Optimum bright-field scanning transmission electron microscopy (OBF STEM) is a recently developed low-dose imaging technique that uses a segmented or pixelated detector. While we previously reported that OBF STEM with a segmented detector has a higher efficiency than conventional STEM techniques such as annular bright field (ABF), the imaging efficiency is expected to be further improved by using a pixelated detector. In this study, we adopted a pixelated detector for the OBF technique and investigated the imaging characteristics. Because OBF imaging is based on the thick weak phase object approximation (tWPOA), a non-zero crystalline sample thickness is considered in addition to the conventional WPOA, where the pixelated OBF method can be regarded as the theoretical extension of single side band (SSB) ptychography. Thus, we compared these two techniques via signal-to-noise ratio transfer functions (SNRTFs), multi-slice image simulations, and experiments, showing how the OBF technique can improve dose efficiency from the conventional WPOA-based ptychographic imaging.

Adapting to environmental changes and formulating behavioral strategies are central to the nervous system, with the prefrontal cortex being crucial. Chronic stress impacts this region, leading to disorders including major depression. This review discusses the roles for prefrontal cortex and the effects of stress, highlighting similarities and differences between human/primates and rodent brains. Notably, the rodent medial prefrontal cortex is analogous to the human subgenual anterior cingulate cortex in terms of emotional regulation, sharing similarities in cytoarchitecture and circuitry, while also performing cognitive functions similar to the human dorsolateral prefrontal cortex. It has been shown that chronic stress induces atrophic changes in the rodent mPFC, which mirrors the atrophy observed in the subgenual anterior cingulate cortex and dorsolateral prefrontal cortex of depression patients. However, the precise alterations in neural circuitry due to chronic stress are yet to be fully unraveled. The use of advanced imaging techniques, particularly volume electron microscopy, is emphasized as critical for the detailed examination of synaptic changes, providing a deeper understanding of stress and depression at the molecular, cellular and circuit levels. This approach offers invaluable insights into the alterations in neuronal circuits within the medial prefrontal cortex caused by chronic stress, significantly enriching our understanding of stress and depression pathologies.

The anisotropic electronic structure of MgB2C2 was studied using soft X-ray emission spectroscopy electron microscopes. MgB2C2 fragments were selected by examining C K-emission profiles. C and B K-emission and Mg L-emission spectra were obtained, revealing common and distinct structures that reflect the mixing of valence orbitals. Since the material is reported to have two-dimensional B-C honeycomb layers, the orientational dependence of these emission spectra was also examined. Experimental data were compared with theoretically calculated partial density of states of the valence bands of the material. The C K-emission profile showed an apparent orientational dependence, while the B K-emission exhibited minimal dependence. This difference originated from the different energy distributions of C-2pz and B-2pz components in the valence bands. The Mg L-emission intensity was very small, likely due to charge transfer from Mg atoms to B-N layers. The Mg L-emission profile showed a peak related to structures in C-K and B-K. An unexpected intensity was observed just above the valence bands, which also showed orientational dependence, possibly due to a small deviation from the ideal composition of Mg:B:C = 1:2:2.

Although modern scanning electron microscope (SEM) possess several electron detectors, it is not clear what kind of information is contained in a SEM image taken by a certain detector. Especially the detectors installed in the objective lens are difficult to know their characters. Thus, we propose a simple method to assess the acceptance of electron detector using a stainless-steel sphere. After taking images under certain conditions, say electron beam energy, working distance etc., the image intensity of each pixel point, which is characterized by coordinate (θ, φ), is evaluated. The advantage of this method is the ease of implementation and the whole information of electron emission from the tilted surfaces is contained in the image. Using this information, the acceptance of the detector can be analyzed systematically. In this paper, the traditional Everhart-Thornley detector is analyzed with this method. It is demonstrated how the sphere image changes according to the measurement condition. The ET image quality is strongly governed by working distance but not so much by the electron beam energy. We propose an alternative method to avoid the ambiguity of working distance. Using a needle type specimen stage, the ET image does not vary so much with WD and the reliability of ET image significantly improves.

