ALBERT

Description: Polysaccharides account for more than 90% of the content of fungal cell walls, but the mechanism underlying the formation of the architecture of the cell walls, which consist of microfibrils embedded in an amorphous wall matrix, remains unknown. We used electron microscopy to investigate ten different fungal cell-wall polysaccharides to determine whether they could self-assemble into the fibrillar or amorphous component of fungal cell walls in a test tube without enzymes. The ultrastructures formed by precipitating β-1,3-glucan and β-1,6-glucan are different depending on the existence of branching in the molecule. Linear β-1,3-glucan and linear β-1,6-glucan precipitate into a fibrillar ultrastructure. Branched β-1,6-glucan, mannan and glycogen precipitates are amorphous. Branched β-1,3-glucan forms a fibrillar plus amorphous ultrastructure. Self-assembly among combinations of different linear and branched cell-wall polysaccharides results in an ultrastructure that resembles that of a yeast cell wall, which suggests that self-assembly of polysaccharides may participate in the development of the three-dimensional architecture of the yeast cell wall.

Description: When considering drug delivery, the amount of drug that can be carried at a particular time and how the drug is incorporated efficiently into cells are important parameters. Transferrin (Tf)-conjugated nanocarriers have been used for the targeted delivery of drugs to cancer cells due to the availability of receptor-mediated clathrin-dependent endocytosis. In general, however, endocytosis seems to differ according to the size and shape of carriers. Large substances are generally internalized into cells by phagocytosis. We studied the internalization mechanism of Tf-conjugated nanoparticles (diameter, 522 nm). Tf-conjugated polystyrene particles were incorporated into cells by receptor-mediated endocytosis with large clathrin-coated vesicles even though their diameter was 〉500 nm and despite that fact that clathrin-coated vesicles have a diameter of 100 nm. This finding suggests that signals for internalization generated by stimulated Tf receptors (TfRs) activate clathrin-mediated endocytosis preferentially. Whether these larger particles could deliver drugs more efficiently than smaller particles was then examined. The toxicity of larger Tf-conjugated biodegradable nanoparticles (poly(lactic-co-glycolic acid)) encapsulating doxorubicin (diameter, 216 ± 38 nm) was appreciably dependent on the number of Tf molecules conjugated on a particle and the number of TfRs expressed on the cell membrane. Larger Tf-conjugated particles delivered drugs to cancer cells expressing many TfRs more selectively than their smaller counterparts (diameter, 56 ± 9 nm) if they were decorated with an appropriate number of Tf molecules.

Description: Structural biology relies on good-quality protein crystals in order for structure determination. Many factors affect the growth process of a protein crystal including the way it nucleates and the types of damage and contamination during its growth. Although the nucleation process and quality of a crystal is vital to structure determination, they are both under-studied areas of research. Our research begins to explore ways of measuring the quality of protein crystals, using TEM, thus overcoming the problems associated with viewing wet specimens in a vacuum. Our current understanding of nucleation is that it is a two-step mechanism involving the formation of nuclei from dense liquid clusters; however; it is still unclear whether nuclei first start as amorphous aggregates or as crystalline lattices. Potentially, electron diffraction may be capable of uncovering this process. Using TEM imaging and diffraction of lysozyme as a model protein crystal, we report the internal two-dimensional strain and the density of crystallites in a protein crystal, at a resolution never seen before. The TEM diffraction shows unique features of crystal mosaicity that can be directly correlated to TEM images.

Description: The cotyledon of legume seeds is a storage organ that provides nutrients for seed germination and seedling growth. The spatial and temporal control of the degradation processes within cotyledons has not been elucidated. Calcium oxalate (CaOx) crystals, a common calcium deposit in plants, have often been reported to be present in legume seeds. In this study, micro-computed tomography (micro-CT) was employed at the SPring-8 facility to examine the three-dimensional distribution of crystals inside cotyledons during seed maturation and germination of Lotus miyakojimae (previously Lotus japonicus accession Miyakojima MG-20). Using this technique, we could detect the outline of the embryo, void spaces in seeds and the cotyledon venation pattern. We found several sites that strongly inhibited X-ray transmission within the cotyledons. Light and polarizing microscopy confirmed that these areas corresponded to CaOx crystals. Three-dimensional observations of dry seeds indicated that the CaOx crystals in the L. miyakojimae cotyledons were distributed along lateral veins; however, their distribution was limited to the abaxial side of the procambium. The CaOx crystals appeared at stage II (seed-filling stage) of seed development, and their number increased in dry seeds. The number of crystals in cotyledons was high during germination, suggesting that CaOx crystals are not degraded for their calcium supply. Evidence for the conservation of CaOx crystals in cotyledons during the L. miyakojimae germination process was also supported by the biochemical measurement of oxalic acid levels.

Description: The electron irradiation damage of MFI-type zeolite was estimated under various accelerating voltages of 100, 200 and 300 kV from successively captured high-resolution transmission electron microscopy (HRTEM) images. To determine the optimal accelerating voltage for HRTEM imaging of electron-sensitive MFI zeolite, the critical dose was estimated from the disappearance of a specific fast Fourier transform spot calculated from experimental images. Based only on the electron dose, a higher voltage was more advantageous. However, taking into account the minimum dose for imaging with a CCD camera, the optimal accelerating voltage for imaging MFI zeolite was 200 kV. The minimum dose for image detection with a CCD camera was surmised from the output/input signal ratio dependence on the accelerating voltage and the contrast range in simulated HRTEM images.

Description: The magnetic field generated by a magnetic recording head is evaluated using electron holography. A magnetic recording head, which is connected to an electric current source, is set on the specimen holder of a transmission electron microscope. Reconstructed phase images of the region around the magnetic pole show the change in the magnetic field distribution corresponding to the electric current applied to the coil of the head. A simulation of the magnetic field, which is conducted using the finite element method, reveals good agreement with the experimental observations.

Description: A new multilayer-coated varied line-spaced grating, JS4000, was fabricated and tested for extending the upper limit of a grating X-ray spectrometer for electron microscopy. This grating was designed for 2–3.8 keV at a grazing incidence angle of 1.35°. It was revealed that this new multilayer structure enables us to take soft-X-ray emission spectra continuously from 1.5 to 4.3 keV at the same optical setting. The full-width at half maximum of Te-L α1,2 (3.8 keV) emission peak was 27 eV. This spectrometer was applied to indium tin oxide particles and clearly resolved Sn-L α (3444 eV) and In-L β1 (3487 eV) peaks, which could not be resolved by a widely used energy-dispersive X-ray spectrometer.

Description: For development of advanced materials, characterization using a scanning transmission electron microscope (STEM) including analysis via X-ray energy-dispersive spectrometry and electron energy-loss spectrometry is essential. Recent advances in aberration-corrected instruments have offered large-scale data acquisition at a high resolution for limited acquisition times both in imaging and in analysis. Further advanced procedures are required to analyze such large-scale datasets more efficiently including quantification. In addition, more simplified tuning procedures are crucial to the best possible resolution in the latest aberration-corrected instruments. In this review article, several approaches to perform advanced electron microscopy, which the author has been developing with his colleague, are described as ‘Microscopy Hacks’. These are (i) quantification and elemental/chemical-imaging procedures, (ii) advanced statistical approaches to handle large-scale datasets and (iii) instrument characterization and tuning procedures including the latest development of an ad hoc autotuning procedure for aberration-corrected STEM imaging.

Description: The crystal structure of a new type of molybdenum oxide crystal encapsulated in a single-walled carbon nanotube (CNT) was examined via diffraction and spectroscopic techniques using both X-rays and electron beams. This new type of molybdenum oxide crystal has a chemical bonding state of MoO 3 , as confirmed by X-ray absorption spectroscopy, and the MoO 3 units exhibit axial symmetry, as clarified by electron diffraction from bundled and individual CNTs encapsulating the crystal. To obtain three-dimensional information on the structure, a cross-sectional sample was prepared using a conventional dimple and ion-mill method. High-resolution transmission electron microscopy images exhibit ring-like shapes that originated from the arrangement of the MoO 3 units inside the CNTs, as observed along the tube axis. The units are spaced 0.36 nm from each other in a ring arrangement and the distance between each ring is 0.391 nm.

