Abstract: Studies visualizing plant tissues and organs in 3D by micro-computed tomography (CT) published since approximately 2015 are reviewed. In this period, the number of publications in the field of plant sciences dealing with micro-CT have increased along with the development of high-performance lab-based micro-CT system as well as the continuous development of cutting-edge technologies at synchrotron radiation facilities. A widespread use of commercially-available lab-based micro-CT systems enabling phase-contrast imaging technique, which is suitable for visualization of biological specimens composed of light elements, appears to have facilitated these studies. Unique features of the plant body, which are particularly utilized for imaging of plant organs and tissues by micro-CT, are having functional air spaces and specialized cell walls, such as lignified ones. In this review we briefly describe the basis of micro-CT technology first and then get down into details of its application to 3D visualization in plant sciences, which are categorized as follows: imaging of various organs, caryopses, seeds, other organs (reproductive organs, leaves, stems and petioles), various tissues (leaf venations, xylems, air-filled tissues, cell boundaries, cell walls), embolisms, root systems, hoping that wide users of microscopes and other imaging technologies will be interested also in micro-CT and obtain some hints for deeper understanding of structure of plant tissues and organs in 3D. Majority of current morphological studies using micro-CT still appear to be at qualitative level. Development of methodology for accurate 3D segmentation is needed for transition of the studies at from qualitative level to quantitative level in the future.

Abstract: We conducted a comprehensive analysis of the surface microtexture of kefir biofilms grown on Theobroma grandiflorum Shum (cupuaçu) juice using atomic force microscopy. Our goal was to investigate the unique monofractal and multifractal spatial patterns of these biofilms to complement the existing limited literature. The biofilms were prepared dispersing four different concentrations of kefir grains in cupuaçu juice. Our morphological analysis showed that the surface of the obtained biofilms is essentially formed by the presence of cupuaçu fibers and microorganisms like lactobacilli and yeast. The topographic height-based parameter analysis reveals that there is a dependence between surface roughness and the concentration of kefir grains used. The strongly anisotropic well-centralized topographical height distribution of the biofilms also exhibited a quasi-symmetrical and platykurtic pattern. The biofilms exhibit comparable levels of spatial complexity, surface percolation and surface homogeneity, which can be attributed to their similar topographic uniformity. This aspect was further supported by the presence of similar multifractality in the biofilms, suggesting that despite their varying topographic roughness, their vertical growth dynamics follow a similar pattern. Our findings demonstrate that the surface roughness of kefir biofilms cultivated on cupuaçu juice is influenced by the concentration of kefir grains in the precursor solution. However, this dependence follows a consistent pattern across different concentrations. Graphical Abstract.

Abstract: We present a review on the development and applications of the ultrafast transmission electron microscopy (UTEM) at RIKEN. We introduce the UTEM system for the pump-probe TEM observation in a wide temporal range. By combining the UTEM and pixelated detector, we further develop five-dimansional scanning TEM (5D STEM) which provides the ultrafast nanoscale movie of physical quantities in nano-materials, such as crystal lattice information and electromagnetic field, by convergent-beam electron diffraction (CBED) and differential phase contrast (DPC) imaging technique. We show our recent results on the nanosecond-to-microsecond magnetic skyrmion dynamics observed by Lorentz TEM (LTEM) and photo-induced acoustic wave generation in picosecond regime by bright-field TEM and electron diffraction measurements by UTEM. We also show the demonstartion of the 5D STEM on the quantitative time (t)-dependent strain mapping by CBED with an accuracy of 4 ps and 8 nm, and the ultrafast demagnetization under zero magnetic field observed by DPC with 10 ns and 400 nm resolution.

Abstract: Diffraction patterns contain useful information about the materials. Recent developments in four-dimensional scanning transmission electron microscopy, the acquisition of the spatial distribution of diffraction patterns have produced significant results. The acquisition of a temporal series of diffractions is achieved for a stationary beam. However, the acquisition of spatiotemporal distribution of diffraction patterns has only established in limited condition. In this study, we developed a simple method that enables the recording of the spatiotemporal distribution of diffraction patterns and applied it to the relaxation time measurement that is robust to sample drift. Mini-Abstract In this article, we suggest a method which enable to obtain a spatiotemporal distribution of diffraction patterns by a combination of 4DCanvas (JEOL Inc.) and Gatan DigiScan II (Gatan Inc). We measured the relaxation time of a metallic glass free from sample drift by using the suggested method.

