimental strategy employs computer-aided imaging, combined with static probes, as an essential interactive tool of investigation for biological analysis. This type of microchemical microscopy facilitates studies in cell physiology and pathophysiology which focus on mechanisms of ionic (elemental) compartmentation, i.e. structure-function correlation at cellular and subcellular levels; it allows investigation of intracellular concentration gradients, of the heterogeneity of cell responses to stimuli, of certain fast physiological events in vivo at ultrastructural resolution, and of events occurring with low incidence or involving cell-to-cell interactions.     

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.     

Backscattered electron imaging of cultured cells: application to electron probe X-ray microanalysis using a scanning electron microscope

We report a simple method to study the elemental content in cultured human adherent cells by electron probe X-ray microanalysis with scanning electron microscopy. Cells were adapted to grow on polycarbonate tissue culture cell inserts, washed with distilled water, plunge-frozen with liquid nitrogen and freeze-dried. Unstained, freeze-dried cultured cells were visualized in the secondary and backscattered electron imaging modes of scanning electron microscopy. With backscattered electron imaging it was possible to identify unequivocally major subcellular compartments, i.e. the nucleus, nucleoli and cytoplasm. X-ray microanalysis was used simultaneously to determine the elemental content in cultured cells at the cellular level. In addition, we propose some improvements to optimize backscattered electron and X-ray signal collection. Our findings demonstrate that backscattered electron imaging offers a powerful method to examine whole, freeze-dried cultured cells for scanning electron probe X-ray microanalysis.     

Characteristic X-ray production cross-sections for standardless elemental analysis in EDX

Measurements have been made of cross-sections for the production of X-rays following ionization of the K-shell by fast electrons for a range of elements and incident electron energies. These data have been fitted to a simple functional expression for the ionization cross-section. This model, the Bethe model, includes two empirical fitting parameters. We found that a single universal pair of parameters may be used to allow the model to predict cross-sections with a mean error of ～ 7% and a maximum error of ～ 20% over the range of conditions considered. A relativistic form of the Bethe model yields a small improvement in accuracy for higher incident electron energies. Measurements have also been made of L-shell production cross-sections. It is expected that the Bethe model will apply also to the L-shell. Comparison of experimental and calculated K/L-shell cross-section ratios generally supports this, but uncertainties in the values in the literature for L-shell fluorescence yields precludes the drawing of any firm conclusions here.     

Beam induced mass loss in high resolution biological microanalysis

Electron beam induced loss of mass from the organic matrix and from higher Z constituents of biological samples was measured by monitoring bremsstrahlung and peak changes in EDS spectra. When any effects of contamination, extraneous X-rays, beam current drift, specimen drift, and specimen shrinkage were monitored and corrected for, the three types of samples gave consistent and similar results at 296 K. Bremsstrahlung losses averaged 45%, 46% and 50% respectively for muscle homogenate, salivary gland sections and albumin. Sulphur losses average 74%, 72% and 86% for the same three sample types. No other elements suffered significant losses. D_l/e for bremsstrahlung averaged 0·14 C/cm². Bremsstrahlung loss at 93 K began approximately one order of magnitude higher in dose, and the extent of loss varied. Sulphur losses, however, were greatly reduced at low temperatures.     

X-ray microanalysis of wet biological specimens in the environmental scanning electron microscope. 1. Reduction of specimen distance under different atmospheric conditions

X-ray microanalysis of non-biological and biological specimens was carried out in the environmental scanning electron microscope (ESEM) under different conditions of specimen distance (the distance travelled by the electron probe within the specimen chamber) and chamber atmosphere. Using both water vapour and argon atmospheres, it was shown that reduction in specimen distance had no effect on atmospheric gas X-ray signal in either case. Unlike water vapour, increased levels of argon (up to 10 torr) caused a marked depression of specimen P/B ratios, with a decrease in both characteristic and background (continuum) counts. These effects in argon were not altered by reduction in specimen distance. Specimen distance was important in relation to beam skirting and elemental analysis. With an extended assembly (short specimen distance), beam skirting in a water-vapour atmosphere was much reduced – leading to enhanced element detectability in a discrete biological specimen (Anabaena cyclindrica).     

Modification of a TEM-goniometer specimen holder to enable beam current measurements in a (S)TEM for use in quantitative X-ray microanalysis

In order to have available a specimen holder suited to measure the beam current as is often required in quantitative electron probe X-ray microanalysis, the rod of a low background beryllium specimen holder of a transmission electron microscope was modified. The tip was electrically insulated from the mass of the microscope and connected electrically to the central contact of a BNC connector mounted on the specimen holder handle. With this modified specimen holder the current absorbed by the specimen and/or the specimen holder could be measured easily and accurately. The modified specimen holder has been used to measure the beam current stability of an analytical electron microscope under various conditions. Data were obtained for tungsten as well as lanthanum hexaboride cathodes. Small changes to other types of specimen tips made it possible to exchange these for the low background tip.     