Description: This review summarizes the recent advances in three-dimensional (3D) imaging techniques and their application to polymer nanostructures, for example, microphase-separated structures of block copolymers. We place particular emphasis on the method of transmission electron microtomography (electron tomography for short; hereafter abbreviated as ET). As a result of recent developments in ET, truly quantitative 3D images of polymer nanostructures can now be obtained with subnanometer resolution. The introduction of scanning optics in ET has made it possible to obtain large amounts of 3D data from micrometer-thick polymer specimens by using conventional electron microscopes at a relatively low accelerating voltage, 200 kV. Thus, ET covers structures over a wide range of thicknesses, from a few nanometers to several hundred nanometers, which corresponds to quite an important spatial range for hierarchical polymer nanostructures. ET provides clear 3D images and a wide range of new structural information that cannot be obtained using other methods. Information traditionally derived from conventional microscopy or scattering methods can be directly acquired from 3D volume data. ET is a versatile technique that is not restricted to only polymer applications; it can also be used as a powerful characterization tool in energy applications such as fuel cells.

Description: The performances of a newly developed 80–200 kV cold field emission gun (CFEG) transmission electron microscope (TEM) integrating a spherical aberration corrector for a TEM image-forming lens have been evaluated. To begin, we show that the stability of both emission and probe currents makes use of this new CFEG much friendlier. The energy spread of electrons emitted from the CFEG has been measured as a function of emission current and shows a very last 0.26 eV energy resolution at 200 kV and even 0.23 eV at 80 kV. The combination of the CFEG and the CEOS™ aberration corrector, associated with enhanced mechanical and electrical stabilities of this new microscope, allows reaching an information transfer below 75 pm at 200 and 80 pm at 80 kV. This unseen resolution at 200 kV has allowed us to study the structure of CoPt nanoparticles by observing direct images of their atomic arrangement along the high indexes zone axis. We have evidenced the presence of defects in these nanostructures that are not parallel to the electron beam. The precise stoichiometry of two iron oxides, FeO and Fe 2 O 3 , has been determined from an analysis of iron valence state that was obtained from a direct analysis of EELS fine structures spectrum of the two oxides.

Description: Data mining from noisy data/images is one of the most important themes in modern science and technology. Statistical image processing is a promising technique for analysing such data. Automation of particle pickup from noisy electron micrographs is essential, especially when improvement of the resolution of single particle analysis requires a huge number of particle images. For such a purpose, reference-based matching using primary three-dimensional (3D) model projections is mainly adopted. In the matching, however, the highest peaks of the correlation may not accurately indicate particles when the image is very noisy. In contrast, the density and the heights of the peaks should reflect the probability distribution of the particles. To statistically determine the particle positions from the peak distributions, we have developed a density-based peak search followed by a peak selection based on average peak height, using multi-reference alignment (MRA). Its extension, using multi-reference multiple alignment (MRMA), was found to enable particle pickup at higher accuracy even from extremely noisy images with a signal-to-noise ratio of 0.001. We refer to these new methods as stochastic pickup with MRA (MRA-StoPICK) or with MRMA (MRMA-StoPICK). MRMA-StoPICK has a higher pickup accuracy and furthermore, is almost independent of parameter settings. They were successfully applied to cryo-electron micrographs of Rice dwarf virus. Because current computational resources and parallel data processing environments allow somewhat CPU-intensive MRA-StoPICK and MRMA-StoPICK to be performed in a short period, these methods are expected to allow high-resolution analysis of the 3D structure of particles.

Description: The sensory nerve endings of the rat tongue, cheek and palate were studied using immunohistochemical staining and transmission electron microscopy analysis. The specimens were fixed in modified Karnovsky solution and embedded in Spurr resin. Substance P, calcitonin gene-related peptide (CGRP)- and protein gene product 9.5 (PGP b9.5)-containing nerve fibers in the rat tongue, cheek and palate were examined by electronic microscopical analysis and immunohistochemical localization. These fibers run very close to the basal lamina of the epithelium and extend into the filliform and fungiform papillae. Numerous plexiform fibers immunoreactive for substance P, CGRP and PGP 9.5 were found in the connective tissue of mucosa. Electron microscopic observations showed clearly immunostained nerve fibers, which are located very close to the basal lamina of epithelial cells. Some electron-dense granules may be observed in the axoplasms of both substance P and CGRP immunoreactive fibers. Several lamellar corpuscles into the subepithelial connective tissue papillae, Merkel corpuscles and numerous thin unmyelinated and myelinated axons were observed. The terminal axons revealed numerous mitochondria, neurofilaments, microtubules and clear vesicles in the base of axoplasmic protrusions. The lamellar cells showed caveolae and interlamelar spaces filled by amorphous substance. Between the lamellar cells and axoplasmic membrane, and in the adjacent lamellae region, desmosome-type junctions were observed. The quantitative and morphometric analysis showed nerve endings with an average area of 4.83 ± 3.4 μm 2 and 19.4 internal mitochondria in this site and the organized corpuscles with an average area of 79.24 ± 27.24 μm 2 and 24.23 internal mitochondria in this place. All the structures observed are involved in the transmission of pain and mechanoreceptors stimulus of these oral mucosae.

Description: The F-actin cytoskeleton of Cryptococcus neoformans is known to comprise actin cables, cortical patches and cytokinetic ring. Here, we describe a new F-actin structure in fungi, a perinuclear F-actin collar ring around the cell nucleus, by fluorescent microscopic imaging of rhodamine phalloidin-stained F-actin. Perinuclear F-actin rings form in Cryptococcus neoformans treated with the microtubule inhibitor Nocodazole or with the drug solvent dimethyl sulfoxide (DMSO) or grown in yeast extract peptone dextrose (YEPD) medium, but they are absent in cells treated with Latrunculin A. Perinuclear F-actin rings may function as ‘funicular cabin’ for the cell nucleus, and actin cables as intracellular ‘funicular’ suspending nucleus in the central position in the cell and moving nucleus along the polarity axis along actin cables.

Description: To evaluate the advantages of combination of two advanced electron microscopic technologies such as serial block-face scanning electron microscopy and double-axis electron beam tomography, we analyzed the three-dimensional morphology of cellular relationships between dendritic and plasma cells in the synovial membrane from patients with rheumatoid arthritis, using the combined approach.

Description: The low-energy Ar-ion milling method was used to prepare ultrathin specimens for transmission electron microscope observation. The samples were thinned initially by a usual focused ion beam technique or typical Ar-ion milling with a high energy of 2–10 keV and were thinned additionally by an Ar-ion beam with an energy less than 1 keV, typically 500–900 eV. This low-energy ion beam was scanned over the specimen, and secondary electrons induced by the ion beam could be detected to form secondary electron images with a resolution of a few micrometre. Because a desired area can be selected and thinned by the low-energy ion beam, redeposition or cross contamination from irradiation of a metal grid that supports the sample can be prevented. It was shown that the low-energy Ar-ion beam thins a surface amorphous damage layer preferentially and effectively rather than a crystal specimen. Images from ultrathin specimens of two different materials revealed a detailed structure.

Description: Off-axis electron holography can be used to measure the inner potential of a specimen from its reconstructed phase image and is thus a powerful technique for materials scientists. However, abrupt reversals of contrast from white to black may sometimes occur in a digitally reconstructed phase image, which results in inaccurate information. Such phase distortion is mainly due to the digital reconstruction process and weak electron wave amplitude in some areas of the specimen. Therefore, digital image processing can be applied to the reconstruction and restoration of phase images. In this paper, fringe reconnection processing is applied to phase image restoration of a crystal structure image. The disconnection and wrong connection of interference fringes in the hologram that directly cause a 2 phase jump imperfection are correctly reconnected. Experimental results show that the phase distortion is significantly reduced after the processing. The quality of the reconstructed phase image was improved by the removal of imperfections in the final phase.

Description: Diabetes causes oxidative stress, which in turn generates excessive free radicals resulting in cellular damage. Vitamin C is an antioxidant that protects tissues and organs from oxidative stress. The thymus is one of the most important lymphoid organs, which regulates T-lymphocyte proliferation and maturation. The aim of this study is to investigate the protective effects of vitamin C on the thymus of streptozotocin (STZ)-induced diabetic rats. The mitotic activity and cell integrity of thymic lymphocytes were explored. Wistar Albino rats were divided into three groups: control (Group 1), STZ-diabetes (Group 2) and vitamin C-treated STZ-diabetics (Group 3). Rats received a single intraperitoneal injection of 45 mg/kg STZ to induce diabetes. Vitamin C (20 mg/kg) was administered intragastrically. Semithin and ultrathin sections were examined under a light or an electron microscope, respectively. Considerable numbers of mitotic lymphocytes were observed in the thymus of control rats. In the diabetic rats, however, numbers of mitotic lymphocytes decreased to ~57% of controls, and cell division abnormalities were observed. Additionally, diabetic rats showed degeneration in the structure of the thymus including trabecular thickening, accumulation of lipid vacuoles, heterochromatic nuclei and loss of mitochondrial cristae. Degradation of medullar and cortical integrity was also detected. In the vitamin C-treated STZ-diabetic group, the structure of the thymus and mitotic activity of the lymphocytes were similar to the control group. These results suggest that vitamin C protects the thymus against injury caused by diabetes and restores thymocyte mitotic activity.

