High Voltage Electron Microscope Research Articles

A nanoscale visual exploration of the pathogenic effects of bacterial extracellular vesicles on host cells

BackgroundBacterial extracellular vesicles (EVs) are pivotal mediators of intercellular communication and influence host cell biology, thereby contributing to the pathogenesis of infections. Despite their significance, the precise effects of bacterial EVs on the host cells remain poorly understood. This study aimed to elucidate ultrastructural changes in host cells upon infection with EVs derived from a pathogenic bacterium, Staphylococcus aureus (S. aureus).ResultsUsing super-resolution fluorescence microscopy and high-voltage electron microscopy, we investigated the nanoscale alterations in mitochondria, endoplasmic reticulum (ER), Golgi apparatus, lysosomes, and microtubules of skin cells infected with bacterial EVs. Our results revealed significant mitochondrial fission, loss of cristae, transformation of the ER from tubular to sheet-like structures, and fragmentation of the Golgi apparatus in cells infected with S. aureus EVs, in contrast to the negligible effects observed following S. epidermidis EV infection, probably due to the pathogenic factors in S. aureus EV, including protein A and enterotoxin. These findings indicate that bacterial EVs, particularly those from pathogenic strains, induce profound ultrastructural changes of host cells that can disrupt cellular homeostasis and contribute to infection pathogenesis.ConclusionsThis study advances the understanding of bacterial EV-host cell interactions and contributes to the development of new diagnostic and therapeutic strategies for bacterial infections.

Cathodoluminescence of CeO2 doped with Gd2O3 in a high voltage electron microscope

Cathodoluminescence of CeO2 doped with Gd2O3 in a high voltage electron microscope

Advances in Characterization of Thick Specimens and Time-Resolved <i>In-situ</i> Observation by Modern High Voltage Electron Microscopy

Advances in Characterization of Thick Specimens and Time-Resolved <i>In-situ</i> Observation by Modern High Voltage Electron Microscopy

Development of an integrated high-voltage electron microscope-gas chromatograph-quadrupole mass spectrometer system for the operando analysis of catalytic gas reactions.

This paper describes the development of a gas chromatography-quadrupole mass spectrometry system attached to a differential-pumping-type environmental cell of the reaction science high-voltage electron microscopy instrument at Nagoya University to distinguish unambiguously between different gas species with the same mass-to-charge ratio. Several model experiments were used to verify the efficacy of the newly proposed system, confirming its ability to analyse the atomic-level structural changes during heterogeneous catalysts and the associated gas-reaction kinetics simultaneously, providing new insights into operando measurements in the field of environmental transmission electron microscopy. Graphical Abstract.

Changes of synaptic vesicles in three-dimensional synapse models by treatment with umbelliferone in scopolamine-induced hippocampal injury model

The neuroprotective effects of umbelliferone (UMB) were visualized in three-dimensional (3D) images on vesicle density changes of organotypic hippocampal slice tissues (OHSCs) induced by scopolamine by high voltage electron microscopy. Observations revealed that the number of vesicles decreased in OHSCs induced by scopolamine, and UMB was found to inhibit scopolamine-induced reduction in vesicles, resulting in an increase in vesicle count. These 3D models provide valuable insight for understanding the increase of synapse vesicles in hippocampal tissues treated with UMB.

Whereabouts of missing atoms in a laser-injected Si (part IV): interaction of plasma Si with dislocations in a laser-injected Si

ABSTRACT The interaction of dislocations with plasma Si in a laser-injected Si was studied comprehensively using a combination of in-situ and ex-situ heating experiments in 200 kV electron microscopes and in a high-voltage electron microscope (HVEM). When dislocations were observed at 200 kV, i.e. virtually in the absence of electron irradiation, no evidence of the interaction between dislocations and plasma Si was obtained even when the observation was carried out at temperatures as high as 1250°C. However, when observed at 1000 kV in an HVEM, definite evidence of interaction was obtained even when the observation was carried out at room temperature. It is concluded that a combination of laser injection and electron irradiation is essential for the interaction to take place.

