Conventional Transmission Electron Microscopy Research Articles

In-situ heating-and-electron tomography for materials research: from 3D (in-situ 2D) to 4D (in-situ 3D).

In-situ observation has expanded the application of transmission electron microscopy (TEM) and has made a significant contribution to materials research and development for energy, biomedical, quantum, etc. Recent technological developments related to in-situ TEM have empowered the incorporation of three-dimensional observation, which was previously considered incompatible. In this review article, we take up heating as the most commonly used external stimulus for in-situ TEM observation and overview recent in-situ TEM studies. Then, we focus on the electron tomography (ET) and in-situ heating combined observation by introducing the authors' recent research as an example. Assuming that in-situ heating observation is expanded from two dimensions to three dimensions using a conventional TEM apparatus and a commercially available in-situ heating specimen holder, the following in-situ heating-and-ET observation procedure is proposed: (i) use a rapid heating-and-cooling function of a micro-electro-mechanical system holder; (ii) heat and cool the specimen intermittently and (iii) acquire a tilt-series dataset when the specimen heating is stopped. This procedure is not too technically challenging and can have a wide range of applications. Essential technical points for a successful 4D (space and time) observation will be discussed through reviewing the authors' example application.

Centriole and transition zone structures in photoreceptor cilia revealed by cryo-electron tomography.

Primary cilia mediate sensory signaling in multiple organisms and cell types but have structures adapted for specific roles. Structural defects in them lead to devastating diseases known as ciliopathies in humans. Key to their functions are structures at their base: the basal body, the transition zone, the "Y-shaped links," and the "ciliary necklace." We have used cryo-electron tomography with subtomogram averaging and conventional transmission electron microscopy to elucidate the structures associated with the basal region of the "connecting cilia" of rod outer segments in mouse retina. The longitudinal variations in microtubule (MT) structures and the lumenal scaffold complexes connecting them have been determined, as well as membrane-associated transition zone structures: Y-shaped links connecting MT to the membrane, and ciliary beads connected to them that protrude from the cell surface and form a necklace-like structure. These results represent a clearer structural scaffold onto which molecules identified by genetics, proteomics, and superresolution fluorescence can be placed in our emerging model of photoreceptor sensory cilia.

GoldDigger and Checkers, computational developments in cryo-scanning transmission electron tomography to improve the quality of reconstructed volumes.

In this work, we present a pair of tools to improve the fiducial tracking and reconstruction quality of cryo-scanning transmission electron tomography (STET) datasets. We then demonstrate the effectiveness of these two tools on experimental cryo-STET data. The first tool, GoldDigger, improves the tracking of fiducials in cryo-STET by accommodating the changed appearance of highly defocussed fiducial markers. Since defocus effects are much stronger in scanning transmission electron microscopy than in conventional transmission electron microscopy, existing alignment tools do not perform well without manual intervention. The second tool, Checkers, combines image inpainting and unsupervised deep learning for denoising tomograms. Existing tools for denoising cryo-tomography often rely on paired noisy image frames, which are unavailable in cryo-STET datasets, necessitating a new approach. Finally, we make the two software tools freely available for the cryo-STET community.

Regulating H2S release from self-assembled peptide H2S-donor conjugates using cysteine derivatives.