Description: We investigated the stability of Pd/ZnO polar interfaces formed by internal oxidation of Pd–Zn alloys by using high-resolution transmission electron microscopy, electron energy-loss spectroscopy and convergent-beam electron diffraction. At 1273 K, a $$(111{)}_{\mathrm{Pd}}/(000\overline{2}{)}_{\mathrm{ZnO}}$$ polar interface defaceted and transformed into a curved interface, while another (111) Pd /(0002) ZnO polar interface retained its flatness. The $$(111{)}_{\mathrm{Pd}}/(000\overline{2}{)}_{\mathrm{ZnO}}$$ polar interface lost some stability over non-polar interfaces at 1273 K, while the (111) Pd /(0002) ZnO polar interface remained stable.

Description: Transmission electron microscope (TEM) observation of light metal hydrides is complicated by the instability of these materials under electron irradiation. In this study, the electron kinetic energy dependences of the interactions of incident electrons with lithium, sodium and magnesium hydrides, as well as the constituting element effect on the interactions, were theoretically discussed, and electron irradiation damage to these hydrides was examined using in situ TEM. The results indicate that high incident electron kinetic energy helps alleviate the irradiation damage resulting from inelastic or elastic scattering of the incident electrons in the TEM. Therefore, observations and characterizations of these materials would benefit from increased, instead decreased, TEM operating voltage.

Description: Synaptic plasticity is the process by which long-lasting changes take place at synaptic connections. The phenomenon itself is complex and can involve many levels of organization. Some authors separate forms into adaptations that have positive or negative consequences for the individual. It has been hypothesized that an increase in the number of synapses may represent a structural basis for the enduring expression of synaptic plasticity during some events that involve memory and learning; also, it has been suggested that perforated synapses increase in number after some diseases and experimental situations. The aim of this study was to analyze whether dopamine depletion induces changes in the synaptology of the corpus striatum of rats after the unilateral injection of 6-OHDA. The findings suggest that after the lesion, both contralateral and ipsilateral striata exhibit an increased length of the synaptic ending in ipsilateral (since third day) and contralateral striatum (since Day 20), loss of axospinous synapses in ipsilateral striatum and a significant increment in the number of perforated synapses, suggesting brain plasticity that might be deleterious for the spines, because this type of synaptic contacts are presumably excitatory, and in the absence of the modulatory effects of dopamine, the neuron could die through excitotoxic mechanisms. Thus, we can conclude that the presence of perforated synapses after striatal dopamine depletion might be a form of maladaptive synaptic plasticity.

Description: Unlike in vitro protein dynamics, intracellular protein dynamics are intricately regulated by protein–protein interactions or interactions between proteins and other cellular components, including nucleic acids, the plasma membrane and the cytoskeleton. Alteration of these dynamics plays a crucial role in physiological phenomena such as gene expression and cell division. Live-cell imaging via microscopy with the inherent properties of fluorescent proteins, i.e. photobleaching and photoconversion, or fluorescence correlation spectroscopy, provides insight into the movement of proteins and their interactions with cellular components. This article reviews techniques based on photo-induced changes in the physicochemical properties of fluorescent proteins to measure protein dynamics inside living cells, and it also discusses the strengths and weaknesses of these techniques.

Description: We have developed a method of atomic force microscopy (AFM)-assisted scanning tunneling spectroscopy (STS) under ambient conditions. An AFM function is used for rapid access to a selected position prior to performing STS. The AFM feedback is further used to suppress vertical thermal drift of the tip–sample distance during spectroscopy, enabling flexible and stable spectroscopy measurements at room temperature.

Description: To elucidate high-temperature plastic deformation (creep) mechanism in materials, it is essential to observe dislocation motion under tensile loading. There are many reports on in situ transmission electron microscopy (TEM) observations in the literature; however, the relationship between the dislocation motion and shear stress in 9Cr steel is still not clear. In this study, in order to evaluate this relationship quantitatively, in situ TEM observations were carried out in conjunction with finite element method (FEM) analysis. A tensile test sample was strained at an elevated temperature (903 K) inside a transmission electron microscope, and the stress distribution in the strained sample was analyzed by FEM. The dislocation behavior was clearly found to depend on the shear stress. At a shear stress of 66 MPa, both the dislocation velocity and mobile dislocation density were low. However, a high shear stress level of 95 MPa caused a noticeable increase in the dislocation velocity and mobile dislocation density. Furthermore, in this article, we discuss the dependence of the dislocation behavior on stress. The results presented here also indicate that the relationship between the microstructure and the strength of materials can be revealed by the methods used in this work.

Description: A dual-axis 360° rotation specimen holder was developed for use in reconstructing the three-dimensional (3D) distribution of a magnetic field using a combination of electron holography and tomography. Pillar-shaped specimens are used to obtain accurate reconstruction without a missing angle. The holder's rotation rod can be turned 〉360°; the pillar is set ±45° to the azimuth for both x - and y -axis rotation. Two rotation series of holograms in individual axes are recorded for vector field tomography. The two vector components of the magnetic field are reconstructed directly from the two series of holograms, and the remaining component is calculated using Maxwell's equation, div B = 0. As a result, all 3D magnetic fields are reconstructed.

Description: A new type of electrochemical cell was developed for in situ transmission electron microscopy observation that enables the electrode materials to be conveniently changed. The electrochemical cell was used to observe the electrochemical growth or dissolution of copper islands on a gold film with simultaneous cyclic voltammetry measurements. The copper islands could be explained by three-dimensional growing model.

Description: Aerosol-assisted CVD synthesis, characterisation and gas-sensing application of gold-functionalised tungsten oxide Journal of Sensors and Sensor Systems, 3, 325-330, 2014 Author(s): F. Di Maggio, M. Ling, A. Tsang, J. Covington, J. Saffell, and C. Blackman Tungsten oxide nanoneedles (NNs) functionalised with gold nanoparticles (NPs) have been integrated with alumina gas-sensor platforms using a simple and effective co-deposition method via aerosol-assisted chemical vapour deposition (AACVD) utilising a novel gold precursor, (NH 4 )AuCl 4 . The gas-sensing results show that gold NP functionalisation of tungsten oxide NNs improves the sensitivity of response to ethanol, with sensitivity increasing and response time decreasing with increasing amount of gold.

Description: Objectifying user attention and emotion evoked by relevant perceived product components Journal of Sensors and Sensor Systems, 3, 315-324, 2014 Author(s): R. Schmitt, M. Köhler, J. V. Durá, and J. Diaz-Pineda A company's aim is to develop products that engage user attention and evoke positive emotions. Customers base their emotional evaluation on product components that are relevant for their perception. This paper presents findings of both identifying relevant product components and measuring emotions evoked by relevant perceived product components. To validate results, the comparison with self-reporting methods identifies similarities and differences between explicit expressed and implicit recorded customer requirements. On the one hand, eye tracking is applied to deduce the attention provoked by perceived product components. In order to link the product strategy with product components, the paper presents results considering the fact that the gaze track is affected by current thoughts. (Köhler et al., 2013, 2014a, b; Köhler and Schmitt, 2012) On the other hand, since self-reporting tools are only useful for obtaining information about the conscious part of customers' emotions, there is a need for measurement methods that measure the changes in physiological signals (bio-signals). Arousal is similar to emotional intensity and is related to the galvanic skin response. Positive or negative emotions are defined by the valence that is measured by facial electromyography. Findings are presented that relate changes in bio-signals on the aesthetical design to the global product impression as well as to emotions and, subsequently, linking changes in physiological signals to the evaluation of semantic concepts and design parameters. The presented approach provides conclusions and valid information about products as well as product components that provoke certain emotions and about product components linked to a certain product concept, which could be part of a product strategy. Consequently, hard facts and special design rules for emotional product design can be deduced.

