Gallium: a new supporting medium for specimen preparation

Gallium offers a useful alternative to conventional plastic and paraffin embedding media. Samples can be inserted into or removed from gallium cleanly and intact in minutes. Gallium can be polished, has good sectioning properties, is electrically conductive, tolerates high beam currents, is usually not a detectable sample component, and probably would not be a solvent for sample constituents. Samples can be immobilized in gallium within a few degrees of room temperature. Gallium has low toxicity, can be obtained in ultrapure form, is not expensive, and is reusable. Gallium has been used to prepare thin and thick sections and to expose bulk sample cross sections for STEM, SEM, microprobe and light microscope analysis. Gallium does not infiltrate; consequently it is not likely to be useful for soft biological samples.     

Cryo-SEM specimen preparation under controlled temperature and concentration conditions

Cryogenic temperature scanning electron microscopy (cryo-SEM) is an excellent technique for imaging liquid and semi-liquid materials of high vapour pressure, which are highly viscous or contain large (>0.5 μm) aggregates, in which nanometric details are to be studied. However, so far there have been no adequate tools for controlled cryo-specimen preparation. The specimen preparation stage is critical, because most of those samples are very sensitive to concentration and temperature changes, leading to nanostructural artefacts in the specimens. We designed and built a system for easy and reliable cryo-SEM specimen preparation under controlled conditions of fixed temperature and humidity. We describe this new methodology, and demonstrate its applicability, by showing imaging data of three liquid material systems. We have studied carbon nanotubes (CNTs) dispersions in superacid. We also characterized a number of systems made of water/isooctane/nonionic and cationic surfactant that showed different microemulsion phases as function of the system composition and temperature. In all of the examples given, we demonstrate artefact- and contamination-free specimens, which have preserved their native nanostructure. Our new system paves the way for a new methodology for the newly emerging field of cryo-SEM.     

Observation particle morphology of colloidal system by conventional SEM with an improved specimen preparation technique

On the basis of our previous report that polymer emulsion with different viscosity can be investigated by conventional scanning electron microscopy (SEM), we have developed an improved specimen preparation technique for obtaining particle morphology and size of colloidal silver, collagen, glutin, and polymer microspheres. In this study, we expect to provide a means for charactering the three-dimensional surface microstructure of colloidal particles. Dilution of the samples with appropriate volatile solvent like ethanol is effective for SEM specimen preparation. At a proper ratio between sample and ethanol, the colloidal particles are dispersed uniformly in ethanol and then deposited evenly on the substrate. Different drying methods are studied to search a proper drying condition, in which the small molecule solvent is removed without destroying the natural particle morphology. And the effects of ethanol in the specimen preparation process are described by analyzing the physicochemical properties of ethanol. The specimen preparation technique is simple and can be achieved in common laboratory for charactering the particle morphology of colloidal system.     

Electron-beam-induced reduction of Fe in iron phosphate dihydrate,ferrihydrite, haemosiderin and ferritin as revealed by electron energy-loss spectroscopy

The effect of high-energy electron irradiation on ferritin/haemosiderin cores (in an iron-overloaded human liver biopsy), its mineral analogue; six-line ferrihydrite (6LFh), and iron phosphate dihydrate (which has similar octahedral ferric iron to oxygen coordination to that in ferrihydrite and ferritin/haemosiderin cores) has been investigated using electron energy-loss spectroscopy (EELS). Fe L_2,3-ionisation edges were recorded on two types of electron microscope: a 200 keV transmission electron microscope (TEM) and a 100 keV scanning transmission electron microscope (STEM), in order to investigate the damage mechanisms in operation and to establish a methodology for minimum specimen alteration during analytical electron microscopic characterisation. A specimen damage mechanism dominated by radiolysis that results in the preferential loss of iron co-ordinating ligands (O, OH and H₂O) is discussed. The net result of irradiation is structural re-organisation and reduction of iron within the iron hydroxides. At sufficiently low electron fluence and particularly in the lower incident energy, finer probe diameter STEM, the alteration is shown to be minimal. All the materials examined exhibit damage which as a function of cumulative fluence is best fitted by an inverse power-law, implying that several chemical and structural changes occur in response to the electron beam and we suggest that these are governed by secondary processes arising from the primary ionisation event. This work affirms that electron fluence and current density should be considered when measuring mixed valence ratios with EELS.     

