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 sample preparation method for biological soft X-ray microscopy: nitrogen-based contrast and radiation tolerance properties of glycol methacrylate-embedded and sectioned tissue

We describe the preparation of a biological tissue for imaging in a transmission soft X-ray microscope. Sections of exocrine pancreas embedded in glycol methacrylate polymer, an embedding medium widely used in visible light and electron microscopy, were examined. Contrast was based primarily on the nitrogen content of the tissue, and dual-wavelength imaging at the nitrogen K-shell absorption edge was used to map the distribution and provide quantitative densitometry of both the protein and embedding matrix components of the sample. The measurements were calibrated by obtaining the absorption spectrum of protein near the nitrogen edge. The contrast was consistent and reproducible, making possible the first large-scale X-ray microscopic study on sections of plastic-embedded soft tissue. At radiation doses of up to 10⁸ Gray, much more than required for routine imaging, no distortion and little mass loss were observed. This sample preparation method should permit routine imaging of tissues in X-ray microscopes, previously a difficult task, as well as multimodal imaging (using visible light, X-ray, electron, and scanned probe microscopies) on the same sample.     

Formation process of β-FeSi2 from amorphous Fe-Si synthesized by ion implantation: Fe concentration dependence

Formation processes of β-FeSi₂ from amorphous Fe-Si layers have been investigated using transmission electron microscopy (TEM). Si(111) substrates were irradiated with 120 keV Fe ions at −150°C to fluences of 1.0 × 10¹⁷ and 4.0 × 10¹⁷ cm^–2. An amorphous Fe-Si layer embedded in an amorphous Si was formed in the low-fluence sample, whereas an amorphous Fe-Si surface layer on an amorphous Si was obtained in the high-fluence one. The amorphous Fe-Si layers were crystallized to β-FeSi₂ after thermal annealing at 800°C for 2 h. Cross-sectional and plan-view TEM observations revealed that, prior to the formation of β-FeSi₂, the amorphous Fe-Si layers crystallized to α-FeSi₂ in the low-fluence sample and to ɛ-FeSi in the high-fluence one. The absence of metastable γ-FeSi₂ which is considered as a precursor of epitaxially grown β-FeSi₂ on Si was attributed to the instability of γ-phase in an amorphous matrix.     

High-pass energy-filtered photoemission electron microscopy imaging of dopants in silicon

Differently doped areas in silicon can show strong electron-optical contrast in dependence on the dopant concentration and surface conditions. Photoemission electron microscopy is a powerful surface-sensitive technique suitable for fast imaging of doping-induced contrast in semiconductors. We report on the observation of Si (100) samples with n- and p-type doped patterns (with the dopant concentration varied from 10¹⁶ to 10¹⁹ cm⁻³) on a p- and n-type substrate (doped to 10¹⁵ cm⁻³), respectively. A high-pass energy filter of the entire image enabled us to obtain spectroscopic information, i.e. quantified photo threshold and related photoyield differences depending on the doping level. Measurements have confirmed the possibility of resolving areas at a high contrast even with the lowest dopant concentration when employing the energy filter. The influence of electron absorption phenomena on contrast formation is discussed.     

Chemical mapping of a block copolymer electrolyte by low-loss EFTEM spectrum-imaging and principal component analysis

Energy-filtered transmission electron microscopy spectrum-imaging (EFTEM SI) in the low electron energy-loss range is a valuable technique for probing the chemical structure of a material with nanoscale spatial resolution using a reduced electron dose. By analyzing EFTEM SI datasets using principal component analysis (PCA), the constituent chemical phases of the material can be identified in an efficient manner without prior knowledge of the specimen. We implement low-loss EFTEM SI together with PCA to investigate thin films of the block copolymer electrolyte poly(styrene-block-ethylene oxide) (PS-b-PEO) blended with a sodium salt. PCA identifies three main phases, the first and second phases corresponding to the two blocks of the copolymer and a third phase corresponding to the salt. The low-loss spectra for these phases are extracted from a noise-reduced EFTEM SI dataset and used to generate a chemical map of the material by multiple linear least square fitting. We validate the results of the low-loss EFTEM SI/PCA technique by applying the method to a control PS-b-PEO sample that does not contain the sodium salt, and by conducting spatially resolved X-ray energy-dispersive spectrometry on the salt-containing PS-b-PEO thin film.     

