Although scanning transmission electron microscopy (STEM) images of individual heavy atoms were reported 50 years ago, the applications of atomic-resolution STEM imaging became wide spread only after the practical realization of aberration correctors on field-emission STEM/TEM instruments to form sub-Ångstrom electron probes. The innovative designs and advances of electron optical systems, the fundamental understanding of electron–specimen interaction processes, and the advances in detector technology all played a major role in achieving the goal of atomic-resolution STEM imaging of practical materials. It is clear that tremendous advances in computer technology and electronics, image acquisition and processing algorithms, image simulations, and precision machining synergistically made atomic-resolution STEM imaging routinely accessible. It is anticipated that further hardware/software development is needed to achieve three-dimensional atomic-resolution STEM imaging with single-atom chemical sensitivity, even for electron-beam-sensitive materials. Artificial intelligence, machine learning, and big-data science are expected to significantly enhance the impact of STEM and associated techniques on many research fields such as materials science and engineering, quantum and nanoscale science, physics and chemistry, and biology and medicine. This review focuses on advances of STEM imaging from the invention of the field-emission electron gun to the realization of aberration-corrected and monochromated atomic-resolution STEM and its broad applications.

The precise structural solution of crystals on a mesostructural scale is challenging due to the difficulties in obtaining electron diffraction and the complicated relationship between the crystal structure factors (CSFs) and the conventional underfocus phase-contrast transmission electron microscopy (TEM) images due to the large unit cell and the complex structures. Here, we present the structural investigation of mesostructured crystals via the combination of electron crystallographic Fourier synthesis and high-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM) that only relies on the mass-thickness contrast. The three-dimensional electrostatic potential is reconstructed from the amplitudes and phases extracted from the Fourier transforms of the corresponding HAADF-STEM images and merged into a set of CSFs. This method is verified on silica scaffolds following a shifted double-diamond surface network with space group I41/amd. The results indicate that electron crystallography reconstruction by HAADF-STEM images is more suitable and accurate in determining the structure in comparison with conventional TEM electron crystallography reconstruction. This approach transfers the contrast of mesostructured crystals to images more accurately and the relationship between the Fourier transforms of HAADF-STEM images and the CSFs is more intuitive. It shows great advantages for the structural solution of crystals on the mesostructural scale.

Atomic mapping of nanomaterials, in particular nanoparticles, using atom probe tomography (APT) is of great interest, as their properties strongly depend on shape, size, and composition. However, APT analyses of nanoparticles are extremely challenging, and there is an urgent need for developing robust and universally applicable sample preparation methods. Herein, we explored a method based on pulse electrodeposition to embed Ag nanoparticles in a Ni matrix and prepare APT specimens from the resulting composite film. By systematically varying the duty cycle during pulse electrodeposition, the dispersion and number density of the nanoparticles within the matrix was significantly enhanced as compared to DC electrodeposition. Several Ag nanoparticles were analyzed with APT from such samples. Shape distortions and biased compositions were observed for the Ag nanoparticles after applying a standard data reconstruction protocol. Numerical simulations of the field evaporation process showed that such artifacts were caused by a difference in the evaporation field of Ni and Ag and a local magnification effect. We expect such detrimental effects to be mitigated by a careful selection of the matrix material, matching the evaporation field of the nanoparticles. Furthermore, we anticipate that the method presented herein can be extended to a wider range of nanomaterials.

Atomically sharpened tips have attracted much interest in the imaging and manufacturing fields due to their high spatial resolutions. Typically, tungsten (W) is mainly used as the material of such a tip, but when the W tip is used in an oxygen environment, a limit is revealed due to corrosiveness stemming from a reaction with the oxygen gas. To solve this problem, methods of depositing a metal on W that does not react with oxygen have been studied. In this study, we introduce a method of depositing iridium (Ir) directly onto an insulating layer without an additional pretreatment to remove the insulating layer remaining on the W surface, forming an Ir-nanopyramid structure at the apex of the W tip by field evaporation and faceting. Field ion microscopy and atom probe tomography were used to analyze the crystal structure and composition at the apex during the faceting process, and the overall tip shape change after faceting was compared and analyzed with transmission electron microscopy. The proposed method does not have a tip heating step when creating an atomically sharp tip such that it can be made easily with a simpler equipment configuration than in the existing method.

