Quantitative analyses of the biomineralization of different hard tissues

The primary crystallites of the different developing hard tissues have an apatite structure. However, they have crystal lattice distortions representing an intermediate state between amorphous and fully crystalline. We have applied energy-filtering transmission electron microscopy in the selected area electron diffraction mode to analyse different stages of crystal formation in dentine, bone, enamel and inorganic apatite mineral. We have obtained quantitative information on the degree of crystal lattice distortion using the paracrystal theory of Hosemann and Bagchi.
We have found that the early formed crystallites of the hard tissues being analysed have a paracrystalline character comparable to biopolymers. However, with maturation, the lattice fluctuations of the crystallites of the hard tissues bone, enamel and dentine decrease to form a typical (para)crystalline character. Also the decrease of the organic proportion in the matrix corresponds to the decrease of the lattice fluctuation of the crystallites in the different hard tissues during maturation.     

Analytical electron microscopy and focused ion beam: complementary tool for the imaging of copper sorption onto iron oxide aggregates

Nanometre‐scale electron spectroscopic imaging has been applied to characterize the operation of a copper filtration plant in environmental science. Copper washed off from roofs and roads is considered to be a major contributor to diffuse copper pollution of urban environments. A special adsorber system has been suggested to control the diffusion of copper fluxes by retaining Cu with a granulated iron hydroxide. The adsorber was tested over an 18‐month period on facade runoff. The concentrations range of Cu in the runoff water was measured between 10 and 1000 p.p.m. and could be reduced by between 96% and 99% in the adsorption ditch. Before the analysis of the adsorber, the suspended material from the inflow was ultracentrifuged onto TEM grids and analysed by energy‐filtered transmission electron microscopy (EFTEM). Copper was found either as small precipitates 5–20 nm in size or adsorbed onto organic and inorganic particles. This Cu represents approximately 30% of the total dissolved Cu, measured by atomic emission spectrometry. To locate where the copper sorption takes place within the adsorber, the granulated iron oxide was analysed by analytical electron microscopy after exposure to the roof run‐off water. A section of the granulated iron hydroxide was prepared by focused ion beam milling. The thickness of the lamina was reduced to 100 nm and analysed by EFTEM. The combination of these two techniques allowed us to observe the diffusion of Cu into the aggregate of Fe. Elemental maps of Fe and Cu revealed that copper was not only present at the surface of the granules but was also sorbed onto the fine particles inside the adsorber.     

Quantitative valence plasmon mapping in the TEM: viewing physical properties at the nanoscale

Using a series of graphitising carbons heat treated at different temperatures, the peak position of the bulk (pi+sigma) plasmon was measured using electron energy loss spectroscopy and observed to shift between 22 and 27eV. Experimental data is presented and discussed showing the effects of the collection conditions and sample orientation upon the observed spectra.We present an empirical technique by which quantitative energy filtered transmission electron microscopy (EFTEM) maps with two energy windows selected in the plasmon region can be readily acquired and processed, the results of which may be interpreted as graphitisation maps and subsequently physical property maps.An experimentally established resolution of approximately 1.6nm makes this technique a very useful tool with which to examine nanoscale properties in microstructural regions of interest in TEM specimens such as fibre/matrix interfaces within carbon-carbon composites, multi-walled carbon nanotubes and graphitic inclusions in carbon steels.Also presented is data demonstrating the unsuitability of pi(*)-related chemical EFTEM maps in both the low-loss region and at the carbon K ionisation edge for mapping bonding in such highly anisotropic media due to the strong orientation dependence of the intensity of the transitions involved. This is followed by suggestions for wider application of the plasmon mapping technique within systems other than those based upon carbon.     