Microstructural Transformations in Solid-State Annealed Al/Ag/Al Diffusion Couples Examined via High-Voltage Electron Microscopy (HVEM)

This study focuses on the practical relevance of the Al-Ag bonding interface in electronic device fabrication, particularly in wire bonding, which is crucial for enhancing component reliability and performance. Experiments involved Al/Ag/Al diffusion couples, annealed at 703 K, revealing two stable intermediate phases, μ and δ. Characterizing the intermediate phases’ compositions and concentration profiles exposed a vital transition at the δ-Al interface. We used high-voltage electron microscopy (HVEM) to examine crystal structure evolution, identifying a (hexagonal close-packed) hcp structure in the intermediate phase between δ and Al, matching the δ phase. Notably, a substantial microstructural transformation occurred within the Ag-Al diffusion couple, as nano-sized precipitates transitioned from spherical to plate-like, along specific {111} planes, reflecting the evolution from off-stoichiometric, disordered phases to ordered ones. Mapping the concentrations of intermediate phases on the Al-Ag phase diagram revealed shifted and narrower solubility ranges compared to the calculations. This study provides insight into the crystal structure and microstructure changes during diffusion in Al/Ag/Al diffusion couples, holding implications for electronic device fabrication. Understanding intermediate phase behavior and evolution is vital in this context, potentially influencing materials development and process optimization in the electronic components industry, and thus, enhancing device performance and reliability.

Formation mechanism of dislocation network of cell structure in cyclically deformed near-[formula omitted] copper single crystals

As the initiation and propagation of fatigue crack is strongly correlated to the dislocations in cell structures of metals, investigating the formation of dislocation cell structures is essential for understanding the fatigue process. Although the cell boundary evolves from the dislocation network, the formation process of the dislocation network of fatigued copper single crystal is not completely realized. In this study, the dislocation structures in near-[1‾11] copper single crystals cyclically deformed at 293 K were investigated to analyze the formation mechanism of the dislocation network. The dislocation networks were characterized using a high-voltage scanning transmission electron microscope, where the virtual-scanning transmission electron microscope technique (virtual-STEM) was employed to conduct the Burgers vector analysis of the dislocations. The low-dislocation-density network was consistent with the three perfect screw dislocations (b = a/2 [1‾01], a/2 [1‾10], and a/2 [011‾]) in the (111) primary slip plane. For the high-dislocation-density region, the perfect and partial dislocations coexisted in the network. As the dislocation density increased, the perfect dislocations dissociated into Shockley partial dislocations (b = a/6 [1‾21‾], a/6 [112‾], and a/6 [2‾11]). This study provides a reference for future applications of the virtual-STEM technique in the field of dislocation analysis.

Effects of estradiol on dopaminergic synapse formation in the mouse olfactory bulb.

Olfactory glomeruli are the sites of initial synaptic integration in olfactory information processing. They are surrounded by juxtaglomerular (JG) cells, which include periglomerular, superficial short axon, and external tufted cells. A subpopulation of JG cells expresses the dopamine synthetic enzymes, tyrosine hydroxylase (TH), and aromatic l-amino acid decarboxylase (AADC). TH cells corelease γ-aminobutyric acid (GABA) and their processes extend to multiple glomeruli forming intra- and interglomerular circuits. It is well established that 17β-estradiol (E2) exerts wide ranging effects in the central nervous system. However, participation of E2 in the modulation of neurotransmission and synaptic plasticity of TH cells in olfactory glomeruli is unknown. To address this, we subcutaneously implanted a 60-day release pellet of E2 or placebo into intact male mice and compared glomerular TH, AADC, and vesicular γ-aminobutyric acid transporter (VGAT) immunoreactivity between them. High-voltage electron microscopy (HVEM) and ultra-HVEM using immunogold revealed significantly increased immunoreactive intensity at the cellular level for TH and AADC after E2 treatment and for VGAT in TH cells. These results indicate that E2 may affect the interplay between dopaminergic and GABAergic systems. Moreover, random-section electron microscopy analysis showed a significant increase in the number of symmetrical synapses from TH cell to mitral/tufted cell dendrites after E2 treatment. This result was supported by quantitative immunofluorescence staining with synapse markers. Together, these data indicate that E2 may regulate inhibition between TH cells and olfactory bulb neurons within the glomerulus via interaction between dopaminergic and GABAergic systems, thereby contributing to neuromodulation of odor information processing.