Self-assembled peptides provide a modular and diverse platform for drug delivery, and innovative delivery methods are needed for delivery of hydrogen sulfide (H2S), an endogenous signaling molecule (gasotransmitter) with significant therapeutic potential. Of the available types of H2S donors, peptide/protein H2S donor conjugates (PHDCs) offer significant versatility. Here we discuss the design, synthesis, and in-depth study of a PHDC containing three covalently linked components: a thiol-triggered H2S donor based on an S-aroylthiooxime (SATO), a GFFF tetrapeptide, and a tetraethylene glycol (TEG) dendron. Conventional transmission electron microscopy showed that the PHDC self-assembled into spherical structures without heat or stirring, but it formed nanofibers with gentle heat (37 °C) and stirring. Circular dichroism (CD) spectroscopy data collected during self-assembly under nanofiber-forming conditions suggested an increase in β-sheet character and a decrease in organization of the SATO units. Release of H2S from the nanofibers was studied through triggering with various thiols. The release rate and total amount of H2S released over both short (5 h) and long (7 d) time scales varied with the charge state: negatively charged and zwitterionic thiols (e.g., Ac-Cys-OH and H-Cys-OH) triggered release slowly while a neutral thiol (Ac-Cys-OMe) showed ∼10-fold faster release, and a positively charged thiol (H-Cys-OMe) triggered H2S release nearly 50-fold faster than the negatively charged thiols. CD spectroscopy studies monitoring changes in secondary structure over time during H2S release showed similar trends. This study sheds light on the driving forces behind self-assembling nanostructures and offers insights into tuning H2S release through thiol charge state modulation.

Uncovering the crystallography and formation mechanism of nanoscale clusters in Sb-rich SPPs of a p-type (Bi, Sb)2Te3 alloy.

Considering that the crystallographic characteristics of the Sb-rich secondary phase particles (SPPs) greatly affect the thermoelectric properties of Bi2Te3 based materials, it is of great significance to explore the mechanism behind the Sb-rich SPPs in the p-type (Bi, Sb)2Te3 material. Here a conventional TEM technique was used to characterize the composition, size and distribution of Sb-rich SPPs in a spark plasma sintered p-type (Bi, Sb)2Te3 alloy. The results indicated that two different morphologies of Sb-rich SPPs including elongated and circular Sb-rich SPPs were frequently observed. Combined with high-resolution transmission electron microscopy, this work provides atomic-scale evidence for the formation mechanism behind the Sb-rich SPPs in the (Bi, Sb)2Te3 material.

Improved conventional TEM sample preparation exploiting the birefringence of materials

Improved conventional TEM sample preparation exploiting the birefringence of materials

An introduction to scanning transmission electron microscopy for the study of protozoans.

Since its inception in the 1930s, transmission electron microscopy (TEM) has been a powerful method to explore the cellular structure of parasites. TEM usually requires samples of <100 nm thick and with protozoans being larger than 1 μm, their study requires resin embedding and ultrathin sectioning. During the past decade, several new methods have been developed to improve, facilitate, and speed up the structural characterisation of biological samples, offering new imaging modalities for the study of protozoans. In particular, scanning transmission electron microscopy (STEM) can be used to observe sample sections as thick as 1 μm thus becoming an alternative to conventional TEM. STEM can also be performed under cryogenic conditions in combination with cryo-electron tomography providing access to the study of thicker samples in their native hydrated states in 3D. This method, called cryo-scanning transmission electron tomography (cryo-STET), was first developed in 2014. This review presents the basic concepts and benefits of STEM methods and provides examples to illustrate the potential for new insights into the structure and ultrastructure of protozoans.

Optimized procedure for conventional TEM sample preparation using birefringence

Specimens for quality transmission electron microscopy (TEM) analyses must fulfil a range of requirements, which demand high precision during the prior preparation process. In this work, an optimized procedure for conventional TEM specimen preparation is presented that exploits the thickness-dependence of interference colors occurring in birefringent materials. It facilitates the correct estimation of specimen thickness to avoid damage or breaking during mechanical thinning and reduces ion-milling times below 30minutes. The benefits of the approach are shown on sapphire and silicon carbide cross-section samples. The presented method is equally suitable for assessing specimen thickness during dimpling and wedge-polishing, and is particularly useful at thicknesses below 20 μm, where the accuracy of mechanical techniques is insufficient. It is precise enough to be employed for a visual thickness estimation during the thinning process, but can be additionally optimized by analyzing the RGB spectrum of the occurring interference colors.

