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.     

Observation of human dentine by focused ion beam and energy-filtering transmission electron microscopy

Molar dentine was sliced into 100 nm ultrathin sections, by means of a focused ion beam, for observation by energy-filtering transmission electron microscopy (EFTEM). Within the matrix, crystals approximately 10 nm wide and 50–100 nm long were clearly observed. When carbon and calcium were mapped in electron spectroscopic images by EFTEM, carbon failed to localize in crystals. However, it was found in other regions, especially those adjacent to crystals. Because carbon localizations were thought to reflect the presence of organic components, carbon concentration in regions near crystals suggested the interaction of crystals and organics, leading to organic control of apatite formation and growth. Ca was present in almost all regions. The majority of Ca localizing in regions other than crystals may be bound to organic substances present in dentine matrix. These substances are thought to both accumulate Ca and act as reservoirs for crystallization of apatite in dentine.     

Intermediate aggregates resulting in the interaction of bile salt with liposomes studied by transmission electron microscopy and light scattering techniques

The interaction of sodium cholate (SC) with phosphatidylcholine liposomes was studied by means of transmission electron microscopy (TEM), changes in the mean particle size (quasielastic light scattering, QELS) and in the static light scattering (SLS) of the system during liposome solubilization. A good correlation was found between the TEM diameter of particles and the mean hydrodynamic diameter (HD) determined by QELS. The intermediate aggregates resulting in this interaction were dependent on the SC concentration in the system. Thus, an initial vesicle growth occurred when the SC concentration in the system was 13.79 mol%. Additional SC amounts (41.17 mol% SC) led to the formation of the largest vesicles (HD 410 nm). Increasing SC amounts led to a slight fall in the vesicle diameter and in the SLS of the system. Thus, for 47.08 mol% SC, TEM images still showed the presence of vesicles albeit with traces of smaller structures and signs of vesicle fusion. When SC concentration exceeded 48 mol% an abrupt decrease in SLS occurred, the size curve starting to show a bimodal distribution. Thus, for 50 mol% SC a sharp distribution curve appeared at 52 nm indicating the formation of small particles and TEM images showed clear signs of vesicle disintegration with formation of tubular structures. The subsequent self organization of these tubular structures (54 mol% SC) led to the formation of open multilayered structures in coexistence with small particles. A gradual increase in the number of these small particles (mixed micelles) led to the complete solubilization of liposomes.     

Direct imaging in a water layer of human chromosome fibres composed of nucleosomes and their higher-order structures by laser-plasma X-ray contact microscopy

X-ray contact microscopy with a 300-ps-duration laser-plasma X-ray source has been used to image hydrated human chromosomes. Clearly imaged are individual nucleosomes and their higher-order particles (superbeads), elementary chromatin fibrils c. 30 nm in diameter and their higher-order fibres of various sizes up to c. 120 nm in diameter. The results demonstrate that X-ray microscopy is now capable of opening a new path of investigation into the detailed structures of hydrated chromosome fibres in their natural state.     

Bundles of hexagonally arranged tubules in timothy grass pollen: Detection of a novel pollen component using anhydrous fixation and image analysis techniques in transmission electron microscopy

Pollen from timothy grass (Phleum pratense L.) was subjected to various aqueous and non‐aqueous fixation and preparation protocols for transmission electron microscopy. Only in the cytoplasm of anhydrously prepared pollen grains were conspicuous inclusions observed that range in size from less than 1 μm up to 8 or 10 μm. These bodies have so far not been described in the literature. Higher magnifications show that these inclusions consist of bundles of hexagonally arranged small tubules. In order to obtain details of the ultrastructure of this novel pollen component, TEM micrographs of ultrathin sections of hexagonally arranged tubules were analyzed using Fourier transform techniques of image analysis. It was found that the tubules form groups with quasi‐periodic hexagonal arrangement, with an average centre‐to‐centre spacing between the neighbouring tubules of approximately 42 nm. Individual tubules are formed by 12 or 13 particles. The outer diameter of the tubules ranges between 22 and 24 nm. From our experiments, we conclude that the quasi‐periodic hexagonally arranged tubules forming conspicuous cytoplasmic inclusions in dry timothy grass pollen grains are structurally similar to microtubules.     

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.     

