Ultra-high Voltage Electron Microscope Research Articles

Structural Basis for Histaminergic Regulation of Neural Circuits in the Mouse Olfactory Bulb.

Odor information is modulated by centrifugal inputs from other brain regions to the olfactory bulb (OB). Neurons containing monoamines, such as serotonin, acetylcholine, and noradrenaline, are well known as centrifugal inputs; however, the role of histamine, which is also present in the OB, is not well understood. In this study, we examined the histaminergic neurons projecting from the hypothalamus to the OB. We used an antibody against histidine decarboxylase (HDC), a synthesizing enzyme of histamine, to identify histaminergic neurons and assess their localization within the OB and the ultrastructure of their fibers and synapses using multiple immunostaining laser microscopy, ultra-high voltage electron microscopy (EM), and EM to confirm their relationships with other neurons. To further identify the origin nucleus of the histaminergic neurons projecting to the OB, we injected the retrograde tracer FluoroGold and analyzed the pathway to the OB anterogradely. HDC-immunoreactive (-ir) fibers were abundant in the olfactory nerve (ON) layer compared to other monoamines. HDC-ir neurons received asymmetrical synapses from ONs and formed synapses containing pleomorphic vesicles with variable postsynaptic densities to non-ON elements, thus forming serial synapses. We also confirmed that histaminergic neurons project from the rostral ventral tuberomammillary nucleus to the granule cell layer of the OB and, for the first time, successfully visualized their axons from the hypothalamus to the OB. These findings indicate that histamine may regulate odor discrimination in the OB, suggesting a regulatory relationship between hypothalamic function and olfaction. We thus elucidate morphological mechanisms with tuberomammillary nucleus-derived histaminergic neurons involved in olfactory information.

Network Tele-Microscopy in Metallic Glasses

Additive manufacturing (AM) can realize the rapid solidification for the formation of ultra-fine solidification microstructures and/or crystal growth reaching the marginal stability condition. The TEM observation of the nucleation and growth behavior of BCC dendrite embedded in Fe-based metallic glasses offers an unique opportunity for clarifying the morphology of the ultra-fine dendrite for the development of AM. The present study reported the network tele-microscopy observation of BCC-Fe dendrites embedded in an Fe-Si-B amorphous matrix obtained by the in-situ heating of TEM specimens using the ultra-high voltage electron microscope Hitachi H-3000.

Dislocation-based high-temperature plasticity of polycrystalline perovskite SrTiO3

Dislocation networks have been demonstrated to substantially enhance functional properties. As-sintered samples are virtually devoid of dislocations, new innovative techniques for introducing sufficiently high dislocation densities into polycrystalline ceramics are needed. While dislocation-based plasticity at high temperatures has been demonstrated for a large range of ceramic single crystals, plasticity in polycrystals is much less understood. Here, we demonstrate plastic strains in excess of several % based on dislocation motion in polycrystalline SrTiO3 at ≈ 1100 °C with 3.9 µm grain size. Ultra-high voltage electron microscopy reveals an associated increase in dislocation density by three orders of magnitude. Achievable strain rates are comparable to creep-based mechanisms and much less sensitive to applied stress than observed for metals. A specialized testing protocol allows quantification of the deformability via stress exponent, activation volume and activation enthalpy giving additional quantification. In conjunction with TEM images, the mechanical data gives insight into the underlying mechanisms.

Microstructure and conductivity of blacklight‐sintered TiO2, YSZ, and Li0.33La0.57TiO3

AbstractRapid densification of ceramics has been realized and its merits were demonstrated through multiple approaches out of which UHS and flash sintering attract recent attention. So far, however, scalability remains difficult. A rise in throughput and scalability is enabled by the introduction of blacklight sintering powered by novel light source technology. Intense illumination with photon energy above the bandgap (blacklight) allows high absorption efficiency and, hence, very rapid, contactless heating for all ceramics. While heating the ceramic directly with light without any furnace promises scalability, it simultaneously offers highly accurate process control. For the technology transfer to industry, attainable material quality needs to be assured. Here, we demonstrate the excellent microstructure quality of blacklight‐sintered ceramics observed with ultrahigh voltage electron microscopy revealing an option to tune nanoporosity. Moreover, we confirm that electronic, electron, oxygen, and lithium‐ion conductivities are equal to conventionally sintered ceramics. This gives the prospect of transmitting the merits of rapid densification to the scale of industrial kilns.