Abstract: The advent of single-molecule atomic-resolution time-resolved electron microscopy (SMART-EM) has created a new field of “cinematic chemistry,” allowing for the cinematographic recording of dynamic behaviors of organic and inorganic molecules and their assembly. However, the limited electron dose per frame of video images presents a major challenge in SMART-EM. Recent advances in direct electron counting cameras and techniques to enhance image quality through the implementation of a denoising algorithm have enabled the tracking of stochastic molecular motions and chemical reactions with sub-millisecond temporal resolution and sub-angstrom localization precision. This review showcases the development of dynamic molecular imaging using the SMART-EM technique, highlighting insights into nanomechanical behavior during molecular shuttle motion, pathways of multistep chemical reactions, and elucidation of crystallization processes at the atomic level.

Abstract: Abstract Microscopy has been essential to elucidate micro- and nano-scale processes in space and time and has provided insights into cell and organismic functions. It is widely employed in cell biology, microbiology, physiology, clinical sciences and virology. While label-dependent microscopy, such as fluorescence microscopy, provides molecular specificity, it has remained difficult to multiplex in live samples. In contrast, label-free microscopy reports on overall features of the specimen at minimal perturbation. Here, we discuss modalities of label-free imaging at the molecular, cellular and tissue levels, including transmitted light microscopy, quantitative phase imaging, cryogenic electron microscopy or tomography and atomic force microscopy. We highlight how label-free microscopy is used to probe the structural organization and mechanical properties of viruses, including virus particles and infected cells across a wide range of spatial scales. We discuss the working principles of imaging procedures and analyses and showcase how they open new avenues in virology. Finally, we discuss orthogonal approaches that enhance and complement label-free microscopy techniques.

Abstract: Morphogenesis is a developmental process that shapes multicellular organisms through complex and cooperative cellular movements. To understand the complex interplay between genetic programs and resulting multicellular morphogenesis, it is essential to characterize the morphologies and dynamics at the single-cell level, with an understanding of how physical forces serve as both signaling components and driving forces of tissue deformations. In recent years, advances in microscopy techniques have led to improvements in imaging speed, resolution, and depth. Concurrently, the development of various software packages has supported large-scale, single-cell-level analyses of challenging images. Although these tools have accelerated comprehensive examination of single-cell-level dynamics and mechanical processes during morphogenesis, sophisticated integration requires further expertise. With this background, this review provides a practical overview of those techniques. First, we introduce microscopic techniques for multicellular imaging and image analysis software tools, with a focus on cell segmentation and tracking. Second, we provide an overview of cutting-edge techniques for mechanical manipulation of cells and tissues. Finally, we introduce recent findings on morphogenetic mechanisms and mechanosensations that were achieved by effectively combining microscopy, image analysis tools, and mechanical manipulation techniques. Mini-abstract In this review, we introduce multicellular imaging and image analysis tools. We also provide an overview of state-of-the-art techniques for mechanical manipulations of cells and tissues and give examples of how the combination of these tools and techniques has contributed to elucidating the mechanobiological aspect underlying morphogenesis.

Abstract: Solid-state batteries have potential to realize a rechargeable Li metal anode. However, several challenges persist in the charging and discharging processes of the Li metal anode, which require a fundamental understanding of Li plating and stripping across the interface of solid-state electrolytes to address. This review overviews studies on Li metal anodes in solid-state batteries using in situ observation techniques with an emphasis on Li electrodeposition and dissolution using scanning electron microscopy (SEM) and solid electrolytes (SEs) such as lithium phosphorus oxynitride (LiPON) and garnet-type compounds such as Li7La3Zr2O12 (LLZ). Previous research is categorized into three topics: (1) Li nucleation, growth, and dissolution at the anode-free interface, (2) Electrochemical reduction of SE, and (3) Short-circuit phenomena in SE. The current trends of each topic are summarized.