Nanowire-based nanocomposite materials are being developed as transparent and flexible electrodes or as stretchable conductors and dielectrics for biosensing. Here, we theoretically investigate the use of scanning dielectric microscopy (SDM) to characterize these materials in a nondestructive way, with a special focus on the achievable spatial resolution and the possibility of detection of the capacitive coupling between nearby nanowires. Numerical calculations with models involving single and multiple buried nanowires have been performed. We demonstrate that the capacitance gradient spread function of a single buried nanowire consists of a modified Lorenzianan with a cubic decay. We show that the achievable spatial resolution can be determined with good accuracy with the help of this spread function. It is shown that, in general, the spatial resolution worsens when any system parameter decreases the maximum of the nanowire spread function or increases its width, or both. Finally, we show that SDM measurements are also sensitive to the capacitive coupling between nearby nanowires. This latter result is of utmost relevance since the macroscopic electric properties of nanowire nanocomposites largely depend on the electric interaction between nearby nanowires. The present results show that SDM can be a valuable nondestructive subsurface characterization technique for nanowire nanocomposite materials.

The fine microstructure resulting from the martensitic transformation drives many of the desired mechanical properties for quench-and-temper steels. The scale of martensite features correlates with austenite grain size, such that finer austenite grains produce finer martensitic structures. Measuring prior austenite grain (PAG) size from observations of the hierarchical martensitic structure using etch-based techniques remains challenging, especially for fine-grained specimens with low C and P content. This has driven an interest in our field in reconstructing prior austenite microstructure from electron backscatter diffraction (EBSD) data collected on tempered martensite. However, reliably reconstructing the data is not straightforward because (1) up to 24 variants can theoretically be observed from a single PAG; (2) the shear-induced plasticity that arises from martensite formation produces significant scatter in the pole figure; (3) there is inherent noise in Hough-indexed EBSD data; (4) annealing twins exist within many PAGs, producing martensite variants of similar orientations when compared with the parent grains; and (5) variation in austenite orientation can occur across a single PAG. To the author's knowledge, the majority of reconstruction algorithms published to date utilize either point-to-point or flood-fill approaches, which can produce artifacts due to the relatively high probability of adjacent EBSD observations falling within any reasonable tolerance angle with 24 variants in a parent and four annealing twin orientations. Therefore, we propose a probabilistic austenite reconstruction technique based on a clustering algorithm known as graph cutting. We demonstrate its application to steels and a binary ferrous alloy, then validate the results against chemical etching and retained austenite orientations. The graph cutting technique utilizes the misorientation distribution of martensitic variants and the potential austenite orientations for each observation, as estimated via Bayesian inference of the experimental orientation relationship and its scatter. It is found that this technique produces accurate reconstructions with identification of annealing twins in the PAGs, even in the presence of a large fraction of poorly indexed data points.

Measurements of highly radioactive actinides such as Np, Pu, Am, or Cm in as-fabricated or irradiated nuclear fuels are crucial to improve the safety and performance of the nuclear fuel cycle, but also necessary to find solutions to the environmental impact of long-lived actinides in nuclear waste. Currently, the most common technique of measuring the quantity and distribution of actinides in nuclear fuel is electron probe microanalysis (EPMA). However, the accurate analysis of actinides by EPMA is accompanied by numerous obstacles such as interferences in the M-line X-ray region, absorption edges, and the lack of reference materials. Therefore, the measurement protocol of radioactive and irradiated samples requires further investigation and most importantly facilitation. Here, we establish a guideline for precise quantification of actinide elements (Th, U, Np, Pu, Am, and Cm) in fabricated or spent nuclear fuels. For this purpose, we have characterized actinide-bearing reference samples to visualize spectral interferences, list peak, and background positions and employ the standardless calibration curve method for the measurements of Am and Cm. This newly derived analytical protocol was successfully tested on an irradiated fuel sample and can be now implemented as an improved guideline for the quantification of actinides.