Description: Challenges for Wireless Mesh Networks to provide reliable carrier-grade services Advances in Radio Science, 9, 377-382, 2011 Author(s): D. von Hugo and N. Bayer Provision of mobile and wireless services today within a competitive environment and driven by a huge amount of steadily emerging new services and applications is both challenge and chance for radio network operators. Deployment and operation of an infrastructure for mobile and wireless broadband connectivity generally requires planning effort and large investments. A promising approach to reduce expenses for radio access networking is offered by Wireless Mesh Networks (WMNs). Here traditional dedicated backhaul connections to each access point are replaced by wireless multi-hop links between neighbouring access nodes and few gateways to the backbone employing standard radio technology. Such a solution provides at the same time high flexibility in both deployment and the amount of offered capacity and shall reduce overall expenses. On the other hand currently available mesh solutions do not provide carrier grade service quality and reliability and often fail to cope with high traffic load. EU project CARMEN (CARrier grade MEsh Networks) was initiated to incorporate different heterogeneous technologies and new protocols to allow for reliable transmission over "best effort" radio channels, to support a reliable mobility and network management, self-configuration and dynamic resource usage, and thus to offer a permanent or temporary broadband access at high cost efficiency. The contribution provides an overview on preliminary project results with focus on main technical challenges from a research and implementation point of view. Especially impact of mesh topology on the overall system performance in terms of throughput and connection reliability and aspects of a dedicated hybrid mobility management solution will be discussed.

Description: Meteor radar measurements of mean winds and tides over Collm (51.3° N, 13° E) and comparison with LF drift measurements 2005–2007 Advances in Radio Science, 9, 335-341, 2011 Author(s): C. Jacobi An all-sky VHF meteor radar (MR) has been continuously operated at Collm (51.3° N, 13° E) since summer 2004. The radar measures meteor parameters, diffusion coefficients, and horizontal winds in the mesopause region. There exists a temporal overlap of the MR wind measurements with co-located low-frequency (LF) ionospheric drift measurements until 2007. Comparison of MR and LF semidiurnal tidal phases allows to empirically determine the virtual height overestimation of LF reflection heights due to the group retardation of LF waves. LF reference heights have to be reduced by up to 20 km to match real heights. Correction of LF heights for group retardation allows to determine the wind underestimation by the LF method compared with meteor radar measurements and opens the possibility to continue long-term trend analysis using mesosphere/lower thermosphere winds.

Description: IP-based mobility management for heterogeneous wireless access Advances in Radio Science, 10, 319-325, 2012 Author(s): H. J. Einsiedler and D. von Hugo Future high quality communication services will be offered in an integrated or converged network infrastructure maintaining both fixed wireless and mobile access via multi-mode user terminals. A support of various scenarios of user and/or terminal mobility within a common IP-based infrastructure requires intelligently designed control protocols. A major challenge is to provide seamless (i.e. lossless and low delay) handover between different radio cells and operator domains to enable continuation of unicast and multicast sessions while using network resources most efficiently. IETF (Internet Engineering Task Force) is specifying related IP mobility management protocols to be applicable also to a flat architecture as envisaged by Next Generation (Mobile) Networks (NGNs/NGMNs). The contribution will describe operator requirements towards such an approach. Both single-domain and multi-domain scenarios will be discussed based on federation ideas. Already existing solutions are taken into consideration and application of solution proposals towards a Distributed Mobility Management (DMM) currently under evaluation within IETF will be outlined.

Description: STF Optimierung von single-bit CT ΣΔ Modulatoren basierend auf skalierten Filterkoeffizienten Advances in Radio Science, 10, 313-317, 2012 Author(s): C. Widemann, C. Zorn, T. Brückner, M. Ortmanns, and W. Mathis Die vorliegende Arbeit beschäftigt sich mit dem Signalübertragungsverhalten von single-bit continuous-time (CT) ΣΔ Modulatoren. Dabei liegt der Fokus der Untersuchung auf dem Peaking der Signaltransferfunktion (STF). Dieser Effekt kann die Performance und die Stabilität des Gesamtsystems negativ beeinflussen, da bei auftretendem STF-Peaking Signale außerhalb des Signalbands verstärkt werden. In dieser Arbeit wird ein neuer Ansatz zur Reduktion des Peakings vorgestellt, der auf der Optimierung der Systemdynamik basiert. Dabei werden die Filterkoeffizienten des Modulators systematisch angepasst. Anhand eines Beispielsystems wird gezeigt, dass der Ansatz genutzt werden kann, um das Übertragungsverhalten des Modulators abhängig vom Ausgangssystem zu verändern. So kann entweder die Systemsperformance verbessert werden, ohne Peaking in der STF zu erzeugen, oder das STF-Peaking reduziert werden, ohne die Systemperformance stark zu beeinflussen.

Description: Differential Algebraic Equations of MOS Circuits and Jump Behavior Advances in Radio Science, 10, 327-332, 2012 Author(s): P. Sarangapani, T. Thiessen, and W. Mathis Many nonlinear electronic circuits showing fast switching behavior exhibit jump effects which occurs when the state space of the electronic system contains a fold. This leads to difficulties during the simulation of these systems with standard circuit simulators. A method to overcome these problems is by regularization, where parasitic inductors and capacitors are added at the suitable locations. However, the transient solution will not be reliable if this regularization is not done in accordance with Tikhonov's Theorem. A geometric approach is taken to overcome these problems by explicitly computing the state space and jump points of the circuit. Until now, work has been done in analyzing example circuits exhibiting this behavior for BJT transistors. In this work we apply these methods to MOS circuits (Schmitt trigger, flip flop and multivibrator) and present the numerical results. To analyze the circuits we use the EKV drain current model as equivalent circuit model for the MOS transistors.

Description: The dry weight of tendon tissue is accounted for mainly by collagen fibers. Accordingly, the tendon-healing process primarily involves repair of collagen fibers. During the remodeling phase of tendon healing, newly proliferating collagen fibers are transformed into a mature repaired tendon. Despite the importance of this phenomenon, the details of fibrous rebuilding have not been reported previously. The aim of this study was to visualize the ultrastructural changes and to obtain a clear understanding of the reorganization of the collagen fibers in the tendon repair site, using rat Achilles tendons. We used scanning electron microscopy (SEM) with cell maceration as the main method of analysis. Pretreatment with cell maceration removed the cellular components successfully. This allowed precise visualization of each collagen fiber and the three-dimensional network of the fibers. This study was the first to apply the cell-maceration/SEM method to observe tendon tissue. Seven days after surgery, new collagen fibers grew extensively in the repair site in a random arrangement. Fourteen days after surgery, the collagen fibers began to form an axial arrangement. Near the tendon stump, this change progressed from the outer layer to the core region. On the other hand, in the middle of the repair site, it progressed from the core to the outer layer. Change in the axial arrangement of collagen fibers contributes to the connection between the repair site and the tendon stump and to the separation of the repair site from the paratenon.

Description: We have used transient electron-beam-induced current (EBIC) to map minority carrier lifetime distributions in multicrystalline Silicon (mc-Si). In this technique, the electron beam from a scanning transmission electron microscope was on–off modulated while the sample was scanned. The resulting transient EBIC was analyzed to form a lifetime map. An analytical function was introduced as part of the analysis in determining this map. We have verified this approach using numerical simulations and have reproduced a lifetime map for an mc-Si wafer.

Description: We study in situ behavior of platinum single atoms on amorphous carbon (a-carbon) using a spherical aberration-corrected transmission electron microscope (AC-TEM). Diffusion of single atoms, bi-atoms, clusters (〈1 nm) and nanoparticles (〈3 nm) was recorded in the same image with a time resolution of 1 s, and such diffusion matches the expected mechanism of Ostwald ripening, which was seen on these samples. In situ AC-TEM shows promise for dynamical observation of single atom diffusion, which is important for understanding nanosized catalysts and ceramic sintering processes. We apply in situ AC-TEM to image platinum (Pt) nanoparticles on a-carbon, which is a model catalyst system for the real Pt electrode catalysts using alloys and core–shell structures supported on carbon/oxide composite materials in the proton exchange membrane fuel cell.

Description: The oviduct is an important organ for successful mammalian reproduction. In this work, human oviducts were inseminated and their explants analyzed using scanning electron microscopy in order to study, at a finer ultrastructual level, the interaction between spermatozoon and oviduct in vitro . Results show unequivocally a spermatozoon tightly attached through the acrosomal region of its head to several cilia of the human tubal epithelial cells. This finding proves that spermatozoa do indeed adhere to the endosalpinx, a fact of utmost relevance for the physiology of the reproductive process, since it supports the idea of a spermatozoa reservoir being formed in the oviduct, which is also briefly discussed.

Description: Mitochondria in all eukaryotes are essential organelles responsible for adenosine triphosphate synthesis, calcium homeostasis and steroidogenesis. Because the structure and distribution of mitochondria are highly diverse depending on their function and cellular conditions, it is important to develop a rapid and accurate method to assess their morphology. In this study, we visualize whole mitochondria in cultured cells using high-voltage electron microscopy (HVEM). Compared with conventional transmission electron microscopic approaches, the present method does not require thin sectioning and thus requires less time for image acquisition and processing. Furthermore, compared with fluorescence-based light microscopic approaches, our method provides more accurate size information. Thus, we propose that HVEM is a useful tool for rapid and accurate analysis of mitochondrial morphology and distribution in a cell.