Personal-computer-based system for electron beam X-ray microanalysis of biological samples

A system based on a personal computer has been developed which provides a relatively inexpensive way to equip an electron microscopy laboratory for quantitative elemental analyses of cryosectioned biological samples. This system demonstrates the feasibility of making an X-ray analyser from a personal computer, together with commercially available hardware and software components. Hardware and software have been assembled to drive the beam in a scanning electron microscope, collect and analyse X-ray spectra, and save, retrieve, and analyse data. Our software provides a menu-controlled user interface to direct spectra acquisition and analysis. Spot analyses, video images, and quantitative elemental images may be obtained and results transferred in ASCII format to other computers. Wet weight, as well as dry weight, concentrations are calculated, if measurements were made of areas of the hydrated sample before it was freeze-dried. Grey-level copies of video and quantitative elemental images may be made on a laser printer.     

Diffraction effects and inelastic electron transport in angle‐resolved microscopic imaging applications

We analyse the signal formation process for scanning electron microscopic imaging applications on crystalline specimens. In accordance with previous investigations, we find nontrivial effects of incident beam diffraction on the backscattered electron distribution in energy and momentum. Specifically, incident beam diffraction causes angular changes of the backscattered electron distribution which we identify as the dominant mechanism underlying pseudocolour orientation imaging using multiple, angle‐resolving detectors. Consequently, diffraction effects of the incident beam and their impact on the subsequent coherent and incoherent electron transport need to be taken into account for an in‐depth theoretical modelling of the energy‐ and momentum distribution of electrons backscattered from crystalline sample regions. Our findings have implications for the level of theoretical detail that can be necessary for the interpretation of complex imaging modalities such as electron channelling contrast imaging (ECCI) of defects in crystals. If the solid angle of detection is limited to specific regions of the backscattered electron momentum distribution, the image contrast that is observed in ECCI and similar applications can be strongly affected by incident beam diffraction and topographic effects from the sample surface. As an application, we demonstrate characteristic changes in the resulting images if different properties of the backscattered electron distribution are used for the analysis of a GaN thin film sample containing dislocations.     

A procedure to prepare cultured cells in suspension for electron probe X-ray microanalysis: application to scanning and transmission electron microscopy

We describe a simple procedure to prepare cultured cells in suspension to analyse elemental content at the cellular level by electron probe X-ray microanalysis. Cells cultured in suspension were deposited onto polycarbonate tissue, culture plate well inserts, centrifuged at low g , washed to remove the extracellular medium, cryofixed and freeze-dried, and analysed in the scanning mode of a scanning electron microscope. We tested the effect of different washing solutions (150 m m ammonium acetate, 300 m m sucrose, and distilled water) on the elemental content of cultured cells in suspension. The results demonstrated that distilled water was the best washing solution to prepare cultured cells. In addition, the low Na content, high K content and high K/Na ratio of the cells indicated that this procedure, based on the centrifugation at low g followed by cryopreparation, constitutes a satisfactory method to prepare cultured cells in suspension. We also investigated the effects of different accelerating voltages on X-ray signal collection. The results showed that moderate accelerating voltages, i.e. 10–11 kV, should be used to analyse whole cells in the scanning mode of the scanning electron microscope. We show that this method of preparation makes it possible to prepare cryosections of the cultured cells, thus permitting analysis of the elemental content at the subcellular level, i.e. nucleus, cytoplasm and mitochondria, using a scanning transmission electron microscope.     

Evaluation of several common standards for the X-ray microanalysis of thin biological specimens

The question of the best type of standard to use for X-ray microanalysis of thin biological specimens remains unanswered. Standards embedded in an organic matrix have the advantage that they resemble biological specimens, but their composition is generally not known exactly. We compared several standards and, surprisingly, inorganic binary salts sprayed onto a supporting film were the most suitable: they corresponded closely with several other methods using organic matrices; they were easily produced; and their composition is known. Glutaraldehydeurea aminoplastic resin thin sections and thin films containing dissolved salts were problematic. The composition of the polymer appears to be variable, and the thin films did not correspond with any other standard tested. Chelex¹⁰⁰ bio-standard beads and flakes loaded with accurately determined concentrations of ions, embedded in epoxy resin and thin sectioned, tended to correspond to the results obtained with the binary salts. However, the results from some bio-standards were inexplicably aberrant. An epoxy resin standard was used for bromine, and was found to agree closely with the binary standards.     