Characterization of dislocation microstructures in near-[formula omitted] single crystal copper using high-voltage scanning transmission electron microscopy

Metallic structures are prone to failure caused primarily by fatigue-related damage to the structural material. Fatigue behavior is strongly correlated with dislocation structures in metals. Therefore, investigating the formation of dislocation structures is essential for understanding the fatigue process. The dislocation structure in face-centered cubic crystals has a strong orientation dependence. However, the detailed characteristics and evolution process of dislocation structures of fatigued [1‾11] single crystal copper are still insufficient. This study investigated dislocation structures in near-[1‾11] single crystal copper cyclically deformed at room temperature under different constant plastic shear strain amplitude (γpl) to analyze the formation mechanism of dislocation structures and their relationship with fatigue behavior. Characteristic dislocation structures were characterized by adopting multiple planes using a high-voltage scanning transmission electron microscope. With increasing shear strain amplitude, a vein-like structure along the (1‾11) plane gradually developed into a wall structure. When γpl ≥ 1 × 10−3, deformation bands and cell bands were formed to accommodate the higher plastic strain. The state of cell-band development significantly affected the cyclic softening of the single crystal copper. A positive linear relationship existed between the shear stress amplitude and the reciprocal of the cell width.

Deep learning model for 3D profiling of high-aspect-ratio features using high-voltage CD-SEM

High-aspect-ratio (HAR) channel holes were developed for competitive cost-per-bit 3D-NAND memory. High-throughput and in-line monitoring solutions for 3D profiling of the HAR features are the key to improving yields. We previously proposed an exponential model to identify the cross-sectional profile of the HAR features using backscattered electron (BSE) images of a high-voltage critical dimension scanning electron microscopy (CD-SEM). However, the 3D profiling accuracy was insufficient when the depth of the HAR features was far greater than the focus depth of the electron beam. To address this issue, we developed a deep learning (DL) model, which takes account of the aperture angle and the aberration of the electron beam, to predict the 3D profile from BSE images. The predicted cross-sections of the HAR holes with different bowing geometries were compared with field-emission SEM measurements. The results show that the DL model provides higher sensitivity than the exponential model does.

A model experiment on nano-particle stability in 12Cr-ODS steel using high-resolution high voltage microscopy

In this work, the microstructure stability of 12Cr-ODS ferritic steel under irradiation is studied using a high-resolution high voltage electron microscope up to 1.5 dpa at 300 and 500 °C. Coherent YTiO3 nanoparticles (NPs) undergo coarsening during electron irradiation and the coarsening rate is positively correlated to irradiation temperature (the third power of NPs size is proportional to the irradiation dose). He pre-implantation can significantly reduce the coarsening rate. A model coupling Ostwald ripening and irradiation enhanced diffusion is used to explain this coarsening. The fitting results between NPs size and irradiation dose show that pre-implanted He causes a lower diffusion of Y in 12Cr-ODS steel compared with the non-implanted sample. In response to the irradiation, plate-like radiation-induced precipitates appear which result in diffraction streaks in fast fourier transform (FFT) pattern. Precipitations are paralleled to [100] or [110] direction of Fe matrix and located near the interface between NPs and matrix. The hypothesis that coherent Cr precipitate along NPs/matrix interface caused diffraction streaks is proposed. Besides, He pre-implantation can also delay this precipitation.