Description: Scanning electron microscopy (SEM) using osmium-maceration methods has been used for analyzing the three-dimensional structure of cell organelles in tissue samples, but it has been quite difficult to observe free and cultured cells with this technique. The present study was performed to develop a method that can be applied to free and cultured cells for SEM studies of intracellular structures after osmium maceration. The method was also applied to light microscopy (LM) and to transmission electron microscopy (TEM). HeLa cells and human leukocytes were fixed with a mixture of 0.5% paraformaldehyde and 0.5% glutaraldehyde followed by an additional fixation with 1% osmium tetroxide. These cells were embedded in low-melting-point agarose. A temperature-responsive dish was also used for collection of cultured cells before embedding. For LM and TEM, the cell-embedded agarose was further embedded in epoxy resin, and semi- and ultrathin sections were examined conventionally. For SEM, the agarose was freeze-fractured in 50% dimethyl sulfoxide, processed for osmium maceration and observed in a high-resolution SEM. Low-melting-point agarose was useful as an embedding medium for SEM, because it was well preserved during prolonged osmication for SEM. Thus, the fine structure of cell organelles was clearly analyzed by SEM after osmium-maceration treatment. These SEM images could also be compared with those of LM and TEM of the agarose-embedded tissues.

Description: The three-dimensional spin structure of the magnetic vortex of FeSiB, an amorphous soft magnetic material, was investigated by holography observation and computer simulation. Magnetization distribution in the neighborhood of the vortex center was estimated from the phase distribution obtained by holography observation. To confirm this magnetization distribution, sample-tilting experiments were performed: when the sample was tilted with respect to the electron beam direction, the phase-image center was found to shift along the tilting axis. Finite-element computer simulation was carried out to estimate the amount of shifts of the phase-image center in the sample tilting from the experimental magnetization distributions in the no sample-tilting conditions. We found that the simulated shifts of the phase-image center were in good agreement with those in the sample-tilting experiment, thus confirming the magnetization distribution near the vortex center obtained by holography observation.

Description: In this study, based on a comprehensive numerical simulation of self-consistent charging, we investigate the formation, evolution and influencing factors of space charge distributions for a grounded insulating thin film of SiO 2 negatively charged by a keV non-penetrating focused electron beam. The simulated space charge presents first positive distributions and then negative ones along both the radial and depth directions because of the difference between electron and hole transports. The variations in distribution occur within a range of the minimum potential acting as a potential barrier for carrier transport. The negative space charge is distributed more widely and deeply, though its peak value in density is usually lower than that of the positive one. Electrons trapped outside the minimum potential range dominate the strength of negative charging. With the increase in potential barrier and the occurrence of leakage current, the space charge eventually reaches equilibrium and exhibits an approximately one-dimensional axial distribution outside the minimum potential range. Distribution features of the space charge density in the equilibrium state correlate with the film and beam parameters via transients of the leakage current. These results and analyses provide new insights into the negative charging effects involved in various electron-beam-based surface microscopic methods, analyses and fabrication techniques.

Description: This study was designed to elucidate details of the structure and formation process of the alternate lamellar pattern known to exist in lamellar bone. For this purpose, we examined basic internal lamellae in femurs of young rats by transmission and scanning electron microscopy, the latter employing two different macerations with NaOH at concentrations of 10 and 24%. Observations after the maceration with 10% NaOH showed that the regular and periodic rotation of collagen fibrils caused an alternation between two types of lamellae: one consisting of transversely and nearly transversely cut fibrils, and the other consisting of longitudinally and nearly longitudinally cut fibrils. This finding confirms the consistency of the twisted plywood model. The maceration method with 24% NaOH removed bone components other than cells, thus allowing for three-dimensional observations of osteoblast morphology. Osteoblasts extended finger-like processes paralleling the inner bone surface, and grouped in such a way that, within a group, the processes arranged in a similar direction. Transmission electron microscopy showed that newly deposited fibrils were arranged alongside these processes. For the formation of the alternating pattern, our findings suggest that: (1) osteoblasts control the collagen fibril arrangement through their finger-like process position; (2) osteoblasts behave similarly within a group; (3) osteoblasts move their processes synchronously and periodically to promote alternating different fibril orientation; and (4) this dynamic sequential deposition of fibrils results in the alternate lamellar (or twisted plywood) pattern.

Description: Three-dimensional (3D) reconstruction experiments were carried out by observing high-resolution 3D electrostatic potential distributions of Pt nanoparticles using off-axis electron holographic tomography. These Pt nanoparticles were mounted on the surfaces of amorphous silicon pillars. In order to realize high-resolution observation, we developed a mechanically stable 3D specimen holder with small specimen drifts and vibrations. From the 3D electrostatic potential distribution data of Pt nanoparticles (2.0 nm in diameter), we obtained the resolution of 1.5 nm.

Description: Brain function is based on proper connectivity between neuronal cells. In the developing brain, neurons extend axons and form synaptic connections with appropriate postsynaptic neurons. Molecular mechanisms underlying establishment of proper synaptic connections are one of the most important topics in the field of developmental neurobiology. Dynamics of synaptic structure and local recruitment of synaptic molecules can be studied by live-cell imaging of neurons expressing fluorescent probes of synaptic molecules. In this review, examples of live-cell fluorescence imaging are presented and their contributions to our understanding about the molecular mechanisms of synapse formation and remodeling are discussed. Imaging of synaptic proteins in living neurons revealed rapid formation of individual synapses within hours and extensive remodeling of synaptic connections. Different types of neurons express unique protrusions from dendrites and axons, which play important roles in synapse formation and maturation. Rapid formation of synaptic structure is associated with continual assembly and disassembly of synaptic scaffolding proteins, which are essential building blocks of the presynaptic active zone and the postsynaptic density (PSD). Quantitative analyses of PSD scaffolding proteins further confirmed their essential roles in maintenance of the synaptic structure. These examples clearly indicate that fluorescence-based live-cell imaging is an indispensable technique in the research on synapse development and its impact will further increase in combination with development of new light microscopic techniques in the future.

Description: High-speed atomic force microscopy (HS-AFM) has been developed as a nano-dynamics visualization technique. This microscopy permits direct observation of structure dynamics and dynamic processes of biological molecules in physiological solutions, at a subsecond to sub-100 ms temporal resolution and an ~2 nm lateral and a 0.1 nm vertical resolution. Importantly, tip–sample interactions do not disturb the biomolecules' functions. Various functioning proteins including myosin V walking on an actin filament and bacteriorhodopsin responding to light have been successfully visualized with HS-AFM. In the quest for understanding the functional mechanisms of proteins, inferences no longer have to be made from static snapshots of molecular structures and dynamic behavior of optical markers attached to proteins. High-resolution molecular movies obtained from HS-AFM observations reveal the details of molecules' dynamic behavior in action, without the need for intricate analyses and interpretations. In this review, I first describe the fundamentals behind the achieved high imaging rate and low invasiveness to samples, and then highlight recent imaging studies. Finally, future studies are briefly described.

Description: This is a personal history of my structural studies of icosahedral viruses that evolved from crystallographic studies, to hybrid methods with electron cryo-microscopy and image reconstruction (cryoEM) and then developed further by incorporating a variety of physical methods to augment the high resolution crystallographic studies. It is not meant to be comprehensive, even for my own work, but hopefully provides some perspective on the growth of our understanding of these remarkable biologic assemblies. The goal is to provide a historical perspective for those new to the field and to emphasize the limitations of any one method, even those that provide atomic resolution information about viruses.

Description: Biological processes occur on a wide range of spatial and temporal scales: from femtoseconds to hours and from angstroms to meters. Many new biological insights can be expected from a better understanding of the processes that occur on these very fast and very small scales. In this regard, new instruments that use fast X-ray or electron pulses are expected to reveal novel mechanistic details for macromolecular protein dynamics. To ensure that any observed conformational change is physiologically relevant and not constrained by 3D crystal packing, it would be preferable for experiments to utilize small protein samples such as single particles or 2D crystals that mimic the target protein's native environment. These samples are not typically amenable to X-ray analysis, but transmission electron microscopy has imaged such sample geometries for over 40 years using both direct imaging and diffraction modes. While conventional transmission electron microscopes (TEM) have visualized biological samples with atomic resolution in an arrested or frozen state, the recent development of the dynamic TEM (DTEM) extends electron microscopy into a dynamic regime using pump-probe imaging. A new second-generation DTEM, which is currently being constructed, has the potential to observe live biological processes with unprecedented spatiotemporal resolution by using pulsed electron packets to probe the sample on micro- and nanosecond timescales. This article reviews the experimental parameters necessary for coupling DTEM with in situ liquid microscopy to enable direct imaging of protein conformational dynamics in a fully hydrated environment and visualize reactions propagating in real time.