Characterization of irradiation-induced dislocation loops and helium bubbles via electron channeling contrast imaging

Characterizing irradiation-induced defects is essential to understand damage mechanisms and develop irradiation-resistant materials. Prevailing strategies rely on transmission electron microscopy (TEM) observations which require rigorous prerequisite to prepare high-quality foils, making it difficult for rapid screening of irradiation-resistant materials. Here, we propose that the electron channeling contrast imaging (ECCI) is expected to provide a novel strategy for characterizing surface irradiation defects of ion-irradiated materials. The comparison between ECCI and conventional cross-sectional TEM for the observation of dislocation loops and helium (He) bubbles is analyzed in a common FeCoNiCr high-entropy alloy. The results indicate that the ECCI images of irradiation-induced defects are optimal at the extra-high tension of 20 kV, with a limit resolution of ∼1.5 nm for visible defects. The irradiation-induced dislocation loops show sharper contrast than bubbles in ECCI images, and the acceptable misorientation was within 4° of sample tilt. These findings offer a promising solution to the challenges for rapid assessment of irradiation damage in ion-irradiated materials.

Dislocations in naturally deformed olivine: Example of a mylonitic peridotite

We have investigated the microstructure of naturally deformed olivine (chemically equilibrated at 1000 °C) by conventional transmission electron microscopy and electron tomography. The peridotite specimen, from Oman ophiolite, has a mylonitic microstructure with remnant, strongly deformed, millimetric porphyroclasts co-existing with small newly formed olivine grains generated by dynamic recrystallization. Imaging by transmission electron microscopy reveals that both newly formed grains and porphyroclasts display [100] and [001] dislocations activity. Subgrain boundaries are composed of either [100] or [001] dislocations. The characterization of this natural sample also permits to identify sporadic [100] dislocation loops, rare [010] dislocation, infrequent melt, and intragranular bubbles or along subgrain boundaries. Electron tomography permits to identify several glide planes, which are similar to previous observations acquired on experimentally deformed polycrystalline olivine, more importantly electron tomography also permits to evidence combination of glide, climb and mixed climb (dislocation moving in an intermediate plane between the plane of glide plane and the plane of pure climb). Our study further infers the diversity of linear defects responsible for plastic deformation of olivine at lithospheric conditions.

3D characterization of abnormal grain growth in nanocrystalline nickel

AbstractAbnormal grain growth, a pervasive phenomenon witnessed during the annealing of nanocrystalline metals, precipitates a swift diminution of the distinctive properties inherent to such materials. Historically, conventional transmission electron microscopy has struggled to efficiently procure comprehensive five‐parameter crystallographic information from a substantial number of grain boundaries in nanocrystalline metals, thus inhibiting a deeper understanding of abnormal grain growth behavior within nanocrystalline materials. In this study, we utilize a high‐throughput characterization method—three‐dimensional orientation mapping in the TEM (3D‐OMiTEM) to characterize the crystallographic five‐parameter character of grain boundaries with an area of over 3.4 × 106 nm2 in an abnormally grown nanocrystalline nickel sample. When coupled with existing theoretical simulation results, it is discerned that the grain boundary population shows a relatively large scatter when it is correlated to the calculated grain boundary energy; the grain boundaries of abnormally grown grains exhibit lower grain boundary energy compared to those that have not undergone abnormal growth. Merging high‐throughput grain boundary information obtained from three‐dimensional orientation mapping data with grain boundary properties derived from high‐throughput theoretical calculations following the concept of materials genome engineering will undoubtedly facilitate further advancements in comprehending and discerning the interfacial behaviors of crystalline materials.

Spatially and Chemically Resolved Visualization of Fe Incorporation into NiO Octahedra during the Oxygen Evolution Reaction.