Effect of Mn addition on the formation of vacancy-type dislocation loops in α-Fe

It has recently been established that vacancy loops of ~100 nm in maximum size could form in hydrogen pre-implanted α-Fe (Huang et al. Fusion Eng. Des., 2010). The formation temperature of these large loops is composition dependent, varying from ~500 °C in α-Fe, to ~450 °C in FeNi and ~ 620 °C in FeCr (Wan et al. J. Nucl. Mater., 2014; Du et al. Acta Metall. Sin., 2019). In this work, the effect of Mn addition upon vacancy loop formation has been investigated. Pure Fe and Fe-1.4 wt% Mn alloy specimens were irradiated with 30 keV H+ ions at room temperature. Radiation-induced loop production, size, population and nature were characterized based on diffraction contrast imaging using a combination of 1 MV high voltage electron microscope (HVEM) and 200 kV conventional transmission electron microscope (CTEM). Results of observation indicate that Mn addition gives rise to the increase of vacancy loop formation temperature in α-Fe, similar to the effect of Cr. A new criterion has been thus proposed, to classify the common alloying elements (Cr, Ni, Mn etc) found in Fe.

Variation in formation and migration of self-interstitial atom clusters in electron irradiated copper with material purity and specimen preparation method

ABSTRACT In situ observation with high-voltage electron microscopy was used to survey effects of impurity atoms on the formation and one-dimensional (1D) migration of self-interstitial atom (SIA) clusters in copper under electron irradiation at 300 K. Specimens were prepared from copper materials of five nominal purities (9N, 6N, 5N, 4N, and 3N) using three methods with different annealing conditions. In standard (STD) specimens prepared through cold rolling and annealing in vacuum, SIA cluster formation and 1D migration depended little on the nominal purity. In non-annealed (NA) specimens prepared from high-purity materials (9N and 6N) using mechanical processing and electropolishing, the defect structure was found to be coarser than in STD specimens. In fact, SIA clusters in NA specimens had number density of more than an order of magnitude lower and an average size more than four times greater. Furthermore, 1D migration had the frequency of about an order of magnitude higher and distance extending more than twice longer. Results of bulk annealing (BA) specimens showed that annealing of as-received block material had minor effects. Distributions of 1D migration distance for all specimens were described using an earlier proposed trap model. These results were discussed assuming that SIA clusters are trapped by impurity atoms existing in as-received materials and those induced by annealing.

Vacancy migration in α-iron investigated using in situ high-voltage electron microscopy

ABSTRACT Modeling the microstructural evolution of irradiated materials requires knowledge of the migration energies of point defects, which results from energetic particle radiation. Herein, we measured the growth rate of self-interstitial atom (SIA) clusters in electron-irradiated α-iron at 275–320 K using in situ high-voltage electron microscopy. To improve the statistical accuracy of the measurement, we used photographic films and video data. This enabled analysis of a considerable amount of data by extracting several SIA clusters and tracking their size growth using image processing techniques. By fitting the temperature-dependent cluster growth rate to the Arrhenius relations derived using rate theory analysis, we obtained vacancy migration energy of eV. The validity of the estimation method was confirmed from the consistency of the dependence of the damage rate on the cluster growth rate using the applied rate theory analysis. We also investigated the possible roles of faster motion of tri-vacancies compared to those of mono- and di-vacancies, as demonstrated by first-principles calculations, on the obtained migration energy using a kinetic analysis model. In addition, the effects of impurities leading to decrease in the cluster growth rate were briefly discussed.