On the measurement of thickness in nanoporous materials by EELS

This work discusses thickness measurements in nanoporous MgO using the log-ratio method in electron energy-loss spectroscopy (EELS). In heterogeneous nanoporous systems, the method can induce large errors if the strength of excitations at interfaces between pores and the matrix is large. In homogeneous nanoporous systems, on the other hand, the log-ratio method is still valid, but the inelastic scattering mean-free-path is no longer equal to that in the same bulk system.     

Interpretation of O K-edge EELS in zircon using a structural variation approach

This work describes an approach to interpret the near-edge fine structure of electron energy-loss spectroscopy (EELS) of O K-edge in zircon using a structural variation method. The positions and intensities of several peaks in the O K-edge EELS spectrum are assigned to specific structural parameters. It suggests that the near-edge structures in EELS can be used to measure atomic structure changes.     

Spectroscopic imaging of electron energy loss spectra using ab initio data and function field visualization

We have devised a technique for spectral imaging using accurate ab initio electron energy loss near edge structure (ELNES) data and function field visualization. The technique is initially applied to a planar defect model in Si with different ring structures and no broken bonds where experimental probes are severely limited. The same model with B doping is also considered. It is shown that specific deviations in different energy ranges of the ELNES spectra are correlated with different structural components of the models.     

A simplified preparation procedure of plant material for elemental analysis by ESI and EELS techniques

Living plant cells were embedded in the water-soluble resin Nanoplast FB 101 and cut into ultrathin sections. Non-vacuolated cells of algae and fungi showed a satisfactory preservation of fine structure. First results of elemental mapping by the ESI technique indicate that phosphorus and sulphur are neither extracted nor dislocated within the cells. The authors are members of the ‘Study Group for Electron Microscopy’ at the Hannover School of Veterinary Medicine.     

Application of polystyrene disk substrates in cellular cultivation methods: generalized specimen preparation protocol for scanning electron microscopy

Sample preparation for scanning electron microscopy (SEM) may vary by cellular type, composition and method of cultivation. It has been proposed here that a generalized method of sample preparation may be applied for the visualization of bacteria, fungi, and human cellular tissue without modification of protocol between cell types. The following protocol was developed to incorporate polystyrene disk substrates in the simplification of sample preparation for SEM and reduce the possibility of processing artefacts. The proposed method of preparation may be applied to samples grown in either liquid or solid cultural medium regardless of cell type. With the proposed protocol, centrifugation, isolation and critical point drying are not required, therefore increasing specimen integrity. The incorporation of polystyrene disks showed positive cellular adhesion and applications in SEM for bacterial, fungal and human neuronal tissue. In addition, the simplicity of the proposed protocol is highly adaptable and may be further incorporated to visually analyse the effects of antifungals, antibiotics and disease pathogenesis through pathogen–host interactions. The proposed method of specimen preparation was incorporated in either liquid or solid state growth mediums during the cultivation of the selected cellular samples and revealed great promise in the preservation and visualization under the scanning electron microscope.     

Electron energy-loss near-edge structure of internal interfaces by spatial difference spectroscopy

Internal interfaces determine the macroscopic mechanical and electronic properties of materials. Their investigation is therefore essential for an understanding of the relationship between microstructure and the materials properties. Besides the atomic structure, the bonding and electronic structure on an atomic scale are of interest. This information is contained in the interface-sensitive component of the electron energy-loss near-edge structure (ELNES). It can be extracted by the spatial difference method. Such spectra have already been interpreted with respect to the bonding and the local atomic environment of the atoms at the interface. Here we assess critically the application of the spatial difference for the investigation of internal interfaces. The method is subjective because a suitable scaling factor has to be chosen. As a guideline we propose to minimize the number of turning points in the difference spectrum. With this hypothesis we successfully determine the ELNES arising from atoms at two internal interfaces between a metal and a ceramic. This reveals qualitatively which type of bonding is present at the interface and quantitatively how many atoms are involved in it.     