The activity of Ni (hydr)oxides for the electrochemical evolution of oxygen (OER), a key component of the overall water splitting reaction, is known to be greatly enhanced by the incorporation of Fe. However, a complete understanding of the role of cationic Fe species and the nature of the catalyst surface under reaction conditions remains unclear. Here, using a combination of electrochemical cell and conventional transmission electron microscopy, we show how the surface of NiO electrocatalysts, with initially well-defined surface facets, restructures under applied potential and forms an active NiFe layered double (oxy)hydroxide (NiFe-LDH) when Fe3+ ions are present in the electrolyte. Continued OER under these conditions, however, leads to the creation of additional FeOx aggregates. Electrochemically, the NiFe-LDH formation correlates with a lower onset potential toward the OER, whereas the formation of the FeOx aggregates is accompanied by a gradual decrease in the OER activity. Complementary insight into the catalyst near-surface composition, structure, and chemical state is further extracted using X-ray photoelectron spectroscopy, operando Raman spectroscopy, and operando X-ray absorption spectroscopy together with measurements of Fe uptake by the electrocatalysts using time-resolved inductively coupled plasma mass spectrometry. Notably, we identified that the catalytic deactivation under stationary conditions is linked to the degradation of in situ-created NiFe-LDH. These insights exemplify the complexity of the active state formation and show how its structural and morphological evolution under different applied potentials can be directly linked to the catalyst activation and degradation.

Reliable identification of the complex or superlattice nature of intrinsic and extrinsic stacking faults in the L12 phase by high-resolution imaging

Planar defect-based deformation mechanisms are becoming increasingly important for understanding and tuning the mechanical properties of superalloys. Typically, to elucidate such deformation mechanisms, the type of planar defect is determined by conventional transmission electron microscopy or by high-resolution imaging along 〈110〉 crystallographic directions. However, an unambiguous identification of the complex or non-complex nature of intrinsic and extrinsic stacking faults in the L12 phase may not be possible by analyzing only one projection. To overcome this limitation, we present a straightforward approach to identify the superlattice or complex character of intrinsic and extrinsic faults in the L12 structure in high-resolution scanning transmission electron microscopy by tilting to a different zone axis and examining the shift of superlattice planes. Using this approach, two key aspects of the Kolbe mechanism for the formation of superlattice extrinsic stacking faults are verified for the first time: the presence of a complex intrinsic fault segment at the leading end and the occurrence of re-ordering.

Comparative of machine learning classification strategies for electron energy loss spectroscopy: Support vector machines and artificial neural networks

Machine Learning (ML) strategies applied to Scanning and conventional Transmission Electron Microscopy have become a valuable tool for analyzing the large volumes of data generated by various S/TEM techniques. In this work, we focus on Electron Energy Loss Spectroscopy (EELS) and study two ML techniques for classifying spectra in detail: Support Vector Machines (SVM) and Artificial Neural Networks (ANN). Firstly, we systematically analyze the optimal configurations and architectures for ANN classifiers using random search and the tree-structured Parzen estimator methods. Secondly, a new kernel strategy is introduced for the soft-margin SVMs, the cosine kernel, which offers a significant advantage over the previously studied kernels and other ML classification strategies. This kernel allows us to bypass the normalization of EEL spectra, achieving accurate classification. This result is highly relevant for the EELS community since we also assess the impact of common normalization techniques on our spectra using Uniform Manifold Approximation and Projection (UMAP), revealing a strong bias introduced in the spectra once normalized. In order to evaluate and study both classification strategies, we focus on determining the oxidation state of transition metals through their EEL spectra, examining which feature is more suitable for oxidation state classification: the oxygen K peak or the transition metal white lines. Subsequently, we compare the resistance to energy loss shifts for both classifiers and present a strategy to improve their resistance. The results of this study suggest the use of soft-margin SVMs for simpler EELS classification tasks with a limited number of spectra, as they provide performance comparable to ANNs while requiring lower computational resources and reduced training times. Conversely, ANNs are better suited for handling complex classification problems with extensive training data.