Freeze-substitution of rabbit tibial articular cartilage reveals that radial zone collagen fibres are tubules

Investigations of the micromorphology of rabbit tibial articular cartilage using scanning and transmission electron microscopy revealed that the collagenous elements in the tissue form fluid-containing tubular structures. The commonly described radial or deep zone longitudinal fibres were found to be tubular structures with internal diameters of 1–2 μm. The walls of the tubules were composed of tightly packed fibrils of collagen. The tangential zone, close to the tibial plateau, was composed mainly of a spongy arrangement of collagen fibrils, containing bunches of tangentially lying small (< 1 μm) diameter tubules. The application of conventional chemical fixation techniques resulted in the fine detail of this tissue being obscured. When the tissue was frozen, followed by cryo-scanning electron microscopy or freeze-drying, prior to observation in the scanning electron microscope the tubule structures were not obviously present. It was only by applying freeze-substitution techniques, followed by critical point drying or resin embedding, that the structure was revealed clearly. Segregation of water into ice crystals did occur during the freezing process, but the formation of those crystals played no part in creating the tubular morphology observed. A similar structure was still revealed following pre-treatment with glycerol, methanol or Triton X-100, provided that concentration of these additives was not too high. The walls of the tubules in the radial region were composed of straight, longitudinally arranged as well as helically arranged, 30 nm diameter fibrils. The lumen of the tubules appears to be lined by a circumferentially arranged array of approximately 10 nm diameter fibres, spaced at regular intervals of 50–70 nm.     

Structural characterization of TiN/NbN multilayers: X-ray diffraction, energy-filtered TEM and Fresnel contrast techniques compared

Two TiN/NbN multilayers with wavelength 13.6 and 6.15 nm have been characterized by X-ray diffraction (XRD), Fresnel contrast analysis (FCA) and energy-filtered transmission electron microscopy (EFTEM). Good agreement between the composition profile obtained by FCA and EFTEM is obtained if the lower resolution of the EFTEM images is taken into account. The relative advantages and disadvantages of the techniques are discussed. Used together the two TEM techniques provide a quantitative characterization that is consistent with, and for some parameters provides more precise values than, that from XRD. The analysis shows that the multilayers have narrow interfaces (< 1 nm) and a composition amplitude close to 95% for the longer wavelength.     

Study of structures at the boundary and defects in organic thin films of perchlorocoronene by high-resolution and analytical transmission electron microscopy

Both the periodic and non-periodic structures of perchlorocoronene (C₂₄Cl₁₂) crystals were characterized by high-resolution transmission electron microscopy (HRTEM), electron diffraction (ED), electron energy-loss spectroscopy (EELS), and energy-filtered transmission electron microscopy (EFTEM). The HRTEM images at the boundary of the C₂₄Cl₁₂ crystals exhibit the flexibility of defect structures, where molecules align to compensate for the discontinuity between two different domains. Emphasized by the filtered images, it was found that the non-periodic regions are created everywhere with a small electron beam irradiation (∼10⁶ electrons nm⁻²) and then spread over the entire regions to completely destroy the periodic structures after a higher electron dose (∼2×10⁶ electrons nm⁻²). The effect of the electron beam irradiation was monitored by ED, EELS, and EFTEM, where periodic structures and content elements are well preserved up to 10⁶ electrons nm⁻², but chlorine atoms decreased with a much higher electron dose. This is explained by the breakage of the C–Cl bond to detach chlorine atoms, confirmed by energy-loss near the edge structures (ELNES) of carbon π? peaks and chlorine loss at the edge of the specimen, as well as by theoretical simulation. The detachment of chlorine is localized at the peripheral edge around a hole confirmed by core-loss EFTEM imaging.     

Analysis of the calcium distribution in predentine by EELS and of the early crystal formation in dentine by ESI and ESD

Predentine is a collagen-rich extracellular matrix between the odontoblasts and the dentine with a width of about 15–20 μm. Electron energy-loss spectroscopy of rat incisors shows a significantly higher calcium content in the predentine at the predentine-dentine border than in the middle region of the predentine. At the predentine-dentine border in the dentine, the calcium and the phosphate groups combine to form apatite crystallites. Electron spectroscopic diffraction with zero-loss filtering revealed that the earliest crystallites contain only Debye-Scherrer rings of apatite, which are fewer in number and more diffuse than the diffraction rings from the mature crystallites. We therefore conclude that the early crystallites still contain lattice defects, which are annealed out to some degree with crystal growth. Electron spectroscopic imaging with zero-loss filtering also showed that the earliest crystallites are chains of dots (or small islands); they build up strands composed of islands, which rapidly acquire a needle-like character and coalesce laterally to form ribbon- or plate-like crystallites. The parallel strands sometimes appear to reinforce the macroperiod of the collagen microfibrils (67 nm) by tiny holes without any crystal-substance lined up perpendicular to the parallel strands of the crystallites.     