Abstract: Liquid-phase transmission electron microscopy (LPTEM) technique has been used to perform a wide range of in situ and operando studies. While most studies are based on the sample contrast change in the liquid, acquiring high qualitative results in the native liquid environment still poses a challenge. Herein, we present a novel and facile method to perform high-resolution and analytical electron microscopy studies in a liquid flow cell. This technique is based on removing the liquid from the observation area by a flow of gas. It is expected that the proposed approach can find broad applications in LPTEM studies.

Abstract: Abstract High-speed atomic force microscopy (HS-AFM) is now a widely used technique to study the dynamics of single biomolecules and complex structures. In the past, it has mainly been used to capture surface topography as structural analysis, leading to important discoveries not attainable by other methods. Similar to conventional AFM, the scope of HS-AFM was recently expanded to encompass quantities beyond topography, such as the measurement of mechanical properties. This review delves into various methodologies for assessing mechanical properties, ranging from semi-quantitative approaches to precise force measurements and their corresponding sample responses. We will focus on the application to single proteins such as bridging integrator-1, ion channels such as Piezo1, complex structures such as microtubules and supramolecular fibers. In all these examples, the unique combination of quantifiable force application and high spatiotemporal resolution allows to unravel mechanisms that cannot be investigated by conventional means.

Abstract: Orthohantaviruses are important zoonotic pathogens responsible for a considerable disease burden globally. Partly due to our incomplete understanding of orthohantavirus replication, there is currently no effective antiviral treatment available. Recently, novel microscopy techniques and cutting-edge, automated image analysis algorithms have emerged, enabling to study cellular, subcellular and even molecular processes in unprecedented detail and depth. To date, fluorescence light microscopy allows us to visualize viral and cellular components and macromolecular complexes in live cells which in turn enables the study of specific steps of the viral replication cycle such as particle entry or protein trafficking at high temporal and spatial resolution. In this review, we highlight how fluorescence microscopy has provided new insights and improved our understanding of orthohantavirus biology. We discuss technical challenges such as studying live infected cells, give alternatives with recombinant protein expression and highlight future opportunities for example the application of super-resolution microscopy techniques, which has shown great potential in studies of different cellular processes and viral pathogens.

Abstract: Time-resolved electron holography was implemented in a transmission electron microscope by means of e-beam gating with a parallel-plate electrostatic deflector. Stroboscopic observations were performed by accumulating gated electron interference images while applying a periodic modulation voltage to the specimen. Electric polarization in an ionic liquid specimen was observed under applied DC and AC fields. While the DC electric field in the specimen was reduced by the polarization of the material, an AC field of 10 kHz was not screened. This indicates that time-resolved electron holography is capable of determining the frequency limit of dynamic response of polarization in materials.

Abstract: We report direct observation by electron holography of the spin polarization of electrons in a vacuum region around a charged SiO2 wire coated with Pt-Pd. Irradiating the SiO2 wire with 300-keV electrons caused the wire to become positively charged due to the emission of secondary electrons. The spin polarization of these electrons interacting with the charged wire was observed in situ using a phase reconstruction process under an external magnetic field. The magnetic field of the spin-polarized electrons was simulated taking into account the distribution of secondary electrons and the effect of the external magnetic field.

Abstract: Small extracellular vesicles (EVs) are characterized by the membrane expression of CD63, CD81 and CD9 tetraspanins. Their size is inferior to 200 nm. They share the same characteristics as the native cells and are found in human fluids. Specific membrane protein biomarkers expressed on small EV are useful for the diagnosis of tumoral pathologies. Clear cells renal cell carcinoma (CCRCC) is diagnosed by imaging examinations and/or tissue biopsy. Carbonic anhydrase IX (CAIX) is a powerful biomarker of CCRCC. The detection of CAIX on small EV from the urine of patients could constitute a liquid biopsy for CCRCC. We have setup a specific protocol for the preparation, immunostaining characterization and the Transmission Electron Microscopy (TEM) observation of small EVs isolated from the urine of CRCC patients. The background labelling was significantly reduced. We successfully detected biomarkers on urinary small EVs from CCRCC patients. This technique could be extended with antibodies directed against other EVs biomarkers for the detection and the monitoring of cancer diseases.

