Thermally Controlled Construction of Fe-Nx Active Sites on the Edge of a Graphene Nanoribbon for an Electrocatalytic Oxygen Reduction Reaction.

Pyrolytically prepared iron and nitrogen codoped carbon (Fe/N/C) catalysts are promising nonprecious metal electrocatalysts for the oxygen reduction reaction (ORR) in fuel cell applications. Fabrication of the Fe/N/C catalysts with Fe-Nx active sites having precise structures is now required. We developed a strategy for thermally controlled construction of the Fe-Nx structure in Fe/N/C catalysts by applying a bottom-up synthetic methodology based on a N-doped graphene nanoribbon (N-GNR). The preorganized aromatic rings within the precursors assist graphitization during generation of the N-GNR structure with iron-coordinating sites. The Fe/N/C catalyst prepared from the N-GNR precursor, iron ion, and the carbon support Vulcan XC-72R provides a high onset potential of 0.88 V (vs reversible hydrogen electrode (RHE)) and promotes efficient four-electron ORR. X-ray absorption fine structure (XAFS) and X-ray photoelectron spectroscopy (XPS) studies reveal that the N-GNR precursor induces the formation of iron-coordinating nitrogen species during pyrolysis. The details of the graphitization process of the precursor were further investigated by analyzing the precursors pyrolyzed at various temperatures using MgO particles as a sacrificial template, with the results indicating that the graphitized structure was obtained at 700 °C. The preorganized N-GNR precursors and its pyrolysis conditions for graphitization are found to be important factors for generation of the Fe-Nx active sites along with the N-GNR structure in high-performance Fe/N/C catalysts for the ORR.

Red-Fluorescent Pt Nanoclusters for Detecting and Imaging HER2 in Breast Cancer Cells.

Overexpression of human epidermal growth factor receptor 2 (HER2) is associated with more frequent cancer recurrence and metastasis. Sensitive sensing of HER2 in living breast cancer cells is crucial in the early stages of cancer and to further understand its role in cells. Biomedical imaging has become an indispensable tool in the fields of early cancer diagnosis and therapy. In this study, we designed and synthesized platinum (Pt) nanocluster bionanoprobes with red emission (Ex/Em = 535/630 nm) for fluorescence imaging of HER2. Our Pt nanoclusters, which were synthesized using polyamidoamine (PAMAM) dendrimer and preequilibration, exhibited approximately 1% quantum yield and possessed low cytotoxicity, ultrasmall size, and excellent photostability. Furthermore, combined with ProteinA as an adapter protein, we developed Pt bionanoprobes with minimal nonspecific binding and utilized them as fluorescent probes for highly sensitive optical imaging of HER2 at the cellular level. More importantly, molecular probes with long-wavelength emission have allowed visualization of deep anatomical features because of enhanced tissue penetration and a decrease in background noise from tissue scattering. Our Pt nanoclusters are promising fluorescent probes for biomedical applications.

Research on nanoprocess of non-equilibrium materials by in situ ultra-high voltage electron microscopy.

Ultra-high voltage electron microscopy is useful for research utilizing high-penetration thickness of electron beam, in situ observation, or irradiation effects by the particle characteristics of electrons. In this review, the importance of non-equilibrium materials science research by a combination with irradiation effects and in situ observation is shown, and examples of some research are introduced. For example, crystal-amorphous-crystalline phase transition in intermetallic compounds, non-equilibrium phase transition in pure metallic nanoparticles and nucleation and growth process of electron irradiation-induced crystallization in amorphous nanoparticles will be discussed. Finally, we want to suggest the importance of exploring non-equilibrium materials science based on dynamic structures which has been unexplored.

Athermal Crystal Defect Dynamics in Si Revealed by Cryo-High-Voltage Electron Microscopy.

Low-temperature crystal defect dynamics in Si has been studied by a newly developed cryo-high-voltage electron microscopy. The planar {113} defects of self-interstitial atoms were introduced at 94 K by 1 MeV electron irradiation with damage higher than 0.42 displacements per atom (dpa), unlike past findings. The defects once grew and then shrunk during the observation. We show that the nucleation and the dissociation dynamics of the {113} defects can be attributed to an athermal process, which is deduced from anomalously fast diffusion of self-interstitial atoms at a low temperature.

Quantum de-trapping and transport of heavy defects in tungsten.