Description: In this review, a non-standard application of high-resolution transmission electron microscope (HRTEM), namely the creation of so-called NanoLaboratory for the nanomaterial property studies within its pole piece, is presented. The most modern research trends with respect to nanotube, graphene and nanowire, as well as electrical, mechanical and electromechanical properties are demonstrated. In addition, the unique possibilities of modeling real technological processes inside HRTEM, for example, the performance of Li-ion batteries, are illustrated. The contribution particularly highlights the recent research endeavors of our Tsukuba group in line with all the above-mentioned directions of in situ TEM.

Description: Inherently unstable, detergent-solubilized membrane protein complexes can often not be crystallized. For complexes that have a mass of 〉300 kDa, cryo-electron microscopy (EM) allows their three-dimensional (3D) structure to be assessed to a resolution that makes secondary structure elements visible in the best case. However, many interesting complexes exist whose mass is below 300 kDa and thus need alternative approaches. Two methods are reviewed: (i) Mass measurement in a scanning transmission electron microscope, which has provided important information on the stoichiometry of membrane protein complexes. This technique is applicable to particulate, filamentous and sheet-like structures. (ii) 3D-EM of negatively stained samples, which determines the molecular envelope of small membrane protein complexes. Staining and dehydration artifacts may corrupt the quality of the 3D map. Staining conditions thus need to be optimized. 3D maps of plant aquaporin SoPIP2;1 tetramers solubilized in different detergents illustrate that the flattening artifact can be partially prevented and that the detergent itself contributes significantly. Another example discussed is the complex of G protein-coupled receptor rhodopsin with its cognate G protein transducin.

Description: In commemoration of the 20th anniversary of the molecular cloning of the gene for the green fluorescent protein from the jellyfish Aequorea victoria , I would like to reflect on the development of new fluorescence imaging technology in the last two decades. As this technology has become increasingly diversified, it has become more and more of a challenge to come up with a comprehensive and exhaustive review of it. Here I will focus on optogenetics and large-scale, three-dimensional reconstruction. Those two technological innovations have been achieved in the neuroscience community owing to the combined efforts of molecular biologists and light microscopists. In addition, modern fluorescence imaging has indeed improved our understanding of the spatiotemporal regulation of fundamental biological functions at cellular level. As an example, I will introduce some findings we made regarding the movement of biomolecules across the nuclear membrane. The above-mentioned imaging approaches are possible today but were impossible two decades ago.

Description: The origins and the recent accomplishments of aberration correction in scanning transmission electron microscopy (STEM) are reviewed. It is remembered that the successful correction of imaging aberrations of round lenses owes much to the successful correction of spectrum aberrations achieved in electron energy loss spectrometers 2–3 decades earlier. Two noteworthy examples of the types of STEM investigation that aberration correction has made possible are shown: imaging of single-atom impurities in graphene and analyzing atomic bonding of single atoms by electron energy loss spectroscopy (EELS). Looking towards the future, a new all-magnetic monochromator is described. The monochromator uses several of the principles pioneered in round lens aberration correction, and it employs stabilization schemes that make it immune to variations in the high voltage of the microscope and in the monochromator main prism current. Tests of the monochromator carried out at 60 keV have demonstrated energy resolution as good as 12 meV and monochromated probe size of ~1.2 Å. These results were obtained in separate experiments, but they indicate that the instrument can perform imaging and EELS with an atom-sized probe 〈30 meV wide in energy, and that an improvement in energy resolution to 10 meV and beyond should be possible in the future.

Description: Novel spherical aberration (Cs) and chromatic aberration (Cc) correctors, which correct aberrations using a new principle, were developed. The asymmetric Cs correctors were designed for use in the probe- and image-forming systems at 300 kV to diminish undesired parasitic aberrations. The correctors composed of non-equivalent multipoles connecting with a demagnifying transfer doublet in the system. The axial aberrations were corrected well up to the fifth order except 6-fold astigmatism ( A 6 ) experimentally. Next, we developed superior Cs correctors for probe- and image-forming systems of low voltage microscope that uses triple dodecapoles to correct 6-fold astigmatism ( A 6 ). An important feature of this system is the rotation of the 3-fold astigmatism azimuth at the second dodecapole. The optimum rotation of the three hexapole fields for the compensation of A 6 was derived from theoretical calculations. The experimental results confirmed the compensation of A 6 and the third-order Cs. Finally, a unique Cc corrector, which utilized the concave lens effect formed by a long quadrupole field, was designed. The performance of the Cc corrector was investigated using a 30-kV transmission electron microscope. The results confirmed that Cc correction was achieved.

Description: Theoretical considerations together with simulations of single-particle electron cryomicroscopy images of biological assemblies in ice demonstrate that atomic structures should be obtainable from images of a few thousand asymmetric units, provided the molecular weight of the whole assembly being studied is greater than the minimum needed for accurate position and orientation determination. However, with present methods of specimen preparation and current microscope and detector technologies, many more particles are needed, and the alignment of smaller assemblies is difficult or impossible. Only larger structures, with enough signal to allow good orientation determination and with enough images to allow averaging of many hundreds of thousands or even millions of asymmetric units, have successfully produced high-resolution maps. In this review, we compare the contrast of experimental electron cryomicroscopy images of two smaller molecular assemblies, namely apoferritin and beta-galactosidase, with that expected from perfect simulated images calculated from their known X-ray structures. We show that the contrast and signal-to-noise ratio of experimental images still require significant improvement before it will be possible to realize the full potential of single-particle electron cryomicroscopy. In particular, although reasonably good orientations can be obtained for beta-galactosidase, we have been unable to obtain reliable orientation determination from experimental images of apoferritin. Simulations suggest that at least 2-fold improvement of the contrast in experimental images at ~10 Å resolution is needed and should be possible.

Description: This paper reviews diverse capabilities offered by modern electron microscopy techniques in studying fine structures of nanoporous crystals such as zeolites, silica mesoporous crystals, metal organic frameworks and yolk-shell materials. For the case of silica mesoporous crystals, new approaches that have been developed recently to determine the three-dimensionally periodic average structure, e.g., through self-consistent analysis of electron microscope images or through consideration of accidental extinctions, are presented. Various structural deviations in nanoporous materials from their average structures including intergrowth, surface termination, incommensurate modulation, quasicrystal and defects are demonstrated. Ibidem observations of the scanning electron microscope and atomic force microscope give information about the zeolite-crystal-growth mechanism, and an energy for unstitching a building-unit from a crystal surface is directly observed by an anatomic force microscope. It is argued how these observations lead to a deeper understanding of the materials.

Description: Environmental transmission electron microscopy and ultra-high resolution electron microscopic observation using aberration correctors have recently emerged as topics of great interest. The former method is an extension of the so-called in situ electron microscopy that has been performed since the 1970s. Current research in this area has been focusing on dynamic observation with atomic resolution under gaseous atmospheres and in liquids. Since 2007, Nagoya University has been developing a new 1-MV high voltage (scanning) transmission electron microscope that can be used to observe nanomaterials under conditions that include the presence of gases, liquids and illuminating lights, and it can be also used to perform mechanical operations to nanometre-sized areas as well as electron tomography and elemental analysis by electron energy loss spectroscopy. The new instrument has been used to image and analyse various types of samples including biological ones.

Description: Spin-polarized scanning electron microscopy (spin-SEM) is a magnetic domain observation method. In spin-SEM, polarization of secondary electrons emitted from a sample in a scanning electron microscope is detected by a spin detector and used as a signal for forming an image. The characteristics of spin-SEM are detection of all three magnetization vector components, which leads to the detection of the magnetization vector direction, high spatial resolution of around 3 nm and applicability to samples with rough or even 3D surfaces. Spin-SEM combined with other imaging methods using an electron probe beam such as scanning Auger electron microscopy for imaging element distribution and electron backscattering diffraction microscopy for imaging crystal direction distribution provides additional information that is important to study the magnetism. Spin-SEM with these excellent characteristics has a broad range of applications from basic research to applied research and developments in various industries.