Development of an optimal protocol for the ultrastructural examination of skin by transmission electron microscopy

The intercellular lipid bilayers of the stratum corneum provide the permeability barrier of the skin. To perform an electron microscopical examination of the ultrastructure of these bilayers, ruthenium tetroxide fixation is required. In this study an optimal fixation protocol was developed and selected upon comparing seven different fixation procedures, using glutaraldehyde (GA) and the postfixatives ruthenium red, osmium tetroxide (OsO₄) and ruthenium tetroxide (RuO₄) in combination with potassium ferrocyanide (K₄Fe(CN)₆) and potassium ferricyanide (K₃Fe(CN)₆). Instead of fixing skin with either OsO₄ or RuO₄, these two fixatives were combined in one protocol. In addition, the use of RuRed was introduced and the influence of both K₄Fe(CN)₆ and K₃Fe(CN)₆ in combination with RuO₄ were examined. Furthermore, we compared two dehydration solvents, methanol and acetone.
The most satisfying results were obtained when the skin was prefixed in GA and postfixed in OsO₄ and RuO₄ with K₃Fe(CN)₆, i.e. with Fe in its highest oxidation state (Fe³⁺). No differences were observed between dehydration in methanol and acetone.     

热电离质谱法测定铀同位素过程中铼灯丝氧化对目标铀离子形成的影响

采用热电离质谱法(thermal ionization mass spectrometry, TIMS)测定铀同位素过程中,质量分馏效应会引起同位素比值测量值偏离真值,一般采用外标校正法对质量分馏效应进行校正,这要求测量过程中标准物质和样品产生一致的质量分馏行为。因此,除考虑点样的一致性外,测量过程中产生单一的目标离子也非常重要。本研究采用热电离质谱技术考察了铼灯丝氧化对铀同位素测定的影响。将1 μg铀以硝酸盐溶液的形式点于铼样品带上,通过监测铀及其氧化物的离子流强度,发现铀主要存在U⁺和UOx⁺(x=1或2)的电离形态,并且灯丝表面氧化程度越高,UOx⁺的产率越高（UOx⁺/U⁺可达到1）。实验结果表明,灯丝去气过程过早使灯丝暴露于大气或点样过程中,以及使用较高的样品蒸干电流都会加剧灯丝的氧化。控制灯丝表面氧化或点样过程中加入石墨,可有效降低UOx⁺的产率,提高目标离子的电离效率。全蒸发测量结果表明,UOx⁺离子流强度大小对测量结果无明显影响,但通过降低UOx⁺的离子流强度,可提高测量结果的重现性,进而提高测量过程中分馏行为的一致性。该研究可为提高铀同位素测定过程中样品的利用率提供理论依据。     

Spatially resolved cathodoluminescence of luminescent materials using an EDX detector

Panchromatic cathodoluminescence (CL) maps were collected in a scanning electron microscope equipped with an EDX (energy dispersive x-ray analysis) detector. These CL maps can readily be correlated with elemental maps obtained by EDX. Although EDX detectors are designed to be insensitive to light and therefore not optimized for high sensitivity CL measurements, high-resolution images can be obtained from luminescent materials without the need for additional hard- or software. The method was tested on highly luminescent BaAl₂S₄ : Eu²⁺ thin films that have a potential use in flat panel displays. The spectral response and linearity of the overall system was determined by means of monochromatic light sources, illuminating the sample through an optical fibre. We studied the response of the EDX detector to the intensity of the incoming light as well as the influence of the detector settings. The observations were explained by numerical simulations.     

Disassembly of DNA–ligand on mica surface: atomic force microscopy studies

Disassembly of DNA–ligands, including DNA–methylene blue (MB) complex and DNA–Co(phen)₃³⁺ complex on mica surface, were investigated by atomic force microscopy (AFM). The disassembly of these complexes occurred after they were immersed in ultra-pure water. AFM results showed that the disassembly depended strongly on bridge ions that were used to immobilize the complex onto mica surface, DNA species and ligands. When Mg²⁺ was used as the bridge ion, the DNA–MB complex was completely disassembled because of the weak interactions between Mg²⁺ and DNA's bases or mica surface. Although if Co²⁺ was used as the bridge ion, the disassembly of the DNA–MB complex mainly depended on the species and shape of DNA. For plasmid DNA pBR 322, plasmid DNA pUC 18 and the linear DNA pBR 322/PstI, the degree of disassembly was gradually increased. Whereas if Co(phen)₃³⁺ was chosen as the ligand, the disassembly of the DNA–Co(phen)₃³⁺ complex was almost blocked because Co(phen)₃³⁺ could hardly diffuse into the ultra-pure water. This obtained information may be useful for practical application of the AFM imaging of biological molecules, especially in liquid.     