The diffusion of defects in crystalline materials1 controls macroscopic behaviour of a wide range of processes, including alloying, precipitation, phase transformation and creep2. In real materials, intrinsic defects are unavoidably bound to static trapping centres such as impurity atoms, meaning that their diffusion is dominated by de-trapping processes. It is generally believed that de-trapping occurs only by thermal activation. Here, we report the direct observation of the quantum de-trapping of defects below around one-third of the Debye temperature. We successfully monitored the de-trapping and migration of self-interstitial atom clusters, strongly trapped by impurity atoms in tungsten, by triggering de-trapping out of equilibrium at cryogenic temperatures, using high-energy electron irradiation and in situ transmission electron microscopy. The quantum-assisted de-trapping leads to low-temperature diffusion rates orders of magnitude higher than a naive classical estimate suggests. Our analysis shows that this phenomenon is generic to any crystalline material.

Empirical determination of transmission attenuation curves in mass-thickness contrast TEM imaging.

The thickness dependence of electron transmission, observed as mass-thickness contrast in transmission electron microscopy (TEM) images, was precisely measured for polystyrene and amorphous carbon. In the early stages of transmission attenuation, a slight increase in the attenuation coefficient was observed, although a constant value is generally expected according to Beer's law. In contrast, as generally known as nonlinear behavior due to multiple scattering, the coefficient decreased during the intermediate stages. In the later stages, an asymptotic behavior in which the transmission approached a constant value was observed. Based on these results, a function containing three parameters was proposed to express the nonlinear transmission attenuation with increasing thickness. Results obtained using this new model and other previously proposed models were compared with experimental data measured over a wide range of conditions: acceleration voltage of 200-3000 kV, objective aperture radius of 0.85-30 nm-1, and thickness of 0.25-10 µm. It was confirmed that our model can well reproduce all of the measurements with a high degree of precision, while the other models were valid only under limited imaging conditions and/or for limited thickness ranges. Thus, a quantitative description of transmission attenuation under normal TEM observation conditions, that is, over a thickness range without physical absorption and a scattering angular range of a few degrees, is finally obtained after more than a century since early studies on ß-rays. Based on a simplified model of multiple scattering, the characteristic behavior of the attenuation curves is intuitively explained.

Ultrafast electron microscopy with relativistic femtosecond electron pulses.

Ultrafast electron microscopy (UEM) with femtosecond temporal resolution is a 'dream machine' that has been long envisioned for the study of ultrafast structural dynamics in materials. For this purpose, we developed a prototype UEM with relativistic femtosecond electron pulses generated by a radio-frequency acceleration-based photoemission gun. TEM images of polystyrene latex particles and gold nanoparticles were observed using approximately 100-fs-long electron pulses with energies of 3.1 MeV. The effect of emittance and the number of pulses on the images were investigated. We demonstrated single-shot imaging with the femtosecond electron pulse at low magnification of approximately 500×.

Development of single nanometer-sized ultrafine oxygen bubbles to overcome the hypoxia-induced resistance to radiation therapy via the suppression of hypoxia-inducible factor­1α.

Radiation therapy can result in severe side-effects, including the development of radiation resistance. The aim of this study was to validate the use of oxygen nanobubble water to overcome resistance to radiation in cancer cell lines via the suppression of the hypoxia-inducible factor 1-α (HIF­1α) subunit. Oxygen nanobubble water was created using a newly developed method to produce nanobubbles in the single-nanometer range with the ΣPM-5 device. The size and concentration of the oxygen nanobubbles in the water was examined using a cryo-transmission electron microscope. The nanobubble size was ranged from 2 to 3 nm, and the concentration of the nanobubbles was calculated at 2x1018 particles/ml. Cell viability and HIF-1α levels were evaluated in EBC­1 lung cancer and MDA­MB­231 breast cancer cells treated with or without the nanobubble water and radiation under normoxic and hypoxic conditions in vitro. The cancer cells grown in oxygen nanobubble-containing media exhibited a clear suppression of hypoxia-induced HIF­1α expression compared to the cells grown in media made with distilled water. Under hypoxic conditions, the EBC­1 and MDA­MB231 cells displayed resistance to radiation compared to the cells cultured under normoxic cells. The use of oxygen nanobubble medium significantly suppressed the hypoxia-induced resistance to radiation compared to the use of normal medium at 2, 6, 10 and 14 Gy doses. Importantly, the use of nanobubble media did not affect the viability and radiation sensitivity of the cancer cell lines, or the non­cancerous cell line, BEAS­2B, under normoxic conditions. This newly created single-nanometer range oxygen nanobubble water, without any additives, may thus prove to be a promising agent which may be used to overcome the hypoxia-induced resistance of cancer cells to radiation via the suppression of HIF-1α.