Description: We have compiled our recent in-situ quantitative environmental transmission electron microscopy (ETEM) studies on typical gold nanoparticulate catalysts for the low-temperature oxidation of CO to describe the issues surrounding the application of ETEM, with a special regard to catalyst chemistry. Thanks to the recent development of high-resolution environmental transmission electron microscopes that can work robustly to accumulate observation data in controlled environments, we can deal with the electron irradiation effects and heterogeneity of real catalysts. We established a structural evolution diagram that summarizes the structure of catalysts under electron irradiation as a function of the electron current density and the electron dose, D . By extrapolating to = 0, D = 0, we could deduce the intrinsic catalysis structure (without electron irradiation) in various environments, including reaction environments. By numerically and statistically analyzing a substantial number of ETEM images of gold nanoparticles, we established a morphology phase diagram that summarizes how the majority of gold nanoparticles change their morphology systematically as a function of the partial pressures of CO and O 2 . Similar diagrams will be helpful in elucidating the phenomena that directly correlate with the catalytic activity determined from ETEM observations. Using these quantitative analyses, we could analyze Cs-corrected ETEM images of the catalysts. The surfaces of gold nanoparticles were structurally reconstructed under reaction conditions, via interactions with CO molecules. CO molecules were observed on the surfaces of catalysts under reaction conditions using high-resolution ETEM. Finally, we discuss the potential of environmental transmission electron microscopy for quantitative in-situ microscopy at the atomic scale.

Description: Optical sectioning using scanning confocal electron microscopy (SCEM) is a new three-dimensional (3D) imaging technique which promises improved depth resolution, particularly for laterally extended objects. Using a stage-scanning system to move the specimen in three dimensions, two-dimensional (2D) images sliced from any plane in XYZ space can be obtained in shorter acquisition times than those required for conventional electron tomography. In this paper, a double aberration-corrected SCEM used in annular dark-field mode was used to observe the 3D structure of SiO 2 hollow spheres fabricated by a carbon template method. The double-shell structure of the sample was clearly reflected in both XY - and XZ -sliced images. However, elongation along the optical axis was still evident in the XZ -sliced images even when double aberration correctors were used. Application of a deconvolution technique to the experimental XZ -sliced images reduced the elongated shell thicknesses of the SiO 2 sphere by 40–50% and the selectivity of information at a certain sample depth was also enhanced. Subsequently, 3D reconstruction by stacking the deconvoluted slice images restored the spherical surface of a SiO 2 sphere.

Description: We report the production of electron vortex beams carrying large orbital angular momentum (OAM) using micro-fabricated spiral zone plates. A series of the spherical waves, focussing onto different positions along the propagating direction of the electron beam, were observed. The n th order vortex beam has an OAM n times larger than that of the first-order vortex beam. We observed an electron vortex with an OAM up to in a high-order diffracted wave. A linear dependence of the diameter of the vortex beam on the OAM was observed, being consistent to numerical simulations.

Description: Alignment of projection images in tomographic reconstruction is a critical process that governs the quality of the reconstructed three-dimensional (3D) image. The most popular alignment method is the marker-based alignment, which typically uses colloidal gold particles added to the specimen (called fiducial markers) to calculate the coordinates of each projection image in the tilt series. This method, however, is not effective when each image contains only a small number of fiducial markers. Therefore, of all the parameters required for alignment, we focussed on the tilt angle and attempted to gage it directly in order to examine whether the acquired angle is accurate enough to perform tomographic reconstruction. We showed that the tilt angle measured using a commercially available capacitive liquid-based inclinometer is more precise than the reading from the monitor of the electron microscope and that it can lead to 3D reconstructions of quality similar to those obtained by the marker-based alignment method.

Description: This study was designed to evaluate liver disease in neonatal piglets with surgical short bowel syndrome causing intestinal failure with partial parenteral nutrition dependence. The short bowel piglets had 75% surgical resection of distal small intestine, including all ileum and cecum, and were compared with sham controls, without resection, and to healthy sow-reared controls. After 18 days of combined parenteral and enteral nutrition in short bowel and sham piglets, liver tissue was collected for quantitative and semi-quantitative histological and ultrastructural evaluation. The short bowel piglets developed biochemical and histological cholestasis, not observed in sham and control piglets. Ultrastructural examination revealed bile canaliculus dilation with bile plugging, microvillus flattening and disappearance, but without abnormalities of the pericanalicular zone. Interestingly, these data are similar to bile canaliculus changes seen in human neonates with IFALD supporting an initial consideration of this model to elucidate the pathogenesis of IFALD.

Description: The filamentous fungus Trichoderma reesei which is known to secrete high amounts of cellulolytic enzymes was found to produce a massive amount of fibrous material at the outer surface of the cell wall as observed by ultrahigh-resolution low-voltage scanning electron microscopy. Using transmission electron microscopy, the cell wall ornamentation of the hyper-cellulosic mutant PC-3-7 was found to be less massive and much thinner than for QM9414. A significant amount of fibrous material was produced in Avicel-grown cultures that were less abundant in glucose-grown cultures and Avicel was occasionally found entangled within the cell wall-associated fibrous layer.

Description: This report is an extension of the study for structural imaging of 5–6 nm thick β-Si 3 N 4 [0001] crystal with a spherical aberration corrected transmission electron microscope by Zhang and Kaiser [2009. Structure imaging of β-Si 3 N 4 by spherical aberration-corrected high-resolution transmission electron microscopy. Ultramicroscopy 109, 1114–1120]. In this work, a local symmetry breaking with an uneven resolution of dumbbells in the six-membered rings revealed in the reported images in the study of Zhang and Kaiser has been analyzed in detail. It is found that this local asymmetry in the image basically is not relevant to a slight mistilt of the specimen and/or a beam tilt (coma). Rather the certain variation of the tetrahedral bond length of Si–N(4) in the crystal structure is found to be responsible for the uneven resolution with a local structural variation from region to region. This characteristic of the variation is also supposed to give a distorted lattice of apparently 2°–2.5° deviations from the perfect hexagonal unit cell as observed in the reported image in the work of Zhang and Kaiser. It is discussed that this variation may prevail only in a thin specimen with a thickness ranging ~≤5–6 nm. At the same time, it is noted that the average of the bond length variation is close to the fixed length known in a bulk crystal of β-Si 3 N 4 .

Description: Metal contacts are regarded as key elements of nanometer-scale electronics. Since gold contacts show quantized conductance even at room temperature, much effort has been devoted to understand their conductance behavior on the nanoscale. However, gold contacts do not always show quantized conductance steps during their thinning process, the reason for which has been an open question. Thus, it is necessary to investigate the relationship between the atomic structure and conductance of gold contacts. We developed a custom-made scanning tunneling microscope combined with an ultra-high vacuum transmission electron microscope to clarify the structural dependence of conductance quantization in gold contacts. We found that [111] and [001] gold contacts with a bottleneck shape showed a gradual decrease in conductance with elastic elongation and successive conductance jumps with periodic plastic deformation. In contrast, [110] gold contacts had a hexagonal prism shape (termed gold [110] nanowires). In the conductance histogram, peaks appeared nearly in steps of the quantum unit. We found that the prominent peaks corresponded to stable gold nanowires with a regular hexagonal cross-section. Following first-principles calculations, we confirmed that very thin gold [110] nanowires were ballistic conductors. The conductance behavior differed depending on the contact shape.

Description: A highly precise determination of lattice parameters using higher-order Laue zone (HOLZ) reflections observed in nano-beam electron diffraction is presented. The introduction of more than 40 HOLZ reflections, whose positions are corrected by considering the aberration of the electron optics and are determined with an accuracy of 0.04 nm –1 , allows us to achieve a remarkable high precision of a 0.02% error, which is four times higher than the precision without HOLZ reflections.

Description: The leaf surface features of the biomass plant Miscanthus sinensis were investigated by electron and ion beam microscopy. Fully developed leaves were collected from the plant, air-dried and examined by field emission scanning electron microscopy. Stomata and longitudinal stripes were present on both the adaxial and abaxial leaf surfaces. Longitudinally aggregated rodlets surrounded the stomata and formed hollow cylinders or chimney-like structures. With varying lengths up to ~10 μm, the rodlets were curved (〈1 μm in diameter) and almost occluded the stomata. As another type of epicuticular wax, platelets were observed in the vicinity of the stomata. The platelets were arranged into rather radially assembled clusters. As a novel approach for cross sectioning native epicuticular waxes, focused ion beam milling allowed precise in situ cutting of the stomata and epicuticular waxes. The longitudinally aggregated rodlets of the stomatal chimneys of M. sinensis were derived from the stomatal guard cells. These results suggest that the epicuticular waxes of M. sinensis can be categorized as stomatal chimneys ( Strelitzia type) and rosettes (Fabales type). Combined electron and ion beam microscopy can help unravel the ultrastructure and vertical profile of epicuticular waxes in a range of plant taxa.