Development of a high energy resolution electron energy-loss spectroscopy microscope

We have developed a high energy resolution electron energy-loss spectroscopy (EELS) microscope, which can take spectra from specified small specimen areas and specified small reciprocal space areas to investigate detailed electronic structures. The EELS microscope is equipped with retarding Wien filters as the monochromator and the analyser. The filters are designed to achieve a stigmatic focus. The energy resolutions are 12 meV and 25 meV for cases without and with a specimen, respectively. Spatial and momentum resolutions are 30–110 nm in diameter and 1.1 nm⁻¹ in angular diameter, respectively. EELS spectra are presented to show the performance of this instrument.     

Structure and morphology of the Alzheimer's amyloid fibril

Amyloid fibrils are deposited in a number of diseases, including Alzheimer's disease, Type 2 diabetes, and the transmissible spongiform encephalopathies (TSE). These insoluble deposits are formed from normally soluble proteins that assemble to form fibrous aggregates that accumulate in the tissues. Electron microscopy has been used as a tool to examine the structure and morphology of these aggregates from ex vivo materials, but predominantly from synthetic amyloid fibrils assembled from proteins or peptides in vitro. Electron microscopy has shown that the fibrils are straight, unbranching, and are of a similar diameter (60-100 A) irrespective of the precursor protein. Image processing has enhanced electron micrographs to show that amyloid fibrils appear to be composed of protofilaments wound around one another. In combination with other techniques, including X-ray fiber diffraction and solid state NMR, electron microscopy has revealed that the internal structure of the amyloid fibril is a ladder of beta-sheet structure arranged in a cross-beta conformation.     

The nacre protein perlucin nucleates growth of calcium carbonate crystals

Atomic force microscopy (AFM) in aqueous solution was used to investigate native nacre of the marine snail Haliotis laevigata on the microscopic scale and the interaction of purified nacre proteins with calcium carbonate crystals on the nanoscopic scale. These investigations were controlled by scanning electron microscopy (SEM), light microscopy (LM) and biochemical methods. For investigations with AFM and SEM, nacre was cleaved parallel to the aragonite tablets in this biogenic polymer/mineral composite. Multilamellar organic sheets consisting of a core of chitin with layers of proteins attached on both sides lay between the aragonite layers consisting of confluent aragonite tablets. Cleavage appeared to occur between the aragonite tablet layer and the protein layer. AFM images revealed a honeycomb‐like structure to the organic material with a diameter of the ‘honeycombs’ equalling that of the aragonite tablets. The walls of the structures consisted of filaments, which were suggested to be collagen. The flat regions of the honeycomb‐like structures exhibited a hole with a diameter of more than 100 nm. When incubated in saturated calcium carbonate solution, aragonite needles with perfect vertical orientation grew on the proteinacous surface. After treatment with proteinase K, no growth of orientated aragonite needles was detected. Direct AFM measurements on dissolving and growing calcite crystals revealed a surface structure with straight steps the number of which decreased with crystal growth. When the purified nacre protein perlucin was added to the growth solution (a super‐saturated calcium carbonate solution) new layers were nucleated and the number of steps increased. Anion exchange chromatography of the water‐soluble proteins revealed a mixture of about 10 different proteins. When this mixture was dialysed against saturated calcium carbonate solution and sodium chloride, calcium carbonate crystals precipitated together with perlucin leaving the other proteins in the supernatant. Thus perlucin was shown to be a protein able to nucleate calcium carbonate layers on calcite surfaces, and in the presence of sodium chloride, is incorporated as an intracrystalline protein into calcium carbonate crystals.     

High-resolution cryo-scanning electron microscopy study of the macromolecular structure of fibronectin fibrils

High-resolution cryo-scanning electron microscopy was used to examine fibronectin fibrils formed in culture by human skin fibroblasts and in a cell-free system by denaturing soluble plasma fibronectin with guanidine. These studies indicate that the conformation of fibrils formed in culture and in a cell-free system appeared to be similar and that fibronectin fibrils have at least two distinct structural conformations. Fibronectin fibrils can be very straight structures with smooth surfaces or highly nodular structures. The average diameter of the nodules in these fibrils is 12 nm. Both conformations can be seen within an individual fibril indicating that they are not different types of fibronectin fibrils but rather different conformational states. Immunolabeling studies with a monoclonal antibody, IST-2, to the heparin II binding domain of fibronectin revealed that the epitope was buried in highly smooth fibrils, but it was readily exposed in nodular fibrils. We propose, therefore, that fibronectin fibrils are highly flexible structures and, depending on the conformation of the fibril, certain epitopes on the surface may be buried or exposed.     