Current generation electron monochromators employed as attachments to scanning transmission electron microscopes (STEM) offer the ability to obtain vibrational information from materials using electron energy-loss spectroscopy (EELS). We show here that in crystals, long- and short-wavelength phonon modes can be probed simultaneously with on-axis vibrational STEM EELS. The long-wavelength phonons are probed via dipole scattering, while the short-wavelength modes are probed via impact scattering of the incident electrons. The localized character of the short-wavelength modes is demonstrated by scanning the electron beam across the edge of a hexagonal boron nitride nanoparticle. It is found that employing convergence angles that encompass multiple Brillouin zone boundaries enhances the short-wavelength phonon contribution to the vibrational energy-loss spectrum much more than that achieved by employing collection angles that encompass multiple Brillouin zone boundaries. Probing short-wavelength phonons at high spatial resolution with on-axis vibrational STEM EELS will help develop a fundamental connection between vibrational excitations and bonding arrangements at atomic-scale heterogeneities in materials.

A reformulated implementation of single-sideband ptychography enables analysis and display of live detector data streams in 4D scanning transmission electron microscopy (STEM) using the LiberTEM open-source platform. This is combined with live first moment and further virtual STEM detector analysis. Processing of both real experimental and simulated data shows the characteristics of this method when data are processed progressively, as opposed to the usual offline processing of a complete data set. In particular, the single-sideband method is compared with other techniques such as the enhanced ptychographic engine in order to ascertain its capability for structural imaging at increased specimen thickness. Qualitatively interpretable live results are obtained also if the sample is moved, or magnification is changed during the analysis. This allows live optimization of instrument as well as specimen parameters during the analysis. The methodology is especially expected to improve contrast- and dose-efficient in situ imaging of weakly scattering specimens, where fast live feedback during the experiment is required.

Cell confluence is an important metric in cell culture, as proper timing is essential to maintain cell phenotype and culture quality. To estimate cell confluence, transparent cells are observed under a phase-contrast or differential interference contrast microscope by a biologist, whose estimations are error-prone and subjective. To overcome the necessity of using the phase-contrast microscope and reducing intra- and inter-observer errors, we have proposed an algorithm that automatically measures cell confluence by using a commonly used brightfield microscope. The proposed method consists of a transport-of-intensity equation-based brightfield microscopic image processing, an image reconstruction method, and an adaptive image segmentation method based on edge detection, entropy filtering, and range filtering. Experimental results have shown that our method has outperformed several popular algorithms, with an F-score of 0.84 ± 0.07, in images with various cell confluence values. The proposed algorithm is robust and accurate enough to perform confluence measurement with various lighting conditions under a low-cost brightfield microscope, making it simple and cost-effective to use for a fully automated cell culture process.

The recent development of electron-sensitive and pixelated detectors has attracted the use of four-dimensional scanning transmission electron microscopy (4D-STEM). Here, we present a precession electron diffraction-assisted 4D-STEM technique for automated orientation mapping using diffraction spot patterns directly captured by an in-column scintillator-based complementary metal-oxide-semiconductor (CMOS) detector. We compare the results to a conventional approach, which utilizes a fluorescent screen filmed by an external charge charge-coupled device camera. The high-dynamic range and signal-to-noise characteristics of the detector greatly improve the image quality of the diffraction patterns, especially the visibility of diffraction spots at high scattering angles. In the orientation maps reconstructed via the template matching process, the CMOS data yield a significant reduction of false indexing and higher reliability compared to the conventional approach. The angular resolution of misorientation measurement could also be improved by masking reflections close to the direct beam. This is because the orientation sensitive, weak, and small diffraction spots at high scattering angles are more significant. The results show that fine details, such as nanograins, nanotwins, and sub-grain boundaries, can be resolved with a sub-degree angular resolution which is comparable to orientation mapping using Kikuchi diffraction patterns.