X-ray microanalytical resolution in frozen-hydrated biological bulk samples

In coated frozen-hydrated gelatin gels the backscattered electron yield does not increase during electron irradiation as it does in uncoated samples. Neither is the backscattered electron yield greater from coated frozen-hydrated gels than that from more conductive organic samples. This is interpreted as indicating that a significant distortion of the electron interaction volume, due to the development of a space charge, does not occur in electron irradiated frozen-hydrated gelatine gels when they are coated with a conducting coat. The depth resolution as estimated from models of biological samples in the form of frozen-hydrated photographic film and frozen-hydrated sections of gelatine gel is consistent with that computed from X-ray depth distribution curves, i.e. close to 2.0 μm at 15 kV. Lateral resolution was estimated from photographic film to be close to 2.0 μm also, at 15 kV.     

SEM electron channelling patterns as a technique for the characterization of ion-implantation damage

Electron channelling patterns (ECPs) formed in back-scattered images in the scanning electron microscope (SEM) have been used occasionally to confirm surface amorphization during ion implantation. In order to place such observations on a more quantitative basis, the study reported here has explored the variation of ECP appearance with both specimen damage levels (and thus subsurface structures) and SEM accelerating voltage (i.e. sampled depth). Polished and annealed (0001) single crystal sapphire discs were implanted to various damage levels up to both subsurface and full surface amorphization. Damage levels were measured independently by Rutherford back-scattering (RBS). Selected-area ECPs were obtained in a Jeol-840 electron microscope operating over the range 5–40 kV in 5-kV steps. Progressive ECP degradation—in terms of high-order line disappearance—was observed with increasing dose, culminating in total pattern loss when full surface amorphization occurred. However, ECP information could still be obtained from the damaged near-surface material even when a subsurface amorphous layer was present, thus demonstrating the shallow retrieval depth of information from the ECP technique. Indeed, because the spatial distribution of damage from ion implantation is both calculable and measurable, these experiments have also allowed us, for the first time, to explore and demonstrate the shallow sample depths from which the majority of ECP contrast originates (< 150 nm in sapphire at an accelerating voltage of 35 kV), even when the beam penetration is considerable by comparison (～ 5 μm). Furthermore, the way in which this sampled depth varies with SEM accelerating voltage is both demonstrated and shown to be a powerful diagnostic technique for studying the distribution of near-surface structural damage.     

Fractographic analysis of fatigue damage in 7000aluminium alloys

In this paper, an attempt is made to correlate the fatigue damage in 7000 aluminium alloys with different impurity contents to the microstructural features and to explain their interdependence through fractographic observations. The Paris constants of these alloys in the form of hot‐forged plates subjected to the overaged T73 temper are evaluated and differences in the fatigue crack growth rate described by striation spacing measurements. Scanning electron microscopy analysis of fatigue fracture surfaces revealed that the type and morphological parameters of coarse intermetallic particles play a critical role in fatigue crack growth behaviour. The elemental distribution determined by means of energy‐dispersive spectroscopy analysis showed that the fractured particles accelerating the crack advances are larger particles of Fe‐rich phases. The fatigue crack growth rate increases considerably with increasing amounts of these particles. The smaller η, S and Mg₂Si particles contribute beneficially to fatigue life.     

Quantitative X-ray imaging of labelled molecules in tissues and cells

Using cisplatin as a model system, we have been able to demonstrate the feasibility of studying the cellular and subcellular distribution of a labelled molecule containing a single atom of platinum per molecule in bone marrow. An X-ray imaging system consisting of a microcomputer, a 4pi system and a software package was interfaced with an electron microscope enabling the computer to control the beam movements as well as receive signals from the STEM and EDS X-ray detectors. X-ray imaging is useful for both tissue and samples in which the population of cells is not homogeneous. Imaging permits elemental distributions to be measured throughout the sample and not in just randomly selected areas as previously done in X-ray microanalysis. Images are created for not only the element labelling the molecule of interest but also other specified elements present.
Three types of maps for imaging labelled molecules are compared and discussed. When the original (collected) data are mapped, the elements of interest are obscured by the continuum. The maps calculated using an internal standard give a concentration distribution on the basis of volume (mmol L⁻¹ of packed cells). The maps calculated using the continuum normalization method according to Hall produces concentration distribution on the basis of mass (mmol kg⁻¹ dry weight). By recalculating using the 'Peak' or 'Hall' method the continuum problem is removed yielding quantitative images of the intracellular distribution of labelled molecules present in low concentrations.     

Determination of UTW kXSi factors for seven elements from oxygen to iron

This study reports k factors for an ultra-thin window (UTW) Si(Li) energy dispersive (ED) X-ray detector which extends the limit for X-ray microanalysis in the analytical electron microscope (AEM) to elements below Na in the periodic table. The oxygen k factors reported for the UTW ED detector suggest that quantitative thin specimen X-ray analysis can be extended to oxygen.