Micron-scale 1D migration of interstitial-type dislocation loops in aluminum

Micron-scale one-dimensional (1D) migration of dislocation loops was discovered by in-situ transmission electron microscope (TEM) observations. Interstitial-type dislocation loops were initially implanted in pure aluminum foils with 30 keV H+. The dynamic behaviour of these loops was investigated in a conventional TEM, operated at 200 kV. During electron irradiation, dislocation loops exhibited super long-range (1.5 μm) 1D migration, featured average speeds over 1 nm/s, and left behind long-lasting tracks up to 60 s. The increase in loop migration speed gave rise to increased track length but showed a subtle impact on the existing time-scale of tracks. Deliberate investigations by varying electron beam positions revealed that the migration distance could be boosted up to micron-scale when exposed to high concentration gradients of self-interstitial atoms built during electron irradiation. Current findings may shed light on the understanding of irradiation damage evolution and bring about new insights into the development of irradiation-resistant materials.

Three-Dimensional Ultrastructural Analysis of the Head-Most Mitochondrial Roots of Mice Spermatozoa Using Focused Ion Beam/Scanning Electron Microscopy (FIB/SEM) Tomography.

This study aimed to clarify the three-dimensional ultrastructure of head-side mice spermatozoa mitochondria. Six 13-week-old male C57BL/6 mice were deeply anesthetized, perfused with 2% paraformaldehyde, and placed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.3) for electron microscopy. After perfusion, the vas deferens was removed, and the specimens were cut into small cubes and subjected to postfixation and en bloc staining. Three-dimensional ultrastructural analysis was performed on five mitochondria on the spermatozoa head using conventional transmission electron microscopy (TEM) and focused ion beam/scanning electron microscopy (FIB/SEM) tomography. Conventional TEM analysis showed that head-side mitochondria were not spiral in morphology but clearly horizontal to the sperm axis. However, this was difficult to evaluate further using conventional TEM. In the FIB/SEM analysis, the first and second head-most mitochondria were flat and straight, with no helix, and shaped as an attachment plug with two electrodes, and their tail side contacted the third mitochondrion. The third mitochondrion was shorter than the fourth and fifth and had a semicircular arching structure. The fourth and fifth mitochondria were spiral-shaped and intertwined. The redundant nuclear envelope encircled the head-most mitochondria. This ultrastructural analysis clarified that the head-most mitochondria have a unique morphology.

Multi-Organ Morphological Findings in a Humanized Murine Model of Sickle Cell Trait.

Sickle cell disease (SCD) is caused by the homozygous beta-globin gene mutation that can lead to ischemic multi-organ damage and consequently reduce life expectancy. On the other hand, sickle cell trait (SCT), the heterozygous beta-globin gene mutation, is still considered a benign condition. Although the mechanisms are not well understood, clinical evidence has recently shown that specific pathological symptoms can also be recognized in SCT carriers. So far, there are still scant data regarding the morphological modifications referable to possible multi-organ damage in the SCT condition. Therefore, after genotypic and hematological characterization, by conventional light microscopy and transmission electron microscopy (TEM), we investigated the presence of tissue alterations in 13 heterozygous Townes mice, one of the best-known animal models that, up to now, was used only for the study of the homozygous condition. We found that endothelial alterations, as among which the thickening of vessel basal lamina, are ubiquitous in the lung, liver, kidney, and spleen of SCT carrier mice. The lung shows the most significant alterations, with a distortion of the general tissue architecture, while the heart is the least affected. Collectively, our findings contribute novel data to the histopathological modifications at microscopic and ultrastructural levels, underlying the heterozygous beta-globin gene mutation, and indicate the translational suitability of the Townes model to characterize the features of multiple organ involvement in the SCT carriers.

Dynamical scattering in ice-embedded proteins in conventional and scanning transmission electron microscopy.