Local analysis of the edge dislocation core in BaTiO3 thin film by STEM-EELS

The a <100> edge dislocation core formed in an epitaxial BaTiO₃ (BTO) thin film grown on a substrate was investigated by scanning transmission electron microscopy combined with electron energy-loss spectroscopy. Elemental analysis using core-loss spectrum indicates that the atomic ratios of O/Ti and Ba/Ti are decreased at the dislocation core. The near-edge fine structure of the oxygen K-edge recorded from the dislocation core differs slightly from that of relaxed BTO region, which suggests that Ba-O bonding is decreased at the dislocation core. The structure of the dislocation core is discussed using a high-angle annular dark-field image and the electron energy-loss spectroscopy results.     

Use of permanent marker to deposit a protection layer against FIB damage in TEM specimen preparation

Permanent marker deposition (PMD), which creates permanent writing on an object with a permanent marker, was investigated as a method to deposit a protection layer against focused ion beam damage. PMD is a simple, fast and cheap process. Further, PMD is excellent in filling in narrow and deep trenches, enabling damage‐free observation of high aspect ratio structures with atomic resolution in transmission electron microscopy (TEM). The microstructure, composition, gap filling ability and planarization of the PMD layer were studied using dual beam focused ion beam, transmission electron microscopy, energy dispersive X‐ray spectroscopy and electron energy loss spectroscopy. It was found that a PMD layer is basically an amorphous carbon structure, and that such a layer should be at least 65 nm thick to protect a surface against 30 keV focused ion beam damage. We suggest that such a PMD layer can be an excellent protection layer to maintain a pristine sample structure against focused ion beam damage during transmission electron microscopy specimen preparation.     

Study of changes in L32 EELS ionization edges upon formation of Ni-based intermetallic compounds

EELS L₃₂ ionization edges in several Ni‐based intermetallic compounds have been studied and interpreted in terms of the distribution of electrons in the valence d‐bands. It is demonstrated that the integral EELS cross‐sections change only slightly upon the formation of intermetallic compounds and therefore the charge transfer between atoms is negligible. On the other hand, the changes in the fine energy‐loss near‐edge structure (ELNES) of the Ni L₃ edge can be readily detected indicating an important redistribution of d‐electrons at the Ni site with alloying. These features are well reproduced by ab‐initio calculations with a FLAPW method in its WIEN97 implementation. In contrast to the drastic effect of chemical environment, structural transformations in the investigated intermetallics result in smaller ELNES changes, which can be detected by only exceptional instruments with a higher energy resolution.     

Revealing local variations in nanoparticle size distributions in supported catalysts: a generic TEM specimen preparation method

The specimen preparation method is crucial for how much information can be gained from transmission electron microscopy (TEM) studies of supported nanoparticle catalysts. The aim of this work is to develop a method that allows for observation of size and location of nanoparticles deposited on a porous oxide support material. A bimetallic Pt‐Pd/Al₂O₃ catalyst in powder form was embedded in acrylic resin and lift‐out specimens were extracted using combined focused ion beam/scanning electron microscopy (FIB/SEM). These specimens allow for a cross‐section view across individual oxide support particles, including the unaltered near surface region of these particles. A site‐dependent size distribution of Pt‐Pd nanoparticles was revealed along the radial direction of the support particles by scanning transmission electron microscopy (STEM) imaging. The developed specimen preparation method enables obtaining information about the spatial distribution of nanoparticles in complex support structures which commonly is a challenge in heterogeneous catalysis.     

Unstained and in vivo fluorescently stained bacterial nucleoids and plasmolysis observed by a new specimen preparation method for high-power light microscopy of metabolically active cells

Microscope slides were coated with a layer of gelatin, the thickness of the gelatin increasing linearly along the long axis. The bacterial suspension is applied to the dried gelatin and covered by a coverslip. The medium is absorbed by the gelatin and thus the cells applied against the coverslip. By this method, cultures of concentrations below 10⁸ cells/ml provide statistically relevant numbers for observation without prior concentration steps. It is easier to apply than the existing methods for the observation of bacterial nucleoids by phase contrast imaging. Because the cells are maintained in growing conditions the method is useful for the vital fluorescence DAPI-staining of various bacterial species and for observations of plasmolysis and its reversal at different physiological conditions and extracellular osmolalities. The previously generally assumed view that the plasmolytic changes of the cell morphology are immediate upon the hyperosmotic shock and are rapidly repaired when the cell is able to metabolize actively was confirmed; this is in contrast to some recent claims.     