In Part I of this diptych, we outline the parallel mode of differential phase contrast (TEM-DPC), which uses real-space distortion of Fresnel images arising from electrostatic or magnetostatic fields to quantify the phase gradient of samples with some degree of structural contrast. We present an analysis methodology and the associated software tools for the TEM-DPC method and, using them together with numerical simulations, compare the technique to the widely used method of phase recovery based on the transport-of-intensity equation (TIE), thereby highlighting the relative advantages and limitations of each. The TEM-DPC technique is particularly suitable for in situ studies of samples with significant structural contrast and, as such, complements the TIE method since structural contrast usually hinders the latter, but is an essential feature that enables the former. In Part II of this work, we apply the theory and methodology presented to the analysis of experimental data to gain insight into two-dimensional magnetic phase transitions.

In Part I of this diptych, we outlined the theory and an analysis methodology for quantitative phase recovery from real-space distortions of Fresnel images acquired in the parallel mode of transmission electron microscopy (TEM). In that work, the properties of the method, termed TEM-differential phase contrast (TEM-DPC), were highlighted through the use of simulated data. In this work, we explore the use of the TEM-DPC technique with experimental cryo-TEM images of a thin lamella of a low-temperature two-dimensional (2D) ferromagnetic material, K2CuF4, to perform two tasks. First, using images recorded below the ordering temperature, we compare the TEM-DPC method with the transport of intensity one for phase recovery and discuss the relative advantages the former has for experimental data. Second, by tracking the induction of the sample as it is driven through a phase transition by heating, we extract estimates for the critical temperature and critical exponent of the order parameter. The value of the latter is consistent with the 2D XY class, raising the prospect that a Kosterlitz–Thoules transition may have occurred.

Amyloid plaques, one of the main hallmarks of Alzheimer's disease (AD), are classified into diffuse (associated with cognitive impairment) and dense-core types (a common finding in brains of people without Alzheimer's disease (non-AD) and without impaired cognitive function) based on their morphology. We tried to determine the usability of gray-level co-occurrence matrix (GLCM) texture parameters of homogeneity and heterogeneity for the differentiation of amyloid plaque images obtained from AD and non-AD individuals. Images of amyloid-β (Aβ) immunostained brain tissue samples were obtained from the Aging, Dementia and Traumatic Brain Injury Project. A total of 1,039 plaques were isolated from different brain regions of 69 AD and non-AD individuals and used for further GLCM analysis. Images of Aβ stained plaques show higher values of heterogeneity parameters and lower values of homogeneity parameters in AD patients, and vice versa in non-AD patients. Additionally, GLCM analysis shows differences in Aβ plaque texture between different brain regions in non-AD patients and correlates with variables that characterize patient's dementia status. The present study shows that GLCM texture analysis is an efficient method to discriminate between different types of amyloid plaques based on their morphology and thus can prove as a valuable tool in the neuropathological investigation of dementia.

The study of the sinking phenomenon of diatom cells, which have a slightly larger specific gravity (~1.3) compared to that of water, is an important research topic for understanding photosynthetic efficiency. In this study, we successfully demonstrated the observation of the sinking behaviors of four different species of diatom using a homemade “tumbled” optical microscope. A homemade 1 mm3 microchamber was employed to decrease the effects of convection currents. In the microchamber, diatom cells were basically settled in a linear manner without floating, although some of the cells were rotated during their sinking. Sinking speeds of the four species of diatom cells, Nitzschia sp., Pheodactylum tricornutum, Navicula sp., and Odontella aurita, were 0.81 ± 5.56, 3.03 ± 10.17, 3.29 ± 7.39, and 11.22 ± 21.42 μm/s, respectively, based on the automatic tracking analysis of the centroids of each cell. Manual analysis of a vector between two longitudinal ends of the cells (two-point analysis) was effective for quantitatively characterizing the rotation phenomenon; therefore, angles and angular velocities of rotating cells were well determined as a function of time. The effects of the cell shapes on sinking velocity could be explained by simulation analysis using the modified Stokes’ law proposed by Miklasz et al.