A simple method that improves the resolution of the phase measurement of differential phase-contrast (DPC) scanning transmission electron microscopy for closed-type environmental cell applications was developed and tested using a model sample simulating environmental cell observations. Because the top and bottom membranes of an environmental cell are typically far apart, the images from these membranes are shifted widely by tilt-series acquisition, and averaging the images after alignment can effectively eliminate undesired signals from the membranes while improving the signal from the object of interest. It was demonstrated that a phase precision of 2π/100 rad is well achievable using the proposed method for the sample in an environmental cell.

Structural observations are essential for the advancement of life science. Volume electron microscopy has recently realized remarkable progress in the three-dimensional analyses of biological specimens for elucidating complex ultrastructures in several fields of life science. The advancements in volume electron microscopy technologies have led to improvements, including higher resolution, more stability, and the ability to handle larger volumes. Although human applications of volume electron microscopy remain limited, the reported applications in various organs have already provided previously unrecognized features of human tissues and also novel insights of human diseases. Simultaneously, the application of volume electron microscopy to human studies faces challenges, including ethical and clinical hurdles, costs of data storage and analysis, and efficient and automated imaging methods for larger volume. Solutions including the use of residual clinical specimens and data analysis based on artificial intelligence would address those issues and establish the role of volume electron microscopy in human structural research. Future advancements in volume electron microscopy are anticipated to lead to transformative discoveries in basic research and clinical practice, deepening our understanding of human health and diseases for better diagnostic and therapeutic strategies.

This report revisits the statistical atom location by channeling enhanced microanalysis (St-ALCHEMI) method, correcting the dopant site occupancy error by applying an appropriate error propagation rule. A revised equation for calculating the uncertainty in the determined dopant fractions is proposed. The revised equation is expected to correct the uncertainty in the determined dopant fractions, which is particularly significant in cases of low dopant concentrations and variable dopant occupancies across inequivalent host atomic sites. The approach is validated using Eu-doped Ca2SnO4 as a typical model system.

We have demonstrated localized surface plasmon (LSP)-enhanced cathodoluminescence (CL) from an atomic layer deposition (ALD)-grown Al2O3/ZnO/Al2O3 heterostructure to develop a bright nanometer-scale light source for an electron beam excitation-assisted (EXA) optical microscope. Three types of metals, Ag, Al, and Au, were compared, and an 181-fold enhancement of CL emission was achieved with Ag nanoparticles (NPs), with the plasmon resonance wavelength close to the emission wavelength energy of ZnO. The enhanced emission is plausibly attributed to LSP/exciton coupling. However, it is also attributed to an increase in coupling efficiency with penetration depth and also to an increase in light extraction efficiency by grading the refractive indices at the heterostructure.

The surface sensitivity of high-resolution secondary electron (SE) imaging is examined using twisted bilayers of MoS2 stacked at an angle of 30-degree. High-resolution SE images of the twisted bilayer MoS2 show a honeycomb structure composed of Mo and S atoms, elucidating the monolayer structure of MoS2. Simultaneously captured annular dark-field scanning transmission electron microscope images from the same region show the projected structure of the two layers. That is, the SE images from the bilayer MoS2 selectively visualize the surface monolayer. It is noted that SE yields from the surface monolayer are approximately 3 times higher than those from the second monolayer, likely attributable to attenuation when SEs emitted from the second layer traverse the surface layer. Mini abstract: The surface sensitivity of atomic resolution secondary electron imaging is examined using MoS2 bilayers, the thinnest system composed of a surface layer and substrate. This study reveals that the secondary electrons visualize the atomic arrangement of the surface monolayer three times more intensely than that of the second layer.