Structure of electrodeposited graded composite coatings of Ni–Al–Al2O3

The structure of electrodeposited composite coatings of Ni–Al–Al₂O₃, with Ni as the matrix and Al and Al₂O₃ as second-phase particles, was investigated using light microscopy and scanning electron microscopy. Ni coatings with no particles, which were used as reference samples, had progressively coarser structures with increasing current density. Co-deposition with Al resulted in refinement of the Ni matrix structure at high (>10 A dm⁻²) current densities. For single-particle baths, the co-deposition of Al₂O₃ was more strongly affected by current density and bath particle content than was the co-deposition of Al. However, for baths containing both Al and Al₂O₃ the amount of incorporated Al₂O₃ no longer depended on current density. With the choice of appropriate conditions, coatings of Ni with up to 39 vol.% Al₂O₃ were made. Similar experiments with Al yielded a maximum of 17.5 vol.% only. Uniform and graded mixed-particle coatings were also produced. When coatings containing Al were annealed, the reaction of the two elements resulted in the formation of either single-phase γ or two-phase γ–γ' alloys, in agreement with the equilibrium phase diagram.     

HREM and STEM of intergranular films at zinc oxide varistor grain boundaries

Grain boundaries in model ZnO–Bi₂O₃ and ZnO–Bi₂O₃–CoO varistors and a commercial multicomponent varistor have been characterized by high-resolution electron microscopy (HREM) and scanning transmission electron microscopy (STEM), in order to determine the relationship between Bi grain boundary segregation and formation of thin intergranular films. By controlling Bi₂O₃ content, applied pressure and temperature, the grain boundary Bi excess has been systematically varied from nearly zero to Γ_Bi = 1 × 10¹⁵ cm⁻² (≈ 1 monolayer), as measured by HB 603 STEM using an area-scan method. HREM shows that intergranular amorphous films are clearly distinguishable in samples with Γ_Bi > 8 × 10¹⁴ cm⁻². These films range in thickness, depending on the Bi excess, from 0.6 to 1.5 nm. Similar films of ≈ 1 nm thickness are widely observed in the commercial varistor. The composition of the films is a ZnO–Bi₂O₃ solid solution, which is in all cases more enriched in ZnO than the bulk eutectic liquid. The Bi-doped grain boundaries in ZnO varistors therefore contain an intergranular amorphous film which has not only an equilibrium thickness, but also a distinct equilibrium composition.     

Studies of cell division (mitosis and cytokinesis) by dynamic secondary ion mass spectrometry ion microscopy: LLC-PK1 epithelial cells as a model for subcellular isotopic imaging

The feasibility of the renal epithelial LLC-PK₁ cell line as a model for cell division studies with secondary ion mass spectrometry (SIMS) was tested. In this cell line, cells undergoing all stages of mitosis and cytokinesis remained firmly attached to the substrate and could be cryogenically prepared. Fractured freeze-dried mitotic cells showed well-preserved organelles as revealed by fluorescence imaging of rhodamine-123 and C₆-NBD-ceramide by confocal laser scanning microscopy. Secondary electron microscopy analysis of fractured freeze-dried dividing cells revealed minimal surface topography that does not interfere in isotopic imaging of both positive (³⁹K, ²³Na, ²⁴Mg, ⁴⁰Ca, etc.) and negative (³¹P, ³⁵Cl, etc.) secondaries with a CAMECA IMS-3f ion microscope. Mitotic cells revealed well-preserved intracellular ionic composition of even the most diffusible ions (total concentrations of ³⁹K⁺ and ²³Na⁺) as revealed by K : Na ratios of approximately 10. Structurally damaged mitotic cells could be identified by their reduced K : Na ratios and an excessive loading of calcium. Quantitative three-dimensional SIMS analysis was required for studying subcellular calcium distribution in dividing cells. The LLC-PK₁ model also allowed SIMS studies of M-phase arrested cells with mitosis-arresting drugs (taxol, monastrol and nocodazole). This study opens new avenues of cell division research related to ion fluxes and chemical composition with SIMS.     