An optical configuration for fastidious STEM detector calibration and the effect of the objective‐lens pre‐field

In the scanning transmission electron microscope, an accurate knowledge of detector collection angles is paramount in order to quantify signals on an absolute scale. Here we present an optical configuration designed for the accurate measurement of collection angles for both image‐detectors and energy‐loss spectrometers. By deflecting a parallel electron beam, carefully calibrated using a diffraction pattern from a known material, we can directly observe the projection‐distortion in the post‐specimen lenses of probe‐corrected instruments, the 3‐fold caustic when an image‐corrector is fitted, and any misalignment of imaging detectors or spectrometer apertures. We also discuss for the first time, the effect that higher‐order aberrations in the objective‐lens pre‐field has on such an angle‐based detector mapping procedure.     

Physical and chemical changes in polystyrene during electron irradiation using EELS in the TEM: contribution of the dielectric function

We have performed electron energy-loss spectroscopy analysis of polystyrene in the analytical transmission electron microscope in order to evaluate the possibility of obtaining both chemical and dielectric information on the polymer at the submicrometre scale. Irradiation has also been studied, particularly the influence of the specimen temperature on the kinetics of degradation.
The main results show that polystyrene is resistant to electron beam with a critical dose of about 10⁴ C m⁻² at 127 K. Spectra could be acquired with doses less than this critical dose. We were thus able to propose an identification of the different chemical bonds of carbon (including the C–H bond), in agreement with previous X-ray absorption near-edge structure experiments. The chemical changes in polystyrene due to severe irradiation damage are also visible in the carbon K-edge near-edge structure.
At the same time, we calculated the dielectric function from the low-loss part of the spectra. Interestingly, this part of the spectrum is the most sensitive to irradiation, since great changes can be seen during exposure.
Lastly, we propose a degradation process, in agreement with all these results.     

Comparison of EFTEM and STEM EELS plasmon imaging of gold nanoparticles in a monochromated TEM

We present and compare two different imaging techniques for plasmonic excitations in metallic nanoparticles based on high energy-resolution electron energy-loss spectroscopy in a monochromated transmission electron microscope. We demonstrate that a recently developed monochromated energy-filtering (EFTEM) approach can be used in addition to the well established scanning technique to directly obtain plasmon images in the energy range below 1 eV. The EFTEM technique is described in detail, and a double experiment performed on the same, triangular gold nanoparticle compares equivalent data acquired by both techniques, respectively.     

Optimized acquisition parameters and statistical detection limit in quantitative EELS

Optimizing the acquisition parameters for EELS recording has to be accomplished simultaneously from the physical and the statistical points of view; the statistical aspect of the question is covered here. Approximate probability density functions of the variables of interest are derived, which provide a global measure of signal-to-noise ratio taking into account every step of the EELS edge area estimation process. Qualitative and quantitative advice is given regarding the critical choice of the estimation and integration energy regions. The notion of visual contrast is presented; it permits the introduction of the concept of statistical detection limit. It is found that for typical experimental conditions, when other factors are equal, the required analysis time for the sample varies approximately as the inverse square of the concentration.     

A simple backscattered electrons and specimen current detection device for the scanning electron microscope

A simple, low-investment device has been developed that allows the collection of backscattered electrons (BSEs) and specimen current (SC) signals for imaging purposes and current measurement. Originally, this system was designed for detection, measurement, and display of specimen current, with a video signal output whose level was modulated by this current. Eventually, a BSE detector was developed, using a graphite disk (about 8 cm in diameter) to collect the BSEs. The disk was mounted on a Philips SEM 5O5, attached and concentrically to the final lens aperture. This configuration gives a large solid angle of collection. The collected charge is further processed by the same electronics used in the aforementioned SC detection system. Electron channeling, topographic contrast with BSE, and material contrast with BSE and SC images can be obtained with reasonably good edge definition.