X-ray microscopy using computed tomography (CT) is an excellent three-dimensional imaging instrument. Three-dimensional X-ray microscopy (3DXRM) is a nondestructive imaging technique used to inspect internal and external structures in units of submicrometers or less. The 3DXRM, although attractive, is mostly used as an observation instrument and is limited as a measurement system in quantitative evaluation and quality control. Calibration is required for use in measurement systems such as coordinate measurement systems, and specific standard samples and evaluation procedures are needed. The certified values of the standard samples must ideally be traceable to the International System of Units (SI). In the 3DXRM measurement system, line structures (LSs) are fabricated as prototype standard samples to conduct magnification calibration. In this study, we evaluated the LS intervals using calibrated cross-sectional scanning electron microscopy (SEM). A comparison of the evaluation results between SEM and 3DXRM for the LS intervals provided the magnification calibration factor for 3DXRM and validated the LSs, whereby the interval methods and feasibility of constructing an SI traceability system were evaluated using the calibrated SEM. Consequently, a magnification calibration factor of 1.01 was obtained for 3DXRM based on the intervals of the LSs evaluated by SEM. A possible route for realizing SI-traceable magnification calibration of 3DXRM has been presented.

The three-dimensional (3D) anatomical structure of living organisms is intrinsically linked to their functions, yet modern life sciences have not fully explored this aspect. Recently, the combination of efficient tissue clearing techniques and light-sheet fluorescence microscopy (LSFM) for rapid 3D imaging has improved access to 3D spatial information in biological systems. This technology has found applications in various fields, including neuroscience, cancer research, and clinical histopathology, leading to significant insights. It allows imaging of entire organs or even whole bodies of animals and humans at multiple scales. Moreover, it enables a form of spatial omics by capturing and analyzing cellome information, which represents the complete spatial organization of cells. While current 3D imaging of cleared tissues has limitations in obtaining sufficient molecular information, emerging technologies such as multi-round tissue staining and super-multicolor imaging are expected to address these constraints. 3D imaging using tissue clearing and light-sheet microscopy thus offers a valuable research tool in the current and future life sciences for acquiring and analyzing large-scale biological spatial information.

In this study, we experimentally analyzed the charging phenomenon when an insulating resist film on a conductive layer formed on bulk glass is irradiated by electron beams. To quantify the charging potential induced, an electrostatic force microscope device was installed in the scanning electron microscope sample chamber, and potential distributions formed under various exposure conditions were obtained. Based on the results obtained, a model for charge accumulation within the sample, explaining positive and negative charging and their transitions, was developed. At an electron beam acceleration voltage of 30 kV, the following observations were made: "global charging" could be avoided by applying -5V to the sample. Regarding "local charging" near the exposure area of the electron beam, at low exposure doses, emission of secondary electrons from the sample surface induced positive charging, while the accumulation of incident electrons within the sample induced negative charging. At exposure doses where the effects of both are balanced, the sample exhibited zero potential, revealing the appearance of the "first zero-cross exposure dose". At higher exposure doses, the sample transitions from negative to positive as the exposure dose increases due to the electron beam induced conduction, resulting in the so-called "second zero-cross exposure dose". The exposure dose dependence of the charging potential distribution at various acceleration voltages was obtained. In particular, we found that at an acceleration voltage of 0.6 kV, the sample surface is not charged even when exposed to small to very large doses of electron beams.

Heteroepitaxial interfaces are important because they determine the performance of devices such that career mobility is sensitive to the distribution of roughness, strain and composition at the interface. High-angle annular dark field imaging in scanning transmission electron microscopy has been utilized to capture them at an atomic scale. For precise identification of atomic column positions, a technique has been proposed to average multiple image frames taken at a high scanning rate by their positional alignment for increasing signal-to-noise ratio. However, the positional alignment between frames is sometimes incorrectly estimated because of the almost perfect periodic structure at the interfaces. Here, we developed an approach for precise positional alignment, where the images are first aligned by two consecutive images and then are aligned more precisely against the integrated image of the first alignment. We demonstrated our method by applying it to the heterointerface of Si0.8Ge0.2 (Si: silicon, Ge: germanium) epitaxial thin films on a Si substrate.