Probing Crystal Dislocations in a Micrometer-Thick GaN Film by Modern High-Voltage Electron Microscopy.

We report on extreme penetration power of relativistic electrons in a micrometer-thick gallium nitride epitaxial film and its application to probing threading dislocations, which were introduced during crystal growth. Maximum usable thickness of the specimen was quantitatively evaluated using high-voltage transmission electron microscopy (TEM) operating at 1 MV. The width of dislocation images was used as a measure for the evaluation of usable thickness. Superior maximum usable thickness was obtained in scanning transmission electron microscopy (STEM) than in TEM mode; the results were 6.9 µm for STEM and 4.4 µm for TEM. In STEM, dislocations can be imaged with an almost constant width of 15-20 nm in a wide thickness range 1-4 µm. The latest high-voltage STEM is thus useful for observing dislocations in micrometer-thick inorganic materials.

Bimetallic M/N/C catalysts prepared from π-expanded metal salen precursors toward an efficient oxygen reduction reaction.

Nonprecious metal electrocatalysts are being explored as alternatives to platinum-group metal electrocatalysts for the oxygen reduction reaction (ORR) which is required for cathode materials in fuel cells. Herein, we describe a new method for preparing bimetallic nitrogen-containing carbon catalysts with high ORR activity using π-expanded M(salen) precursors. The M/N/C and bimetallic FeM/N/C ORR catalysts were obtained by pyrolysis of a mixture of a carbon support (Vulcan XC-72R) and the metal complex as a precursor. The bimetallic FeCu catalyst prepared from Fe and Cu complexes with the N,N'-bis(2-hydroxy-1-naphthylidene)-1,2-phenylenediamine ligand (2NAPD) is found to have an onset potential of 0.87 V, which is positively shifted by 50 mV from that of the catalyst prepared from the monometallic Fe(2NAPD) complex. The FeCu/N/C catalyst promotes efficient four-electron reduction in the ORR. High-resolution transmission electron microscopy studies reveal that both Fe and Cu metals together with pyridinic nitrogen species are highly dispersed within the carbonaceous structure in FeCu/2NAPD@VC, suggesting that the N-coordinated Fe and Cu sites promote efficient four-electron reduction of O2. This new methodology facilitates design of nonprecious bimetallic carbon catalysts with excellent ORR activity.

Au-Protected Ag Core/Satellite Nanoassemblies for Excellent Extra-/Intracellular Surface-Enhanced Raman Scattering Activity.

Silver nanoparticles (AgNPs) and their assembled nanostructures such as core/satellite nanoassemblies are quite attractive in plasmonic-based applications. However, one biggest drawback of the AgNPs is the poor chemical stability which also greatly limits their applications. We report fine Au coating on synthesized quasi-spherical silver nanoparticles (AgNSs) with few atomic layers to several nanometers by stoichiometric method. The fine Au coating layer was confirmed by energy-dispersive X-ray spectroscopy elemental mapping and aberration-corrected high-angle annular dark-field scanning transmission electron microscopy. The optimized minimal thickness of Au coating layer on different sized AgNSs (22 nm Ag@0.9 nm Au, 44 nm Ag@1.8 nm Au, 75 nm Ag@2.9 nm Au, and 103 nm Ag@0.9 nm Au) was determined by extreme chemical stability tests using H2O2, NaSH, and H2S gas. The thin Au coating layer on AgNSs did not affect their plasmonic-based applications. The core/satellite assemblies based on Ag@Au NPs showed the comparable SERS intensity and uniformity three times higher than that of noncoated Ag core/satellites. The Ag@Au core/satellites also showed high stability in intracellular SERS imaging for at least two days, while the SERS of the noncoated Ag core/satellites decayed significantly. These spherical Ag@Au NPs can be widely used and have great advantages in plasmon-based applications, intracellular SERS probes, and other biological and analytical studies.