High-resolution image-based simulation reveals membrane strain concentration on osteocyte processes caused by tethering elements

Osteocytes are vital for regulating bone remodeling by sensing the flow-induced mechanical stimuli applied to their cell processes. In this mechanosensing mechanism, tethering elements (TEs) connecting the osteocyte process with the canalicular wall potentially amplify the strain on the osteocyte processes. The ultrastructure of the osteocyte processes and canaliculi can be visualized at a nanometer scale using high-resolution imaging via ultra-high voltage electron microscopy (UHVEM). Moreover, the irregular shapes of the osteocyte processes and the canaliculi, including the TEs in the canalicular space, should considerably influence the mechanical stimuli applied to the osteocytes. This study aims to characterize the roles of the ultrastructure of osteocyte processes and canaliculi in the mechanism of osteocyte mechanosensing. Thus, we constructed a high-resolution image-based model of an osteocyte process and a canaliculus using UHVEM tomography and investigated the distribution and magnitude of flow-induced local strain on the osteocyte process by performing fluid–structure interaction simulation. The analysis results reveal that local strain concentration in the osteocyte process was induced by a small number of TEs with high tension, which were inclined depending on the irregular shapes of osteocyte processes and canaliculi. Therefore, this study could provide meaningful insights into the effect of ultrastructure of osteocyte processes and canaliculi on the osteocyte mechanosensing mechanism.

Room-Temperature Plasticity of a Nanosized GaN Crystal.

GaN wurtzite crystal is commonly regarded as eminently brittle. However, our research demonstrates that nanodeconfined GaN compressed along the M direction begins to exhibit room-temperature plasticity, yielding a dislocation-free structure despite the occurrence of considerable, irreversible deformation. Our interest in M-oriented, strained GaN nanoobjects was sparked by the results of first-principles bandgap calculations, whereas subsequent nanomechanical tests and ultrahigh-voltage (1250 kV) transmission electron microscopy observations confirmed the authenticity of the phenomenon. Moreover, identical experiments along the C direction produced only a quasi-brittle response. Precisely how this happens is demonstrated by molecular dynamics simulations of the deformation of the C- and M-oriented GaN frustum, which mirror our nanopillar crystals.

Planar cell polarity induces local microtubule bundling for coordinated ciliary beating

Multiciliated cells (MCCs) in tracheas generate mucociliary clearance through coordinated ciliary beating. Apical microtubules (MTs) play a crucial role in this process by organizing the planar cell polarity (PCP)-dependent orientation of ciliary basal bodies (BBs), for which the underlying molecular basis remains elusive. Herein, we found that the deficiency of Daple, a dishevelled-associating protein, in tracheal MCCs impaired the planar polarized apical MTs without affecting the core PCP proteins, causing significant defects in the BB orientation at the cell level but not the tissue level. Using live-cell imaging and ultra-high voltage electron microscope tomography, we found that the apical MTs accumulated and were stabilized by side-by-side association with one side of the apical junctional complex, to which Daple was localized. In vitro binding and single-molecule imaging revealed that Daple directly bound to, bundled, and stabilized MTs through its dimerization. These features convey a PCP-related molecular basis for the polarization of apical MTs, which coordinate ciliary beating in tracheal MCCs.