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).     

An ultrastructural study of cryofractured myocardial cells with special attention to the relationship between mitochondria and sarcoplasmic reticulum

A comparative study of various cryofracturing techniques has been conducted on the mammallian myocardial cell. Quench freezing of fresh or fixed tissue in melting Freon 22 resulted in severe cellular damage due to ice crystallization. Fixation with Karnovsky's fixative prior to quenching had no modifying effect on the size and distribution of the ice crystals. The crystals were orientated primarily in the direction of the long axis of the myofibrils, manifested as empty tube-like structures in the scanning electron microscope (SEM). Regular cross-bridging often seen at the Z-band levels indicated that ice crystals, at least in some portions of the cells, were confined within the sarcomere. Within the same cell the size of the ice crystals could vary considerably. Treatment of the tissue with polyvinylpyrrolidone (PVP) prior to rapid freezing had no noticeable cryoprotective effect. The surface of the thin layer of PVP surrounding the freeze dried tissue appeared amorphous in the SEM. However, the first evidence of ice crystallization was found a few micrometres under the surface. The freezing artefacts were completely circumvented if the cryofracturing was carried out on ethanol-impregnated or on critical point dried material. While the first method resulted in a smooth fracture plane passing through the cell structures, the intracellular fracture plane of the critical point dried material followed the surface of the cell organelles. Separation of the cell organelles caused by freezing or by critical point drying revealed thread-like structures extending from the mitochondrial surface. Re-examination of SEM-processed material in the transmission electron microscope (TEM) revealed that these structures were part of the sarcoplasmic reticulum (SR), and that a close contact between the SR and the outer mitochondrial membrane existed. TEM of conventional prepared material revealed that strands of electron-dense material, here named ‘mito-reticular junctional fibres’, bridged the narrow gap between the mitochondrial surface and the SR. It is suggested that these fibres have a specific anchoring function.     

Assessment of collagen fibril spacing in relation to selected region of interest (ROI) on electron micrographs--application to the mammalian corneal stroma

AIMS: To evaluate measurements of collagen fibril spacing using different shaped regions of interest (ROI) on transmission electron micrograph (TEM) images of rabbit corneal stroma. METHODS: Following glutaraldehyde fixation and phosphotungstic acid staining, TEM images of collagen fibrils in cross section were projected at a final magnification close to 250,000 × to obtain overlays. Interfibril distances (IFDs; center‐to‐center spacing) were measured within different ROIs of the same nominal area (0.25 μm²) but different shape (with the length to width, L:W, ratio from 1:1 to 6:1). The IFD distribution was analyzed, and the 2D organization assessed using a radial distribution analysis. RESULTS: The fibrils had an average diameter of 35.3 ± 3.8 (SD) nm, packing density of 393 ± 4 fibrils / μm² and a fibril volume fraction of 0.39 ± 0.02. IFDs ranged from 29 to 1400 nm depending on the shape of the ROI, with average values ranging from 263 to 443 nm. By artificially selecting IFD data only to a radial distance of 250 nm, the average IFDs were just 145–157 nm. The radial distributions, to 250 nm, all showed a nearest neighbors first peak which shifted slightly from predominantly at 45–54 nm with more rectangular ROIs. The radial distribution profiles could be shown to be statistically different if the ROI L:W ratio was 2:1 or greater. CONCLUSION: Selection of an ROI for assessment of packing density and interfibril distances should be standardized for comparative assessments of TEMs of collagen fibrils. Microsc. Res. Tech., 2011. © 2011 Wiley‐Liss, Inc.     

Microstructural investigations in Cf–SiC composites in as-sintered state and after creep experiments

The high interest in ceramic matrix composites during the last decade has led to a considerable number of studies devoted to their thermomechanical properties and damage processes. Despite their sensitivity to oxygen partial pressure, carbon fibres appear to possess higher stability and better mechanical properties if they are treated under protective atmospheres than other ceramic fibres (especially classical silicon carbide fibres). The aim of this investigation is to characterize at the nanoscale the main microstructural parameters of C_f–SiC composites provided by the SEP (Division of SNECMA, Bordeaux, France). This material was fabricated from a 2.5D preform made of high strength polyacrylonitrile (PAN)-based carbon fibres densified according to the chemical vapour infiltration process. A pyrocarbon (PyC) interphase was deposited on the fibre prior to the β-SiC matrix infiltration. A careful high resolution electron microscopy (HREM) microstructural investigation focused on the fibre microstructure as well as on the different interfaces in the material: pyrocarbon/fibre and matrix/pyrocarbon interfaces. All these observations have been realized in longitudinal and transverse sections of the specimen. These observations are found in good agreement with Guigon's model for high strength ex-PAN carbon fibres. The PyC interphase texture was strongly anisotropic at the fibre/interphase and interphase/matrix interfaces over a mean thickness of 8–15 nm. Tensile creep tests were performed under partial pressure of argon between 1273 and 1673 K for stress levels ranging from 110 to 220 MPa. Scanning electron microscopy and high resolution electron microscopy were used to study the microstructural modifications inside the fibres and at the different interfaces. A discussion of the possible creep mechanisms based on the microstructural investigation and the creep results is presented.     