The testis of bamboo shark is characterized by diametric development leading to zonation architecture. Here, we investigated the staining pattern of 12 lectins in 6 groups of differential binding specificities within the germ, somatic, and interstitial cells of each zone. The neutral mucopolysaccharides appeared in the interstitial tissue in all the zones and became more significant in the spermatozoal–Sertoli cell junction. The cellular localization of the lectins varies in testicular zones and cell types. There was a gradual increase in glycosylation toward the degenerative zone. The increased intensity of most lectins in the interstitial cells indicates the association of glycoconjugates in their androgen-secreting activity. Statistical analyses showed a significant correlation between the groups of lectins and each lectin used, stronger response to lectins in the interstitial cells (ICs) than other cell types. Moreover, the response to glucosamine (GlcNAc), galactosamine (GalNAc), and fucose tended to be higher than glucose and galactose. Furthermore, the intensity of response was increased toward the degenerative zone. In addition, we can use peanut agglutinin (PNA) as an acrosomal marker in combination with other marker proteins for studying shark spermatogenesis. These findings refer to the crucial role of glycoconjugates in spermatogenesis in the bamboo shark testis.

In insects, the number, cytological and histological structures, and the spherocrystals of the Malpighian tubules (MTs) can vary considerably in different insect groups. These differences are considered important because they can be used as taxonomic characters. For this purpose, the ultrastructure of the MT epithelial cells in Leptophyes albovittata (Kollar, 1833) (Orthoptera, Tettigoniidae) was examined by light microscopy, scanning electron microscopy, and transmission electron microscopy. The wall of each tubule consists of a single layer of cells. These cells have round-shaped nuclei. Two different cell types were demonstrated in the tubule cell. These are cells that have electron-dense cytoplasm and electron-lucent cytoplasm. It was observed that the cytoplasm of these cells has many spherocrystals. The chemical composition of the spherocrystals was found to be high in carbon, phosphorus, and manganese in tubule cells.

Static stretching provides benefits to the range of motion, modulates intramuscular connective tissue, and is incorporated into warm-up exercises. In this study, we present the effects in the motor endplate and belly muscle resulting from previous static stretching to climbing training. Twenty-four adult male Wistar rats were divided into four groups (n = 6 each): Sedentary (Sed), Climbing (Clb), Static stretching (Ss), and Static stretching prior to climbing (Ssc). The animals (Clb, Ss, and Ssc groups) were subjected to a training protocol 3×/week for 8 weeks, and the Ssc group was subjected to the Ss and Clb protocols in the same session. Samples from the animals were processed for immunostaining, histochemistry, and light microscopy. The Clb group presented a higher motor endplate; the Ss group presented no changes in the motor endplate; and the Ssc group demonstrated a higher compactness. We concluded that static stretching prior to the climbing protocol maintained the density of the motor endplate and increased the compactness of the neuromuscular junction structure. Also, there was a reduction in the myofibers’ diameter (Type I and IIa), an increase in myofibrillar densities (Type I and IIx, and total), and the reorganization of the myonuclei and the interstitium.

ITGB1 (Integrin β1, CD29) is a member of the integrin family and has a role as a major adhesion receptor. Gastric cancer (GC) is an important cause of mortality worldwide, especially in China. As a potential cancer enhancer, the role ITGB1 plays in GC progression remains unclear. In the current study, our assay on the databases of tumoassociated gene expression and interaction found that the high expression of ITGB1 was closely correlated with the poor prognosis of GC patients. To explore the roles, ITGB1 plays in GC progression, and an ITGB1-deleted cell line (ITGB1−/−SGC7901) was generated using the CRISPR/Cas9 method. The tumor malignancy-associated cell behaviors and microstructures were detected, imaged, and analyzed using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), wound healing, transwell, scanning electron microscopy, laser scanning confocal microscopy, and others. The results indicated that ITGB1 deletion decreased the GC cell proliferation and motility, and inhibited motility-relevant microstructures, such as pseudopodia and filopodia, markedly in ITGB1-deleted SGC7901 cells. The analysis of STRING database and western blots indicated that ITGB1 contributes to the malignancy of GC mediated by Src-mediated FAK/PI3K/Akt signaling pathways. Taken together, the results showed that ITGB1 may be a potential targeting marker for GC diagnosis and therapy in the future.