Description: Three-dimensional (3D) protein structures facilitate the understanding of their biological functions and provide valuable information for developing medicines. Single-particle analysis (SPA) from electron microscopy (EM) is a structure determination method suitable for macromolecules. To achieve a high resolution using combinations of several SPA software packages, ‘workflow’ optimization and comparative evaluation by scoring results are essential. Two-dimensional (2D) averaging is a key step for 3D reconstruction. The integrated convergence-evaluation oriented system (IC-EOS) proposed here provides an effective tool for customizing 2D averaging. This assesses the behavior and characteristics of workflows and evaluates the convergence of iteration steps without human intervention. We chose five base measurements for quantifying convergence: resolution, variance, similarity, shift-distance and rotation-angle. Curve fitting to history graphs scored their stability. We call this score ‘fluctuation’. The number of particle images discarded from the library and the number of classification groups were examined to see their effects on optimization levels and fluctuation of measurements, allowing the IC-EOS to select the most appropriate workflow for the target. A case study using a bacterial sodium channel and a simulation study using GroEL showed that resolution of 2D averaging improved with relatively stricter particle selection. With fewer groups, resolutions of class averages improved, but similarities between class-averages and their constituent particle images degraded. Fluctuation was useful for selecting adequate conditions, even when achieved values alone were not conclusive. The vote method, using fluctuation, was robust against noise and enabled a decision without exhaustive search trials. Thus, the IC-EOS is a step toward full automation of SPA.

Description: We quantitatively analyzed the contrast degradation and blur of 20-nm gold nanoparticles adsorbed on the top of amorphous silicon films of thicknesses of 0.54, 1.09, 1.63 and 2.2 μm in bright-field transmission electron microscope (TEM) images taken at accelerating voltages of 0.5, 1, 2 and 3 MeV. The thickness dependence of the transmission was well explained and consistent with our calculations. The blur function, derived by assuming that the TEM image of a thick specimen can be reproduced by convolving the TEM image of a very thin specimen with it, was found to be expressed by a two-dimensional Lorentzian function. Considering the two characteristics of the Lorentzian function, a sharp peak around the center and a long tail, we concluded that, for TEM observations of thick specimens, the image contrast is degraded predominantly by inelastic scattering and the image is blurred predominantly by multiple elastic scattering.

Description: Immunoelectron microscopy is a powerful method to diagnose viral diseases and to study the distribution of the viral agent within plant cells and tissues. Nevertheless, current protocols for the immunological detection of viral diseases with transmission electron microscopy (TEM) in plants take between 3 and 6 days and are therefore not suited for rapid diagnosis of virus diseases in plants. In this study, we describe a method that allows rapid cytohistochemical detection of tobacco mosaic virus (TMV) in leaves of tobacco plants. With the help of microwave irradiation, sample preparation of the leaves was reduced to 90 min. After sample sectioning, virus particles were stained on the sections by immunogold labelling of the viral coat protein, which took 100 min. After investigation with the TEM, a clear visualization of TMV in tobacco cells was achieved altogether in about half a day. Comparison of gold particle density by image analysis revealed that samples prepared with the help of microwave irradiation yielded significantly higher gold particle density as samples prepared conventionally at room temperature. This study clearly demonstrates that microwave-assisted plant sample preparation in combination with cytohistochemical localization of viral coat protein is well suited for rapid diagnosis of plant virus diseases in altogether about half a day by TEM.

Description: An accurate method using image sharpness to determine the best focusing is proposed for ultra-high-voltage electron microscopy. This method maximizes image sharpness for adjusting the focus. Five images with different defocus values are used to calculate the image sharpness. To obtain the best focus value that produces greatest image sharpness, fitting the quasi-Gaussian function to five image sharpness is a suitable alternative. This method, which maximizes image sharpness, gives better accuracy than the wobbler method for the ultra-high-voltage electron microscope. The focusing area can be selected without moving the field of view, because the focusing area can be selected at almost any area in the image.

Description: The cross-sectioned profiles of differentiating preameloblasts, inner enamel secretory ameloblasts and outer enamel secretory ameloblasts at the distal terminal web were quantitatively compared. First, the angles of each line constituting the sectioned cell polygons were measured, and the patterns of angular distribution histograms were compared. Second, all groups of line angles from one differentiating preameloblast population, two inner enamel secretory ameloblast and one outer enamel secretory ameloblast populations at the distal terminal web were compared statistically by the 2 -test using the multiple comparison method. The results showed that cell shapes between differentiating preameloblasts and inner enamel secretory ameloblasts were similar, but that those between differentiating preameloblasts and outer enamel secretory ameloblasts and between inner enamel secretory ameloblasts and outer enamel secretory ameloblasts were significantly different. Third, F-actin fluorescence microscopy in the distal terminal web was performed and was consistent with the angular distribution. These results suggest that cell shapes of inner enamel secretory ameloblasts and differentiating preameloblasts at the distal terminal web are specialized for sideways cell movement during decussating tooth enamel formation.

Description: Growing a GaN film on a patterned Al 2 O 3 substrate is one of the methods of reducing threading dislocations (TDs), which can significantly deteriorate the performance of GaN-based LEDs. In this study, the microstructural details of the GaN film grown on a cone-shaped patterned Al 2 O 3 substrate were investigated using high-resolution transmission electron microscopy and weak-beam dark-field techniques. Various defects such as misfit dislocations (MDs), recrystallized GaN (R-GaN) islands and nano-voids were observed on the patterned Al 2 O 3 surfaces, i.e. the flat surface (FS), the inclined surface (IS) and the top surface (TS), respectively. Especially, the crystallographic orientation of R-GaN between the GaN film and the inclined Al 2 O 3 substrate was identified as $[\overline 1 2\overline 1 0]_{{\rm GaN}} \hbox{//}[\overline 1 101]_{{\rm R - GaN} \,{\rm on}\,{\rm IS}} \hbox{//}[\overline 1 100]_{ {{\rm Al}} _{\rm 2} {\rm O}_{\rm 3}} $ , $(\overline 1 012)_{{\rm GaN}} \hbox{//}(1\overline 1 02)_{{\rm R - Ga}\,{\rm Non}\,{\rm IS}} \hbox{//}(\overline {11} 26)_{ {{\rm Al}} _{\rm 2} {\rm O}_{\rm 3}} $ . In addition, a rotation by 9° between $(10\overline 1 1)_{{\rm R - GaN}} $ and $(0002)_{{\rm GaN}} $ and between $(10\overline 1 1)_{{\rm R - GaN}} $ and $(0006)_{ {{\rm Al}} _{\rm 2} {\rm O}_{\rm 3}} $ was found to reduce the lattice mismatch between the GaN film and the Al 2 O 3 substrate. Many TDs in the GaN film were observed on the FS and TS of Al 2 O 3 . However, few TDs were observed on the IS. Most of the TDs generated from the FS of Al 2 O 3 were bent to the inclined facet rather than propagating to the GaN surface, resulting in a reduction in the dislocation density. Most of the TDs generated from the TS of Al 2 O 3 were characterized as edge dislocations.

Description: Alzheimer's disease (AD), one of the most prevalent neurodegenerative brain diseases, has been extensively researched for years. However, its synaptic structure, which is a basis for understanding neurodegenerative disorders, has not yet been understood clearly. Defining the structures of neurons and their synaptic connections is the significant goal of brain research. To study synaptic connectivity, three-dimensional (3D) reconstructions of the nervous system are very helpful. In this study, the 3D structure of brain synapses in the Drosophila melanogaster Swedish amyloid precursor protein (APP) mutant, which is characterized by early onset AD, was analyzed using focused ion beam/scanning electron microscopy (FIB/SEM). This technique is one of the most useful for 3D reconstruction, as the process of obtaining serial images is fully automated and thus avoids the problems inherent in hand-operated ultrathin serial sectioning. The 3D images of normal and AD brains reported in this study reveal characteristic features of AD such as appearance of autophagy, abnormal axon formation and increased mitochondrial size. This 3D analysis reveals structural change as a basis for understanding neurodegenerative disorder.

Description: The coupled surface plasmon-polariton (SPP) modes excited in an Al/SiO 2 /Al multilayer structure were analyzed using angle-resolved electron energy-loss spectroscopy (AREELS) with a relativistic electron probe. The dispersion relations for the coupled SPP modes were then directly observed and compared with predicted relations obtained via calculations. Good agreement was noted between the experimental and calculated results. In the multilayer structures, the dispersion relation for the coupled SPP modes was found to be sensitive to the thickness of each film, which could be interpreted qualitatively by the electron energy-loss probability calculated for thin aluminum (Al) films and narrow Al gaps using Kröger's formula. It was demonstrated that significant differences in the excitation probability for SPPs could be observed depending on the coupling modes.