Immunoglobulin binding (B1) domain mediated antibody conjugation to quantum dots for in vitro and in vivo molecular imaging.

A facile method for the preparation of antibody-quantum dot (QD) conjugates using the immunoglobulin binding (B1) domain of protein G is presented. The utility of antibody-QD conjugates using the B1 domain is demonstrated for fluorescence imaging of breast tumor cells in vitro and in vivo.

The Impact of the Polymer Chain Length on the Catalytic Activity of Poly(N-vinyl-2-pyrrolidone)-supported Gold Nanoclusters.

Poly(N-vinyl-2-pyrrolidone) (PVP) of varying molecular weight (M w = 40-360 kDa) were employed to stabilize gold nanoclusters of varying size. The resulting Au:PVP clusters were subsequently used as catalysts for a kinetic study on the sized-dependent aerobic oxidation of 1-indanol, which was monitored by time-resolved in situ infrared spectroscopy. The obtained results suggest that the catalytic behaviour is intimately correlated to the size of the clusters, which in turn depends on the molecular weight of the PVPs. The highest catalytic activity was observed for clusters with a core size of ~7 nm, and the size of the cluster should increase with the molecular weight of the polymer in order to maintain optimal catalytic activity. Studies on the electronic and colloid structure of these clusters revealed that the negative charge density on the cluster surface also strongly depends on the molecular weight of the stabilizing polymers.

Immunohistochemical analysis of dentin matrix protein 1 (Dmp1) phosphorylation by Fam20C in bone: implications for the induction of biomineralization.

Dmp1 is an acidic phosphoprotein that is specifically expressed in osteocytes. During the secretory process, the full-length, precursor Dmp1 is cleaved into N- and C-terminal fragments. C-terminal Dmp1 is phosphorylated, becoming a highly negatively charged domain that may assist in bone mineralization by recruiting calcium ions and influencing subsequent mineral deposition. It has been recently reported that the Golgi-localized protein kinase Fam20C phosphorylates Dmp1 in vitro. To investigate this phosphorylation in situ, we determined the locations of phosphorylated Dmp1 and Fam20C in rat bones using immunohistochemistry. During osteocytogenesis, osteoblastic, osteoid, and young osteocytes (but not old osteocytes) express Dmp1 mRNA and contain Dmp1 protein in the Golgi apparatus. These Dmp1-producing cells were distributed across the surface layer of cortical bone. Using immunofluorescence, we found that N- and C-terminal Dmp1 fragments were predominantly distributed along the lacunar walls and canaliculi of mineralized bone, respectively, but were not present in the osteoid matrix. We also found that Fam20C and its substrate, C-terminal Dmp1, colocalized in the Golgi of osteoblastic, osteoid, and young osteocytes. Furthermore, phosphorylated C-terminal Dmp1 was present in the Golgi of young osteocytes. Double-labeling immunoelectron microscopy revealed that phosphorylated C-terminal Dmp1 localized to the canalicular wall in mineralized bone. These findings suggest that C-terminal Dmp1 is phosphorylated within osteocytes and then secreted into the pericanalicular matrix of mineralized bone. Phosphorylated, negatively charged C-terminal Dmp1 in the pericanalicular matrix may play an important role in bone mineralization by recruiting calcium ions.

Fast, vacancy-free climb of prismatic dislocation loops in bcc metals.

Vacancy-mediated climb models cannot account for the fast, direct coalescence of dislocation loops seen experimentally. An alternative mechanism, self climb, allows prismatic dislocation loops to move away from their glide surface via pipe diffusion around the loop perimeter, independent of any vacancy atmosphere. Despite the known importance of self climb, theoretical models require a typically unknown activation energy, hindering implementation in materials modeling. Here, extensive molecular statics calculations of pipe diffusion processes around irregular prismatic loops are used to map the energy landscape for self climb in iron and tungsten, finding a simple, material independent energy model after normalizing by the vacancy migration barrier. Kinetic Monte Carlo simulations yield a self climb activation energy of 2 (2.5) times the vacancy migration barrier for 1/2〈111〉 (〈100〉) dislocation loops. Dislocation dynamics simulations allowing self climb and glide show quantitative agreement with transmission electron microscopy observations of climbing prismatic loops in iron and tungsten, confirming that this novel form of vacancy-free climb is many orders of magnitude faster than what is predicted by traditional climb models. Self climb significantly influences the coarsening rate of defect networks, with important implications for post-irradiation annealing.