The spatial coherence and the axial brightness of a cold field emission gun, a Schottky field emission gun and a $\mathrm{LaB}_6$ thermionic gun are precisely measured. By analyzing the Airy pattern from a selected area aperture, various parameters including the spatial coherence length are determined. Using the determined coherence length, the axial brightness of the field emission guns is estimated using the equation which we previously derived based on the discussion of the Wigner function of an electron beam. We also make some extensions in the method to be applicable to the measurements of the thermionic gun, which has anisotropic intensity distribution in most case unlike the field emission guns. Not conventional average brightness but the axial brightness measured for the three kinds of emitters are compared accurately and precisely without influenced by the measurement conditions.

Cell membrane structures are supramolecular complexes that require the ordered assembly of membrane proteins and lipids. The morphology of various cell adhesion structures in multicellular organisms, such as those between epithelial cells, neural synapse and immune synapses, was initially described through electron microscopic analyses. Subsequent studies aimed to catalog their constituent proteins, which encompass transmembrane cell adhesion molecules, cytoskeletal proteins, and scaffolding proteins that bind the two components. However, the diversity of plasma membrane lipids and their significance in the organization of cell adhesion structures were underappreciated until recently. It is now understood that phase separation of lipids and liquid-liquid phase separation of proteins are important driving forces for such self-assembly. In this review, we summarize recent findings on the role of lipids as scaffolds for supramolecular complexes, using tight junctions in epithelial cells as an example.

Scanning Transmission Electron Microscopy (STEM) enables direct determination of atomic arrangements in materials and devices. However, materials such as battery components are weak for electron beam irradiation and low electron doses are required to prevent beam-induced damages. Noise removal is thus essential for precise structural analysis of electron beam sensitive materials at atomic resolution. Total square variation (TSV) regularization is an algorithm that exhibits high noise removal performance. However, the use of the TSV regularization term leads to significant image blurring and intensity reduction. To address these problems, we here propose a new approach adopting L2 norm regularization based on higher-order total variation. An atomic-resolution STEM image can be approximated as a set of smooth curves represented by quadratic functions. Since the third-degree derivative of any quadratic function is 0, total third-degree variation (TTDV) is suitable for a regularization term. The application of TTDV for denoising the atomic-resolution STEM image of CaF2 observed along the [001] zone axis is shown, where we can clearly see the Ca and F atomic columns without compromising image quality.

Sandwich freezing is a method of rapid freezing by sandwiching specimens between two copper disks and has been used for observing exquisite close-to-native ultrastructure of living yeast and bacteria. Recently, this method has been found to be useful for preserving cell images of glutaraldehyde-fixed cultured cells, as well as animal and human tissues. In the present study, this method was applied to observe the fine structure of living Arabidopsis plant tissues and was found to achieve excellent ultrastructural preservation of cells and tissues. This is the first report of applying the sandwich freezing method to observe plant tissues.

A new configuration for near-field ptychography using a full-field illumination with a structured electron beam is proposed. A structured electron beam illuminating the entire field of view is scanned over the specimen, and a series of in-line holograms formed in the near-field region below the specimen are collected. The structured beam is generated by a conductive film with random openings, which ensures high stability and coherence of the beam. Observation in the near-field region reduces the beam concentration that occurs in the far-field region, which contributes to accurate recording of the beam intensity with a finite dynamic range of the detectors. The use of full-field illumination prevents the accumulation of errors caused by concatenating the local structures, which is the method used in conventional reconstruction. Since all holograms are obtained from the entire field of view, they have uniform multiplicity in terms of specimen information within the field of view. This contributes to robust and efficient reconstruction for a large field of view. The proposed method was tested using both simulated and experimental holograms. For the simulated holograms, the reconstruction of the specimen transmission function was achieved with an error less than 1/3485 of the wavelength. The method was further validated using experimental holograms obtained from MgO particles. The reconstructed phase transmission function of the specimen was consistent with the specimen structure and was equivalent to a mean inner potential of V on the MgO particle, which is in close agreement with previously reported values.