Electron diffraction from micro- and nanoparticles of hydroxyapatite

Hydroxyapatite (HAP) obtained from aqueous solutions under different conditions has been examined by high-resolution transmission electron microscopy (HRTEM) and electron diffraction, including selected-area electron diffraction (SAED) and microdiffraction. A Philips CM300 field-emission gun electron microscope with a Schottky W/ZrO field-emission tip and a spherical aberration constant of 0.65 mm was used at 300 kV. The HAP crystals had different sizes, ranging from a few nanometres to a few micrometres. Single-crystal diffraction patterns have been obtained from the largest microcrystals using the conventional SAED technique. Assemblies of nanoparticles gave only broad diffuse rings. Nevertheless, microdiffraction with electron microprobes 3.5–10 nm in diameter clearly indicated the crystalline character of the nanoparticles in these assemblies. Experimental HRTEM images, Fourier transforms and calculated images exhibited the fine structure of the HAP crystals.     

Electrically conducting polymers: preparation and investigation of oxidized poly(acetylene) by EFTEM

Energy-filtering transmission electron microscopy (EFTEM) was used to demonstrate that the oxidation of poly(acetylene), necessary to produce a highly conducting phase, proceeds homogeneously on a scale of 100 nm to several micrometres. The distribution of the anions BF₄⁻ and PF₆⁻ serves as indicator for the penetration of poly(acetylene) by the oxidizing agent. The sample preparation necessitates prevention of sample degradation by air and moisture. It was possible to synthesize, orientate, oxidize, embed and section poly(acetylene) samples under inert conditions. Only mounting of the grids into the sample holder of the electron microscope was performed under ambient conditions. EFTEM was possible for phosphorus and boron, but not for fluorine owing to irradiation damage leading to loss of fluorine from the sample. Polyvinylbutyral was chosen as the embedding medium because it allows embedding of the sample prior to oxidation. Embedding of a previously oxidized poly(acetylene) sample leads to migration of the anions from the interior of the sample into the embedding medium.     

Analytical electron microscopy as a tool for accessing colloid formation process in natural waters

Analytical electron microscopy was used to characterize aquatic iron‐rich colloids. We focused our attention on a redox transition medium in the drainage water of a peat soil. In the anoxic peat water, observations by transmission electron microscopy and associated energy dispersive analyses (TEM‐EDS) highlight the presence of spherical entities (～100–600 nm), containing only traces of iron. The increase of dissolved oxygen concentration favours the formation of iron oxy(hydr)oxides. In the oxygenated drain, particles with the same morphology and size range are present. Statistical TEM‐EDS analyses show that they represent the only colloidal form of iron in the drain samples. Nevertheless, although Fe–K peaks appear clearly on EDS spectra, the proportion of iron in these colloids reaches at most 4% at. (whereas C + O > 90% at.). Structural information completes this study. Both electron spectroscopic imaging (ESI) and electron energy‐loss spectroscopy (EELS) reveal the disparity between element distributions within the drain entities. Iron and calcium are preferably distributed on the outer sphere of the particle, whereas carbon and oxygen follow the theoretical variation of the signal intensity within a plain sphere. The implication of organic matter as nucleation site for iron precipitation is spectacularly demonstrated by the presence of nanometre‐sized iron‐rich phases highlighted by EELS line scans.     

Structure of nanometre-sized Xe particles embedded in Al crystals

The structure and lattice parameters of Xe particles about 1 nm to about 6 nm in size embedded in Al were investigated with off‐Bragg condition high‐resolution transmission electron microscopy. An Xe particle about 1 nm in size had different structural properties from those 2–6 nm in sizes. Some 1‐nm Xe particles had an face‐centred cubic (f.c.c.) structure with the same orientation as the Al matrix, whereas others of the same size had a non‐f.c.c. structure. The lattice parameters of a 1‐nm f.c.c. Xe particle were about 20% smaller than the average value obtained from electron diffraction, i.e. the particle was compressed by about 80%. The lattice parameters of Xe crystals about 2 nm to about 6 nm in size were almost the same as those obtained from diffraction results. One of the reasons for the extra compression seen with a 1‐nm Xe particle is the increase in pressure inside an Xe particle with decreasing particle size.     