In-situ observation of damage structure in Cu-Cr-Zr and Cu-Cr alloy during 1.25 MeV electron irradiation

The sink strengths of precipitate/matrix interfaces in Cu alloys, were investigated by performing in-situ observation of electron irradiation in high-voltage electron microscopy (HVEM). Fine black spot defects, which seemed to be dislocation loops were observed at irradiation temperature ranging between RT and 373 K in Cu–Cr–Zr, Cu–Cr and GlidCop Al–60 alloys with low number density. In Cu–Cr, loops formed on a part of precipitates interfaces. Hence, a part of precipitate/matrix interfaces is a sink with interstitial bias. Whereas, most of the interfaces are neutral sinks for point defects. In the case of Cu–Cr–Zr and GlidCop Al–60, black spots formed around dislocation, which were induced during two-step heat treatment. The results showed that the precipitate/matrix interface of Cr-rich precipitates in Cu alloys serve as a strong sink for point defects.

HVEM in situ study of fracture of Al2O3/Nb sandwich specimens

Abstract In situ straining experiments on α-Al2O3/Nb/α-Al2O3 single crystal sandwiches have been performed in a high-voltage electron microscope in order to observe the dislocation motion connected with the fracture of these samples. The tricrystals had two different orientation relationships at the interfaces, both possessing high fracture energies. The in situ studies showed that the fracture occurs along the interfaces but more frequently inside the Nb sheet. The fracture is connected with strong plastic deformation. The crack opening and the dislocation processes are analysed.

A Model for Dose Dependence of the Void Swelling in Electron-Irradiated Alloys

Understanding the void swelling dependence on irradiation dose for structural materials is critical for the design and operation of advanced nuclear reactors. Due to their easy accessibility in high-voltage transmission electron microscopes, electron beams have been frequently employed to investigate the void swelling mechanisms. Here, we build a general model to describe the radiation-induced swelling produced by energetic electrons. Based on this model, we develop a quantitative relation between void swelling and irradiation dose, which is in good agreement with experimental data. By extrapolating to high-dose swelling in electron-irradiated alloys, our model validation is consistent with available experiments. Furthermore, the model is well supported by our phase-field simulations.

Evolution of dislocation loops and effect of annealing temperature on hydrogen-ion-implanted Fe-based binary alloys

Reduced activation ferritic/martensitic (RAFM) steels have been considered as a family of prime candidate structural materials for fusion reactors due to low radioactivity and good resistance to irradiation swelling. Various types of defects such as dislocation loops can form in these materials during irradiation. Effects of alloying elements in iron on the formation and migration of dislocation loops have been widely investigated. However, most studies dealt with interstitial-type dislocation loops in iron alloys, while very few focused on vacancy-type dislocation loops. Previous high voltage electron microscope (HVEM) studies from the authors' group have shown that interstitial loops are fully eliminated in hydrogen-ion-implanted α-Fe at 500 ℃, only vacancy loops remain and can achieve up to 100 nm in size. The addition of Ni in α-Fe can reduce the formation temperature of vacancy-type dislocation loops (<i>T</i><sub>c</sub>) to ~450 ℃, while the addition of Cr can increase the temperature to above 600 ℃. However, these experiments are usually difficult to perform due to the scarce resource of HVEM facilities. In this work, in-situ observations by conventional transmission electron microscope (CTEM, 200 kV) are systematically carried out on the hydrogen-ion-implanted α-Fe and Fe-based binary alloys (Fe-3wt.%Cr, Fe-1.4wt.%Ni and Fe-1.4wt.%Mn). The evolutions of morphology and average size of dislocation loops under different annealing temperatures are investigated. The formation temperatures of vacancy-type dislocation loops are determined from the change of average loop size with annealing temperature. The results are consistent with previous studies by HVEM. The effect of Mn atoms in α-Fe is similar to that of Cr atoms, which leads to <i>T</i><sub>c</sub> increase, and the addition of Ni in α-Fe can reduce <i>T</i><sub>c</sub>. Furthermore, the results of D thermal desorption spectrum analysis show that <i>T</i><sub>c</sub> is affected by the binding and release process of hydrogen isotopes to vacancies in α-Fe. Alloying element Ni promotes the binding and release of hydrogen isotopes to vacancies, which leads to<i> T</i><sub>c</sub> decrease. Cr and Mn inhibit the binding and release of hydrogen isotopes to vacancies, causing <i> T</i><sub>c</sub> to increase.