Structure determination of biological macromolecules using cryogenic electron microscopy is based on applying the phase object (PO) assumption and the weak phase object (WPO) approximation to reconstruct the 3D potential density of the molecule. To enhance the understanding of image formation of protein complexes embedded in glass-like ice in a transmission electron microscope, this study addresses multiple scattering in tobacco mosaic virus (TMV) specimens. This includes the propagation inside the molecule while also accounting for the effect of structural noise. The atoms in biological macromolecules are light but are distributed over several nanometres. Commonly, PO and WPO approximations are used in most simulations and reconstruction models. Therefore, dynamical multislice simulations of TMV specimens embedded in glass-like ice were performed based on fully atomistic molecular-dynamics simulations. In the first part, the impact of multiple scattering is studied using different numbers of slices. In the second part, different sample thicknesses of the ice-embedded TMV are considered in terms of additional ice layers. It is found that single-slice models yield full frequency transfer up to a resolution of 2.5 Å, followed by attenuation up to 1.4 Å. Three slices are sufficient to reach an information transfer up to 1.0 Å. In the third part, ptychographic reconstructions based on scanning transmission electron microscopy (STEM) and single-slice models are compared with conventional TEM simulations. The ptychographic reconstructions do not need the deliberate introduction of aberrations, are capable of post-acquisition aberration correction and promise benefits for information transfer, especially at resolutions beyond 1.8 Å.

Nano-refinement of the face-centered cubic Zr(Fe,Cr)2 secondary phase particles in Zircaloy-4 alloy via localized-shearing/bending-driven fracture under high-temperature compression

Nanoparticles are extensively introduced to improve the mechanical, physical, and chemical properties of alloys. In the present study, the underlying nano-refinement mechanisms of face-centered cubic Zr(Fe, Cr)2 secondary phase particles (SPPs) that precipitated in Zircaloy-4 alloy under high-temperature compression were investigated in detail by utilizing high-resolution transmission electron microscopy (HRTEM) and conventional TEM techniques. The frequently observed Zr(Fe, Cr)2 SPPs were incoherent with the matrix and exhibited brittle fracture behaviors without measurable plasticity. HRTEM observations revealed two mechanisms underlying the nano-refinement of incoherent micro-sized SPPs via localized shear fracture on {112¯}SPP and nanoprecipitate-assisted bending fracture, respectively. The latter was, for the first time, found to occur when the movements of large SPPs were blocked by nanometer-sized SPP during alloy deformation. Accordingly, two force models were proposed to visualize their potential nano-refinement processes. The knowledge attained from this study sheds new light on the deformation behaviors of Zr(Fe, Cr)2 SPPs and their associated size refinement mechanisms under high-temperature compression, and is expected to greatly benefit the process optimization of zirconium alloys to achieve precipitate nano-refinement.

A New Cell Line Derived from the Caudal Fin of the Dwarf Gourami (Trichogaster lalius) and Its Susceptibility to Fish Viruses.

The detection of megalocytiviruses, especially the infectious spleen and kidney necrosis virus (ISKNV), in ornamental fish has increased with the rapid growth of the ornamental fish industry. In this study, dwarf gourami fin (DGF) cells derived from the caudal fin of the dwarf gourami (Trichogaster lalius), which is highly susceptible to red sea bream iridovirus (RSIV) and ISKNV, were established and characterized. The DGF cells were grown at temperatures ranging from 25 °C to 30 °C in Leibovitz's L-15 medium supplemented with 15% fetal bovine serum and were subcultured for more than 100 passages, predominantly with epithelial-like cells. DGF cells had a diploid chromosome number of 2n = 44. Although the initial purpose of this study was to establish a cell line for the causative agents of red sea bream iridoviral disease (RSIV and ISKNV), DGF cells were also susceptible to rhabdoviruses (viral hemorrhagic septicemia virus, hirame rhabdovirus, and spring viraemia of carp virus), exhibiting a significant cytopathic effect characterized by cell rounding and lysis. Additionally, viral replication and virion morphology were confirmed using virus-specific conventional polymerase chain reaction and transmission electron microscopy. Furthermore, both RSIV and ISKNV were replicated at high concentrations in DGF cells compared to other cell lines. Notably, the DGF cells maintained a monolayer during ISKNV infection, indicating the possibility of persistent infection. Thus, DGF can be used for viral diagnosis and may play a critical role in advancing our understanding of ISKNV pathogenesis.