Quantitative comparison of energy-filtering transmission electron microscopy and atom probe tomography

Whereas transmission electron microscopy (TEM) is a well established method for the analysis of thin film structures down to the sub-nanometer scale, atom probe tomography (APT) is less known in the microscopy community. In the present work, local chemical analysis of sputtered Fe/Cr multilayer structures was performed with energy-filtering transmission electron microscopy (EFTEM) and APT. The single-layer thickness was varied from 1 to 6 nm in order to quantify spatial resolution and chemical sensitivity. While both the methods are able to resolve the layer structure, even at 2 nm thickness, it is demonstrated that the spatial resolution of the APT is about a factor of two, higher in comparison with the unprocessed EFTEM data. By calculating the influence of the instrumental parameters on EFTEM images of model structures, remaining interface roughness is indicated to be the most important factor that limits the practical resolution of analytical TEM.     

Reduction of charging in protein electron cryomicroscopy

Charging causes a loss of resolution in electron cryomicroscopy with biological specimens prepared without a continuous carbon support film. Thin conductive films were deposited onto catalase crystals prepared across holes using ion-beam sputtering and thermal evaporation and evaluated for the effectiveness of charge reduction. Deposits applied by ion-beam sputtering reduced charging but concurrently resulted in structural damage. Coatings applied by thermal evaporation also reduced charging, and preserved the specimen structure beyond 5 Å resolution as judged from electron diffraction patterns and images of glucose-embedded catalase crystals tilted to 45° in the microscope. This study demonstrates for the first time the feasibility of obtaining high-resolution data from unstained, unsupported protein crystals with a conductive surface coating.     

An electron microscopy study of the phase Al3Fe

The phase Al₃Fe (monoclinic C2/m, a = 1·549 nm, b = 0·808 nm, c = 1·248 nm, β = 107·8°) has been studied by transmission electron microscopy (TEM) and high resolution electron microscopy (HREM). Crystals were obtained from a direct chill-cast ingot of an Al-0·25 wt% Fe-0·13 wt% Si alloy. Extracted crystals were prepared by dissolving the aluminium phase in butanol and filtering off the particles. The extracted Al₃Fe crystals were mainly (100) platelets. The monclinic lattice was confirmed by tilt experiments and the mirror plane was confirmed by convergent beam electron diffraction. Experimental HREM images from the [100] and [110] projection agreed with images calculated by the multislice method. The interpretation of images in terms of a projected crystal structure is discussed. Common defects in Al₃Fe crystals were: twins on (100) and faults on (001). The (001) faults could be described by a displacement 1/2·[100] on a fault plane at z = 0·5 in the unit cell. A model for (001) faults, based on multiple twinning, is proposed.     

Mapping of ELNES on a nanometre scale by electron spectroscopic imaging

The amorphous interfacial layer between Si substrates and diamond films grown by plasma-assisted chemical vapour deposition has been studied by electron spectroscopic imaging. The amorphous layer consists mainly of carbon, which can only be distinguished from the diamond film by analysis of the near-edge structure (ELNES) of the carbon K edge. Series of electron spectroscopic images were acquired across the carbon K edge and were analysed in order to reveal the presence of the π*- and σ*-excitations. After background removal from the corresponding images, phase maps for the distribution of sp² and sp³ hybridized carbon can be obtained. From the whole series of images, electron energy-loss spectra can be extracted for any given area in the images. The results show that the amorphous layer covers large areas along the interface and that regions with only 1–2 nm layer thickness can clearly be analysed. The results obtained with the electron spectroscopic imaging technique will be compared with results obtained on a field emission gun scanning transmission electron microscope.     