Donor and acceptor-like self-doping by mechanically induced dislocations in bulk TiO2

Dislocations have been recently introduced as a novel tool to tailor the conductivity of functional ceramics. However, tuning strategies suffer from poor insight into the structural complexity of dislocations and their networks. Here, we demonstrate that dislocations can be used to both enhance and reduce the overall conductivity in the same ceramic material. Accurate control of the arrangement of dislocations within the dislocation network enables tailoring TiO2 bulk samples to behave like being chemically modified either with an acceptor or donor dopant. Our approach combines ultra-high voltage electron microscopy, oxygen partial pressure, and temperature dependent electrical conductivity measurements combined with time-of-flight secondary ion mass spectrometry. This allows us to focus on mechanically tailored interaction of next neighbor dislocations and to differentiate between percolating conductive pathways and separated charge carrier zones. This seemingly simple approach purposefully tailors the conductivity of TiO2, opening new avenues to engineer functional ceramics beyond common chemical doping strategies.

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.

Influence of dislocations on thermal conductivity of strontium titanate

Recently, several creative processing techniques yielded thermoelectrics with reduced thermal conductivity and, thereby, an enhanced figure or merit. These were based on engineered complex microstructures with attendant dislocation structures. In this study, we implement highly controlled mesoscopic dislocation structures into the model thermoelectric SrTiO3 in order to quantify phonon scattering at dislocations. Both single crystals and polycrystalline material have been furnished with enhanced dislocation densities increased by a factor of 150–300 by plastic deformation. Thermal conductivity was measured using laser flash analysis between room temperature and 325 °C. Etch pit techniques and ultra-high voltage electron microscopy afford quantification of dislocation density. Experimental results were compared to predictions by the Debye-Callaway model. The latter revealed that dislocation densities of 1015 m−2 would be necessary for the reduction of thermal conductivity of SrTiO3 in the investigated temperature range, which could not be realized using the plastic deformation mechanism applied.

Application of ultra-high voltage electron microscope tomography to 3D imaging of microtubules in neurites of cultured PC12 cells.

Electron tomography methods using the conventional transmission electron microscope have been widely used to investigate the three-dimensional distribution patterns of various cellular structures including microtubules in neurites. Because the penetrating power of electrons depends on the section thickness and accelerating voltage, conventional TEM, having acceleration voltages up to 200 kV, is limited to sample thicknesses of 0.2 µm or less. In this paper, we show that the ultra-high voltage electron microscope (UHVEM), employing acceleration voltages of higher than 1000 kV (1 MV), allowed distinct reconstruction of the three-dimensional array of microtubules in a 0.7-µm-thick neurite section. The detailed structure of microtubules was more clearly reconstructed from a 0.7-µm-thick section at an accelerating voltage of 1 MV compared with a 1.0 µm section at 2 MV. Furthermore, the entire distribution of each microtubule in a neurite could be reconstructed from serial-section UHVEM tomography. Application of optimised UHVEM tomography will provide new insights, bridging the gap between the structure and function of widely-distributed cellular organelles such as microtubules for neurite outgrowth. LAY DESCRIPTION: An optimal 3D visualisation of microtubule cytoskeleton using ultra-high voltage electron microscopy tomography The ultra-high voltage electron microscope (UHVEM) is able to visualise a micrometre-thick specimen at nanoscale spatial resolution because of the high-energy electron beam penetrating such a specimen. In this study, we determined the optimal conditions necessary for microtubule cytoskeleton imaging within 0.7-µm-thick section using a combination with UHVEM and electron tomography method. Our approach provides excellent 3D information about the complex arrangement of the individual microtubule filaments that make up the microtubule network.