Focused ion beam sample preparation of continuous fibre-reinforced ceramic composite specimens for transmission electron microscopy

The microanalysis of interfaces in fibre-reinforced composite materials is dependent on the successful preparation of specimens suitable for transmission electron microscope (TEM) inspection. Ideal samples should possess large amounts of structurally intact and uniform thin area in the fibre/matrix interface regions of the samples. Because fibre/matrix interfaces in this class of materials are often designed to fail under mechanical stress, conventionally prepared samples are prone to interfacial failure and differential thinning, both of which preclude detailed TEM microanalysis. These effects were seen in a conventionally dimpled and ion-beam-thinned specimen prepared from a continuous fibre reinforced ceramic composite composed of CaWO₄-coated Nextel 610^TM fibres in an alumina matrix. The dimpled specimen showed large amounts of interfacial failure, with only thick regions of the specimen left intact. To overcome these limitations, a focused ion beam (FIB) technique was applied to this same material. The superiority of the FIB-produced sample is evident in both the morphology and scanning transmission electron microscopy analyses of the sample.     

Membrane ion channels as physiological targets for local Ca2+ signalling

Ionized calcium plays a central role as a second messenger in a number of physiologically important processes determining smooth muscle function. To regulate a wide range of cellular activities the mechanisms of subcellular calcium signalling should be very diverse. Recent progress in development of visible light-excitable fluorescent dyes with high affinity for Ca²⁺ (such as oregon green 488 BAPTA indicators, fluo-3 and fura red) and confocal laser scanning microscopy provides an opportunity for direct visualization of subcellular Ca²⁺ signalling and reveals that many cell function are regulated by the microenvironment within small regions of the cytoplasm ('local control' concept). Here confocal imaging is used to measure and locate changes in [Ca²⁺]_i on a subcellular level in response to receptor stimulation in visceral myocytes. We show that stimulation of muscarinic receptors in ileal myocytes with carbachol leading to activation of inositol 1,4,5-trisphosphate receptors (IP₃Rs) accelerates the frequency of spontaneous calcium sparks (discharged via ryanodine receptors, RyRs) and gives rise to periodic propagating Ca²⁺ waves oscillating with a frequency similar to that of carbachol-activated cationic current oscillations. Furthermore, by combining the whole-cell patch clamp technique with simultaneous confocal imaging of [Ca²⁺]_i in voltage-clamped vascular myocytes we demonstrate that calcium sparks may lead to the opening of either Ca²⁺-activated Cl⁻ channels or Ca²⁺-activated K⁺ channels, and the discharge of a spontaneous transient inward current (STIC) or a spontaneous transient outward current (STOC), respectively.     

Soft-X-ray damage to biological samples

X-ray damage to biological samples was investigated in the wavelength region of 2.7–5 nm, which overlaps the so-called 'water window', the wavelength range of 2.4–4.3 nm usually used in X-ray microscopy. Yeast cells and myofibrils were chosen as representatives of whole cell samples and motile protein systems, respectively. The samples were exposed to X-rays using an apparatus composed mainly of a laser-plasma X-ray source, a Wolter mirror condenser, and a sample cell. The yeast cells lost their dye exclusion ability when the X-ray flux was higher than 1 × 10⁶ photons μm⁻², while the myofibrils lost contractility when the X-ray flux was higher than 4 × 10⁵ photons μm⁻². These X-ray fluxes are lower than the flux required for the X-ray microscope observation of biological samples at a resolution higher than that of light microscopes.     

Active coatings for SiC particles to reduce the degradation by liquid aluminium during processing of aluminium matrix composites: study of interfacial reactions

The application of a surface coating on SiC particles is studied as an alternative means of solving problems of reactivity between SiC reinforcements and molten aluminium and problems of low wetting which limit the application of casting routes for fabrication of Al–SiC_p composites. The selected active barrier was a ceramic composed of SiO₂, which was generated by controlled oxidation of the SiC particles. The coating behaves as an active barrier, preventing a direct reaction between molten aluminium and SiC to form Al₄C₃ as the main degradation product. At the same time, the SiO₂ provokes other interfacial reactions, which are responsible for an improvement in wetting behaviour.
Composites were prepared by mixing and compacting SiC particles with Al powders followed by melting in a vacuum furnace, and varying the residence time. Transmission electron microscopy (TEM), high resolution electron microscopy (HREM) and field emission TEM were employed as the main characterization techniques to study the interfacial reactions occurring between the barrier and the molten aluminium. These studies showed that the SiO₂ coating behaves as an active barrier which reacts with the molten Al to form a glassy phase Al–Si–O. This compound underwent partial crystallization during the composite manufacture to form mullite. The formation of an outer crystalline layer, composed mainly of Al₂O₃, was also detected. Participation of other secondary interface reactions inside the active barrier was also identified by HREM techniques.     