Influence of Ca content and oxygen partial pressure on microstructural evolution of (Co,Ca)O at elevated temperatures

Ca‐doped (1, 1.7, 5 and 10 mol% CaO) cobalt oxide single‐crystal samples, with an [001] orientation, were annealed at elevated temperatures of 1000–1200 °C for different times and at different oxygen partial pressures. The microstructure was examined by means of transmission light and electron microscopy. High‐temperature X‐ray diffractometry was used, with the aim of determining the temperature of the CoO ? Co₃O₄ transition in these materials. Extensive precipitation of Ca‐free Co₃O₄ spinel crystals was observed with increasing Ca content and oxygen activity. It is suggested that the electrical conductivity changes in this material may be related to this precipitation, because it changes the electronic state of cobalt cations.     

Contribution of energy-filtering TEM to the detection of calcium: Application to mast cells

The ultrastructural distribution and quantification of calcium in mast cells prepared by anhydrous processing was investigated by energy-filtering transmission electron microscopy (EFTEM) using a Zeiss 902 electron microscope. Optimal conditions for calcium detection were determined using inorganic (calcium phosphate) and organic (calcium-loaded chelex beads) standards with known amounts of calcium. Electron energy-loss spectroscopy (EELS) revealed calcium at the L_2,3 edge and also at the M_2,3 edge for all specimens examined. Comparison with X-ray microanalysis confirmed the results obtained with EELS. Electron spectroscopic imaging (ESI) was applied for mapping calcium both in standards and in cells and we showed that mast cell granules were the main site of calcium localization. Although, results have shown that a combination of analytical techniques is required to obtain reliable results.     

Application of transmission electron microscopes to nanometre-sized fabrication by means of electron beam-induced deposition

Electron beam‐induced deposition was carried out using a scanning transmission electron microscope with a field emission gun to fabricate nanometre‐sized structures. A small amount of a metal–organic gas was introduced near the substrate in the microscope chamber, and focused electron beams were irradiated. Two‐ and three‐dimensional structures were fabricated by scanning the beam position. The minimum line width of the freestanding structures was 8 nm at a constant gas flux used. This line width of 8 nm is considered to be achieved by employing a high accelerating voltage, which leads to a small probe size, and the optimum scanning speed.     

In-situ observation of shape and atomic structure of Xe nanocrystals embedded in aluminium

An imaging technique to determine in situ the shape and atomic structure of nanosized Xe crystals embedded in Al is described using high-resolution transmission electron microscopy (HRTEM). The Xe nanocrystals, with sizes less than 5 nm were prepared by the implantation of 30 keV Xe⁺ into Al at room temperature. The fcc Xe nanocrystals are mesotactic with the Al lattice and have a lattice parameter ≈ 50% larger than that of Al. HRTEM images of the Xe were not clear in [110] zone axis illumination because of the small number of Xe atoms relative to Al atoms in any atom column. An off-axial imaging technique that consists of tilting the specimen several degrees from a zone axis and defocusing to suppress the Al lattice fringes is employed for the 110 projection of the Xe/Al system and the structure of the Xe nanocrystals is successfully imaged. The Xe images clearly represent projections of cuboctahedra with faces parallel to eight Al {111} planes truncated by six {100} planes. The results of multislice image simulations using a three-dimensional atomic model agreed well with the results obtained by the off-axial imaging technique. The usefulness of the technique is demonstrated with observations of crystal defects introduced into the Xe under intense 1000 keV electron irradiation.     

Polyphosphate at the Streptomyces lividans cytoplasmic membrane is enhanced in the presence of the potassium channel KcsA

The distribution of polyphosphate (polyP) within the cytoplasmic membrane of Streptomyces lividans hyphae or protoplasts has been determined at high spatial resolution by elemental mapping using energy‐filtered electron microscopy (EFTEM). The results revealed that polyP was best traceable after its interaction with lead ions followed by their precipitation as lead sulphide. Concomitant studies of the S.lividans wildtype (WT) strain and its co‐embedded mutant ΔK (lacking a functional kcsA gene) were conducted by labelling as the surface matrix of either one was labelled by cationic colloidal thorium dioxide. Within the WT strain, additional polyP was found to accumulate distinctly at the inner face of the cytoplasmic membrane. After removal of the cell wall (within protoplasts), the polyP‐derived lead‐sulphide (PbS) precipitate formed clusters of fibrillar material extending up to 50 nm into the cytoplasm. This feature was absent in the ΔK mutant strain. Together the results revealed that the presence of the KcsA channel and the structured polyP coincide.