New crystallography using relativistic femtosecond electron pulses

Ultrafast electron microscopy (UEM) with femtosecond temporal resolution has long been a cherished dream tool for scientists wishing to study ultrafast structural dynamics in materials, appealing to researchers from across a wide range of speciality areas. Associate Professor Jinfeng Yang, from the Institute of Scientific and Industrial Research, at Osaka University in Japan, leads a team working on ultrafast electron diffraction (UED) and ultrafast electron microscopy (UEM) development. 'Through the study of ultrafast phenomena with the UEM, we hope to gain a deeper understanding of materials and their physical properties and achieve a novel breakthrough in materials science,' he highlights. 'We fully expect to facilitate new knowledge and discoveries as a result of our work.' The team's work on relativistic UEM has led to the creation of unprecedented innovative technology that enables femtosecond atomic-scale imaging using just a single shot measurement. This will pave the way for the study of dynamics of irreversible processes within materials sciences. Not only does the group's work represent a huge step forward in innovative technology for researchers working across a number of scientific fields, but it is also progress in developing a very compact, ultra-high voltage electron microscopy. It can also be used in a variety of settings such as general research institutions and laboratories. In addition, through its provision of a solution to the problem of femtosecond temporal resolution our technology is breaking new ground in electronic microscopy developments,' says Yang.

Achievements and Future Development by Ultra-High Voltage Electron Microscopes and Cryo-TEM in Nanotechnology Open Facilities of Research Center for Ultra-High Voltage Electron Microscopy, Osaka University

Achievements and Future Development by Ultra-High Voltage Electron Microscopes and Cryo-TEM in Nanotechnology Open Facilities of Research Center for Ultra-High Voltage Electron Microscopy, Osaka University

Effects of Helium Implantation and Subsequent Electron Irradiation on Microstructures of Fe-11 wt.% Cr Model Alloy**Supported by the National Natural Science Foundation of China under Grant Nos U1832133, 11475229 and 91426301.

Helium effects on dislocation and cavity formation of Fe-11 wt.% Cr model alloy are investigated. Single-beam (electron) and dual-beam (He+/e−) irradiations are performed at 350°C and 400°C using an ultra-high voltage electron microscope combined with ion accelerators. In-situ observation shows that the growth rate of dislocation loops is reduced in the helium pre-injected specimen. The mean size of cavities decreased in the helium pre-injected specimen. The possible mechanisms are discussed.

Fast in Situ Ultrahigh-Voltage Electron Microscopy Observation of Crystal Nucleation and Growth in Amorphous Antimony Nanoparticles

Electron-irradiation-induced crystallization processes and the mechanisms in amorphous antimony nanoparticles have been investigated by microsecond temporal and picometer spatial resolution in situ observations. Electron irradiation experiments and the simultaneous in situ observations were carried out with an ultrahigh-voltage electron microscope operating at an accelerating voltage of 1 MV, which has a temporal resolution of 625 μs per frame. At the early stage of the crystallization in approximately 20 nm amorphous nanoparticles, a small crystal nucleus on the surface repeats between formation and annihilation. When the nucleus size becomes more than the critical size of 6.3 nm in diameter, crystal growth takes place in the whole nanoparticle. The crystal growth rate estimated was approximately 20 μm s–1. The growth rate depends on the particle size, and it was confirmed that the smaller the particle size, the faster the growth rate. It was suggested that the crystallization driven by long-range elastic interaction due to small crystal nucleus formation in amorphous nanoparticles is induced by short-range atomic rearrangements.

Microstructure change of duplex stainless steels after thermal aging and electron beam irradiation

Duplex stainless steel has been used as a structural material for light water reactors. It is well known that the thermal aging during operation causes spinodal decomposition to Cr-rich (α') phase and Fe-rich (α) phase in the ferrite phase, resulting in brittlement. In order to understand the mechanism of this phenomena and the effect of irradiation, a duplex model alloy (Fe-25Cr-10Ni-2.5Mo-1Mn) prepared by arc melting and thermally aged in the appropriate condition was subjected to accelerated irradiation by a multi-beam ultra-high voltage electron microscope and partially an ion accelerator in this study. Spinodal decomposition of Fe and Cr was confirmed in the model alloy aged at 450 °C. In addition, the electron beam irradiation to aged model alloy resulted in the decrease in the amplitude of spinodal decomposition. From this experiment, it was suggested that spinodal decomposition could be suppressed by accelerated irradiation.