Off-axis electron holography of patterned magnetic nanostructures

Magnetization reversal processes in lithographically patterned magnetic elements that have lateral dimensions of 70–500 nm, thicknesses of 3–30 nm and a wide range of shapes and layer sequences have been followed in situ using off-axis electron holography in the transmission electron microscope. This technique allows domain structures within individual elements and the magnetic interactions between them to be quantified at close to the nanometre scale. The behaviour of 30 nm-thick Co elements was compared with that of 10 nm-thick Ni and Co elements, as well as with Co/Au/Ni trilayers. The hysteresis loops of individual elements were determined directly from the measured holographic phase images. The reproducibility of an element's domain structure in successive cycles was found to be affected by the out-of-plane component of the applied magnetic field and by the exact details of its initial magnetic state. Close proximity to adjacent elements led to strong intercell coupling, and remanent states with the in-plane magnetic field removed included domain structures such as solenoidal (vortex) states that were never observed during hysteresis cycling. Narrow rectangular bars reversed without the formation of end domains, whereas closely separated magnetic layers within individual elements were observed to couple to each other during field reversal.     

Direct evidence of radial and tangential morphology of high-modulus aromatic polyamide fibres

Previous work, at foreign laboratories, essentially based on electron microscopy of longitudinal sections, has suggested a radial morphology for the aromatic poly-amide high modulus fibres; the present paper gives direct evidence of such a morphology, thanks to a special preparation technique which allows a great improvement in the quality of the cross-sections of these fibres. This is demonstrated for both a commercial ‘Du Pont de Nemours’ yarn sample ‘Kevlar 29’, and an experimental high modulus aramid RPT (Rhǒne Poulenc Textile) yarn. In the first case, Ag₂S insertion technique was used and permitted one to see, on the cross-sections, an alternation of dark and clear radial bands, again with a tendency towards tangential splitting. In the second case the fibres were included into an amorphous polymer, a sample preparation technique which greatly enhances the quality of the cross-sections; optical microscopy showed the radial morphology fairly well; dark-field transmission electron microscopy—using the equatorial doublet of the electron diffraction pattern—allowed satisfactory resolution: both the radial, and occasionally the tangential, splitting, and the size of the cross-sectioned crystallites were easily revealed: these crystallites appear as isodiametric bright particles c. 15 nm in lateral size.     

Three-dimensional morphometry in scanning electron microscopy: a technique for accurate dimensional and angular measurements of microstructures using stereopaired digitized images and digital image analysis

A method for accurate dimensional and angular measurements of microstructures analysed in the scanning electron microscope is described. The method considers central and parallel projections and involves (a) digital image acquisition of stereopaired images from the scanning electron microscope's photodisplay, (b) generation of 3D-image representations, (c) setting of measuring points in the digitized stereopaired images, (d) computation of exact space coordinates ( x / y / z ) from the corresponding point coordinates ( x _L/ y _L; x _R/ y _R), (e) determination of distances and angles between consecutive corresponding points using vector equations, and (f) transfer of computed data into spreadsheets of the data analysis software using dynamic data exchange with simultaneous graphical display of the frequency distribution of variables.
Measurements performed on specimens with known dimensions (grid with 10 μm wide square meshes, polystyrene beads with 0.33 μm diameter) and angles (synthetic crystals of K(Al,Cr)[SO₄], CuSO₄.5H₂O and NaCl) revealed a high accuracy in dimensional as well as angular measurements (total error 1 ± 0.5%).
In Monte Carlo experiments the overall error was found to depend strongly on the size of the measured structure relative to the size of the measurement field (field width).     