Delineation of six species of the primitive algal genus Glaucocystis based on in situ ultrastructural characteristics.

The field of microbiology was established in the 17(th) century upon the discovery of microorganisms by Antonie van Leeuwenhoek using a single-lens microscope. Now, the detailed ultrastructures of microorganisms can be elucidated in situ using three-dimensional electron microscopy. Since the availability of electron microscopy, the taxonomy of microscopic organisms has entered a new era. Here, we established a new taxonomic system of the primitive algal genus Glaucocystis (Glaucophyta) using a new-generation electron microscopic methodology: ultra-high-voltage electron microscopy (UHVEM) and field-emission scanning electron microscopy (FE-SEM). Various globally distributed Glaucocystis strains were delineated into six species, based on differences in in situ ultrastructural features of the protoplast periphery under UHVEM tomography and in the mother cell wall by FE-SEM, as well as differences in the light microscopic characteristics and molecular phylogenetic results. The present work on Glaucocystis provides a model case of new-generation taxonomy.

A new type of 3-D peripheral ultrastructure in Glaucocystis (Glaucocystales, Glaucophyta) as revealed by ultra-high voltage electron microscopy.

The coccoid glaucophyte genus Glaucocystis is characterized by having a thick cell wall, which has to date prohibited examination of the native ultrastructural features of the protoplast periphery. Recently, however, the three-dimensional (3-D) ultrastructure of the protoplast periphery was revealed in two divergent Glaucocystis species, with the world's most powerful ultra-high voltage electron microscope (UHVEM). The two species exhibit morphological diversity in terms of their 3-D ultrastructural features. However, these two types do not seem to encompass actual ultrastructural diversity in the genetically diverse genus Glaucocystis. Here, we report a new type of peripheral 3-D ultrastructure resolved in "G. incrassata" SAG 229-2 cells by 3-D modeling based on UHVEM tomography using high-pressure freezing and freeze-substitution fixation. The plasma membrane and underlying flattened vesicles in "G. incrassata" SAG 229-2 exhibited grooves at intervals of 200-600 nm, and the flattened vesicles often overlapped one another at the protoplast periphery. This 3-D ultrastructure differs from those of the two types previously reported in other species of Glaucocystis. The possibility of classification of Glaucocystis species based on the 3-D ultrastructure of the protoplast periphery is discussed.

Ultra-high voltage electron microscopy of primitive algae illuminates 3D ultrastructures of the first photosynthetic eukaryote.

A heterotrophic organism 1-2 billion years ago enslaved a cyanobacterium to become the first photosynthetic eukaryote, and has diverged globally. The primary phototrophs, glaucophytes, are thought to retain ancestral features of the first photosynthetic eukaryote, but examining the protoplast ultrastructure has previously been problematic in the coccoid glaucophyte Glaucocystis due to its thick cell wall. Here, we examined the three-dimensional (3D) ultrastructure in two divergent species of Glaucocystis using ultra-high voltage electron microscopy. Three-dimensional modelling of Glaucocystis cells using electron tomography clearly showed that numerous, leaflet-like flattened vesicles are distributed throughout the protoplast periphery just underneath a single-layered plasma membrane. This 3D feature is essentially identical to that of another glaucophyte genus Cyanophora, as well as the secondary phototrophs in Alveolata. Thus, the common ancestor of glaucophytes and/or the first photosynthetic eukaryote may have shown similar 3D structures.

Recombinant protein (EGFP-Protein G)-coated PbS quantum dots for in vitro and in vivo dual fluorescence (visible and second-NIR) imaging of breast tumors.

We report a one-step synthetic strategy for the preparation of recombinant protein (EGFP-Protein G)-coated PbS quantum dots for dual (visible and second-NIR) fluorescence imaging of breast tumors at the cellular and whole-body level.

Aqueous synthesis of glutathione-coated PbS quantum dots with tunable emission for non-invasive fluorescence imaging in the second near-infrared biological window (1000-1400 nm).

Glutathione-coated PbS quantum dots with tunable emission were synthesized in the aqueous phase and used for non-invasive tissue imaging in the second near-infrared biological window.