The Motion of An Inv Nodal Cilium: a Realistic Model Revealing Dynein-Driven Ciliary Motion with Microtubule Mislocalization

Background/Aims: Nodal cilia that rotate in the ventral node play an important role in establishing left-right asymmetry during embryogenesis; however, inv mutant cilia present abnormal movement and induce laterality defects. The mechanism of their motility, which is regulated by dynein activation and microtubule arrangement, has not been fully understood. This study analyzed the dynein-triggered ciliary motion in the abnormal ultrastructure of the inv mutant, aiming to quantitatively evaluate the influence of microtubule mislocalization on the movement of the cilium. Methods: We established a realistic 3-D model of an inv mutant cilium with an ultrastructure based on tomographic datasets generated by ultra-high voltage electron microscopy. The time-variant activation of the axonemal dynein force was simulated by pairs of point loads and embedded at dynein-mounted positions between adjacent microtubule doublets in this mathematical model. Utilizing the finite element method and deformable grid, the motility of the mutant cilium that is induced by various dynein activation hypotheses was investigated and compared to experimental observation. Results: The results indicate that for the inv mutant, simulations of the ciliary movement with the engagement of dyneins based on the distance-controlled pattern in the partially activation scenario are broadly consistent with the observation; the shortening of the microtubules induces smaller movement amplitudes, while the angles of the mislocalized microtubules affect the pattern of the ciliary movement, and during the ciliary movement, the microtubules swing and twist in the mutant ciliary body. Conclusion: More generally, this study implies that dynein engagement is sensitive to subtle geometric changes in the axoneme, and thus, this geometry greatly influences the integrity of a well-formed ciliary rotation.

Ultra-high voltage electron microscopy investigation of irradiation induced displacement defects on AlGaN/GaN HEMTs

Irradiation effects produced by high energy heavy ions and electrons on AlGaN/GaN high electron mobility transistor (HEMT) were investigated using ultra-high voltage electron microscopy (HVEM), optical measurements, and device characteristics analysis. The dislocation loop created on a thick (1.5μm) sample irradiated by 18MeV Ni ions was observed by HVEM. The distribution of the defects matched the calculated distribution obtained using the stopping and range of ions in matter (SRIM) code. In-situ dislocation loop generation was observed in the sample during a high energy (2MeV) electron irradiation by using HVEM. Device characteristics were found to remain stable, no increase in leakage current or electric field intensity being noticed up to a high radiant fluence of Ni and electron irradiation. These results demonstrate a high radiation tolerance of the device for space applications.

Automatic system for electron tomography data collection in the ultra-high voltage electron microscope

In this study, we report an automatic system for collection of tilt series for electron tomography based on the ultra-HVEM in Osaka University. By remotely controlling the microscope and reading the observation image, the system can track the field of view and do focus in each tilt angle. The automatic tracking is carried out with an image matching technique based on normalized correlation coefficient. Auto focus is realized by the optimization of image sharpness. A toolkit that can expand the field of view with technique of image stitching is also developed. The system can automatically collect the tilt series with much smaller time consumption.

Three-dimensional fine structure of the organization of microtubules in neurite varicosities by ultra-high voltage electron microscope tomography.

Neurite varicosities are highly specialized compartments that are involved in neurotransmitter/ neuromodulator release and provide a physiological platform for neural functions. However, it remains unclear how microtubule organization contributes to the form of varicosity. Here, we examine the three-dimensional structure of microtubules in varicosities of a differentiated PC12 neural cell line using ultra-high voltage electron microscope tomography. Three-dimensional imaging showed that a part of the varicosities contained an accumulation of organelles that were separated from parallel microtubule arrays. Further detailed analysis using serial sections and whole-mount tomography revealed microtubules running in a spindle shape of swelling in some other types of varicosities. These electron tomographic results showed that the structural diversity and heterogeneity of microtubule organization supported the form of varicosities, suggesting that a different distribution pattern of microtubules in varicosities is crucial to the regulation of varicosities development.