Lamellar subcomponents of the cuticular cell membrane complex of mammalian keratin fibres show friction and hardness contrast by AFM

There is a substantial body of information indicating that 18‐methyleicosanoic acid (18‐MEA) is covalently linked to the outer surface of all mammalian keratin fibres and also forms the outer β‐layer of the cuticular cell membrane complex (CCMC) which separates the cuticle cells from each other. Low cohesive forces are expected between the lipid‐containing outer β‐layer and the δ‐layer of the CCMC, thus providing a weak point for cuticular delamination and presenting a fresh layer of 18‐MEA to the newly exposed surface. We have used lateral force microscopy and force modulation atomic force microscopy (AFM) to examine human hair fibres in which the non‐covalently linked fatty acids have been removed. Examination of the lateral force images of new cuticle surfaces revealed by the attrition of overlying cuticle layers showed three separate zones of clearly defined frictional contrast. These are thought to correspond with the δ‐layer, the proteinaceous epicuticle and outer β‐layers of the CCMC. The δ‐layer was found to have a thickness of 16 nm (SD = 1 nm, n = 25), comparable to the 18.0 nm thickness measured from transverse cross‐sections of fibres with transmission electron microscopy. Force modulation AFM showed that the outer β‐layer was softer than the epicuticle and the δ‐layer. The frictional contrast was removed following treatment with methanolic KOH (0.1 mol dm^?3) at 25 °C for 30 min, suggesting the hydrolysis of the thioester linkage and removal of 18‐MEA from the surface.     

The fine structure of fenestrated adrenocortical capillaries revealed by in-lens field-emission scanning electron microscopy and scanning transmission electron microscopy

Cell biologists probing the physiologic movement of macromolecules and solutes across the fenestrated microvascular endothelial cell have used electron microscopy to locate the postulated pore within the fenestrae. Prior to the advent of in-lens field-emission high-resolution scanning electron microscopy (HRSEM) and ultrathin m et al coating technology, quick-freeze, platinum-carbon replica and grazing thin-section transmission electron microscopy (TEM) methods provided two-dimensional or indirect imaging methods. Wedge-shaped octagonal channels composed of fibrils interwoven in a central mesh were depicted as the filtering structures of fenestral diaphragms in images of platinum replicas enhanced by photographic augmentation. However, image accuracy was limited to replication of the cell surface. Subsequent to this, HRSEM technology was developed and provided a high-fidelity, three-dimensional topographic image of the fenestral surface directly from a fixed and dried bulk adrenal specimen coated with a 1 nm chromium film. First described from TEM replicas, the “flower-like” structure comprising the fenestral pores was readily visualized by HRSEM. High-resolution images contained particulate ectodomains on the lumenal surface of the endothelial cell membrane. Particles arranged in a rough octagonal shape formed the fenestral rim. Digital acquisition of analog photographic recordings revealed a filamentous meshwork in the diaphragm, thus confirming and extending observations from replica and grazing section TEM preparations. Endothelial cell pockets, first described in murine renal peritubular capillaries, were observed in rhesus and rabbit adrenocortical capillaries. This report features recent observations of fenestral diaphragms and endothelial pockets fitted with multiple diaphragms utilizing a Schottky field-emission electron microscope. In-lens staging of bulk and thin section specimens allowed tandem imaging in HRSEM and scanning TEM modes at 25 kV.     

Microscopical investigations on the epicuticle of mammalian keratin fibres

The existence of a thin chemically resistant layer, the epicuticle, close to the surfaces of all undamaged mammalian keratin fibres has been known since 1916. The identification of such a specific structure within the fibre cuticle has remained elusive. Now, through transmission electron microscope investigations of stained transverse sections of hairs from various animal species, the epicuticle has been tentatively identified as a sharply defined and continuous layer approximately 13 nm thick covering the entire outwardly facing intracellular surface of every cuticle cell. The staining behaviour of the epicuticle leads one to suppose that it is rich in cystine and that thioester-bound lipids might be present within its bulk. With the atomic force microscope it was established that the undamaged outer surface of all mammalian keratin fibres, even including those from the monotremes, were longitudinally striated. The lateral spacing of the striations was always in the range 0.29–0.39 µm. Striations only occurred on the freely exposed outer surfaces of the original undamaged fibres; evidently arising by some, as yet undefined, interaction in the follicle with the cuticle of the inner root sheath. By stripping off fatty acids known to be covalently attached to the fibre's outer surface, the striations were shown to reflect a corresponding irregularity of the epicuticle's surface.