Quantitative electron spectroscopic imaging studies of microelectronic metallization layers

The combination of focused ion beam (FIB) sample preparation and quantitative electron spectroscopic imaging is an ideal tool for the investigation of layered structures used in microelectronic metallization schemes. In the present work, Si₃N₄/Cu/Si₃N₄/SiO₂/Si and Al/TiN/Ti/SiO₂/Si metallization layers produced by physical vapour deposition are investigated. We apply series of energy filtered images in the low loss region for a mapping of the sample thickness which makes it possible to refine the parameters of the FIB process. We also show how series of energy filtered images in the core loss region can be used to obtain elemental distribution images and chemical bonding information on these samples on a nanometre scale. For materials with a small grain size and/or a strong variation in Bragg orientation, the intensity distribution of the elemental map is strongly influenced by the superimposed Bragg contrast. This detrimental effect can be reduced greatly by using hollow cone illumination, as is demonstrated for polycrystalline Cu. One striking feature observed in Cu layers prepared with FIB is strong, regularly arranged contrast variations caused by subsurface defects in the Cu grains. We suppose that these defects are a consequence of a strong interaction of Ga atoms from the FIB with Cu.     

Accurate structure determinations of very small (4–20 nm) areas, using refinement of dynamic electron diffraction data

It is shown that accurate structure refinements are possible from electron diffraction data obtained using areas 4–20 nm in diameter and 4–20 nm thick. To obtain accurate atomic positions it is essential to take the dynamic diffraction fully into account, for which new software was developed. Examples (La₃Ni₂B₂N₃, Ba₂Ca₃Cu₅O_10+δ and Ce₅Cu₁₉P₁₂) are given of the structure refinements of known and unknown structures with R -values in the range 2–6%. The procedure reported in this paper opens the way to structure determination of particles in many materials of industrial and scientific interest which cannot be solved by conventional structure determination (e.g. because of small size, heavily twinned materials or small fractions in polyphase materials).     

Secondary ion mass spectrometry imaging of the fixation of 15N-labelled NO in pollen grains

We used secondary ion mass spectrometry to image cellular targets of nitrogen oxides (widespread air pollutants) in pollen grains of birch ( Betula verrucosa Ehrh.) and cockfoot ( Dactylis glomerata L.). The pollen samples were exposed to air supplemented with high doses of ¹⁵NO. The pollen grains were then fixed, dehydrated using a newly developed 'vapour phase' preparation method and embedded in LRW resin. Semithin sections were then analysed. Imaging was performed in scanning mode. As usual, the two isotopes ¹⁴N and ¹⁵N were imaged as ¹²C¹⁴N⁻ and ¹²C¹⁵N⁻, respectively. The isotopic percentages of ¹⁵N were quantitatively determined either by image processing or by direct analysis. We show that the preferential areas of NO fixation in the pollen cell are the sporoderm and discrete intracytoplasmic structures that we tentatively describe as globoid-like structures similar to those encountered in seeds.     

Optimization of a FIB/SEM slice-and-view study of the 3D distribution of Ni4Ti3 precipitates in Ni–Ti

The 3D morphology and distribution of lenticular Ni₄Ti₃ precipitates in the austenitic B2 matrix of a binary Ni₅₁Ti₄₉ alloy has been investigated by a slice-and-view procedure in a dual-beam focused ion beam/scanning electron microscope system. Due to the weak contrast of the precipitates, proper imaging conditions need to be selected first to allow for semi-automated image treatment. Knowledgeable imaging is further needed to ensure that all variants of the precipitates are observed with equal probability, regardless of sample orientation. Finally, a volume ratio of 10.2% for the Ni₄Ti₃ precipitates could be calculated, summed over all variants, which yields a net composition of Ni_50.27Ti_49.73 for the matrix, leading to an increase of 125 degrees for the martensitic start temperature. Also, the expected relative orientation of the different variants of the precipitates could be confirmed.