<i>In-situ</i> study of one-dimensional motion of interstitial-type dislocation loops in hydrogen-ion-implanted aluminum

The one-dimensional (1D) glide motion of dislocation loops along the direction of Burgers vector in various metallic materials has attracted considerable attention in recent years. During the operation of nuclear fusion reactor, component materials will be bombarded by high energy neutrons, resulting in production of radiation defects such as self-interstitial-atoms (SIAs), vacancies and their clusters. These defects feature large difference in migration energy, which may lead to concentration imbalance between SIAs and vacancies, and eventually irradiation damages such as swelling and embrittlement. Generally speaking, the mobility of a defect cluster is lower than that of a point defect. However, fast 1D motion may also take place among SIA clusters in the form of prismatic dislocation loops. This increases the transport efficiency of SIAs towards grain boundaries, surface and interface sites in the material, in favour of defect concentration imbalance and damage accumulation. To date, most literature works have found that the 1D motion of dislocation loops exhibited short-range (nanometer-scale) character. In addition, such experimental studies were generally conducted in pure metals using high voltage electron microscopes (HVEM) operated at acceleration voltages ≥1000 kV. However, for pure aluminum (Al), the maximum transferable kinetic energy from 200 keV electrons is 19.5 eV, while the displacement threshold energy is only 16 eV. Therefore, the observation and mechanistic investigation of 1D motion of dislocation loops in Al should also be possible with conventional transmission electron microscopes (C-TEM), as it may also exhibit the effects of beam heating and point defect production in HVEM. In view of the shortage of HVEM, this work reports the 1D motion of dislocation loops in pure Al implanted with hydrogen ions using C-TEM. Simultaneous dislocation loop motion in opposite directions of Burgers vector 1/2<inline-formula><tex-math id="Z-20211226170459">\begin{document}$\left\langle {110} \right\rangle $\end{document}</tex-math><alternatives><graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="1-20211229_Z-20211226170459.jpg"/><graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="1-20211229_Z-20211226170459.png"/></alternatives></inline-formula>has been captured, as well as the collective 1D motion of an array of dislocation loops in the direction of Burgers vector 1/3<inline-formula><tex-math id="Z-20211226170340">\begin{document}$\left\langle {111} \right\rangle $\end{document}</tex-math><alternatives><graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="1-20211229_Z-20211226170340.jpg"/><graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="1-20211229_Z-20211226170340.png"/></alternatives></inline-formula> under 200 keV electron irradiation. In addition, 1D motion of dislocation loops up to micron-scale range along the direction of Burgers vector 1/3<inline-formula><tex-math id="Z-20211226170427">\begin{document}$\left\langle {111} \right\rangle $\end{document}</tex-math><alternatives><graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="1-20211229_Z-20211226170427.jpg"/><graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="1-20211229_Z-20211226170427.png"/></alternatives></inline-formula>, and up to a few hundred nanometers range along the direction of Burgers vector 1/2<inline-formula><tex-math id="Z-20211226170442">\begin{document}$\left\langle {110} \right\rangle $\end{document}</tex-math><alternatives><graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="1-20211229_Z-20211226170442.jpg"/><graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="1-20211229_Z-20211226170442.png"/></alternatives></inline-formula> have been found, which is different from previous literature works. A characteristic migration track would form behind the moving dislocation loop, lasting for about tens of seconds. The more rapid the dislocation loop motion, the longer the migration track length is. The concentration gradient of SIAs by electron irradiation and the redistribution of hydrogen atoms caused by the moving dislocation loops may account for the observed micron-scale 1D motion of dislocation loops and the migration tracks.