Abstract: To improve the performance of organic light emitting diodes (OLEDs), it is essential to understand and control the electric potential in the organic semiconductor layers. Electron holography (EH) is a powerful technique for visualizing the potential distribution with a transmission electron microscope (TEM). However, it has a serious issue that high-energy electrons may damage the organic layers, meaning that a low- dose EH is required. Here, we used a machine learning technique, three-dimensional (3D) tensor decomposition, to denoise electron interference patterns (holograms) of bilayer OLEDs composed of N,N'-di-[(1-naphthyl)-N,N'-diphenyl]-(1,1'-biphenyl)-4,4'- diamine (α-NPD) and tris-(8-hydroxyquinoline)aluminum (Alq3), acquired under a low dose rate of 130 e- nm-2 s-1. The effect of denoising on the phase images reconstructed from the holograms was evaluated in terms of both the phase measurement error and the peak signal-to-noise ratio (PSNR). We achieved a precision equivalent to that of a conventional measurement that had an exposure time 60 times longer. The electric field within the Alq3 layer decreased as the cumulative dose increased, which indicates that the Alq3 layer was degraded by the electron irradiation. On the basis of the degradation of the electric field, we concluded that the tolerance dose without damaging the OLED sample is about 1.7 × 105 e- nm-2, which is about 0.6 times that of the conventional EH. The combination of EH and 3D tensor decomposition denoising is capable of making a time-series measurement of an OLED sample without any effect from the electron irradiation.

Abstract: In situ transmission/scanning transmission electron microscopy (TEM/STEM) measurements have taken a central stage for establishing structure-chemistry-property relationship over the past couple of decades. The challenges for realizing 'a lab-in-gap', i.e., gap between the objective lens pole-pieces, or 'a lab-on-chip', to be used to carry out experiments are being met through continuous instrumental developments. Commercially available TEM columns and sample holder that have been modified for in situ experimentation have contributed to uncovering structural and chemical changes occurring in the sample when subjected external stimulus such as temperature, pressure, radiation (photon, ions, electrons), environment (gas, liquid, magnetic or electrical field), or a combination thereof. Whereas atomic resolution images and spectroscopy data are being collected routinely using TEM/STEM, temporal resolution is limited to millisecond. On the other hand, better than femto second temporal resolution can be achieved using an ultrafast electron microscopy (UEM) or dynamic electron transmission electron microscopy (DTEM), but the spatial resolution is limited to sub-nanometers. In either case, in situ experiments generate large datasets that need to be transferred, stored, and analyzed. The advent of artificial intelligence (AI), especially machine learning (ML) platforms are proving crucial to deal with this big data problem. Further developments are still needed in order to fully exploit our capability to understand, measure, and control chemical and/or physical processes. We present the current state of instrumental and computational capabilities and discuss future possibilities.

Abstract: Nanosized precipitates have a critical role in increasing the strength of metallic alloys. There are many reports that the initial precipitates are metastable phases holding a different composition and crystal structure from the equilibrium precipitate. The metastable precipitate transforms to its stable phase during heat treatment. A transmission electron microscope enables researchers to study the phase transition of metastable precipitates to stable phases due to its fine resolution in identifying crystal structure and chemical composition. This review introduces the various phase transformation mechanisms of metastable precipitates to stable phases obtained from the analysis using a transmission electron microscope. The role of dislocation movement in the phase transition is further discussed.

Abstract: Hydrogen is attracting attention as an energy carrier for realizing a low-carbon society, because it can directly convert the energy obtained from chemical reactions into electrical energy without carbon dioxide emissions. This paper presents in situ transmission electron microscopic (TEM) observations related to hydrogen storage in metal and metal hydrides, hydrogen embrittlement of metallic materials used for storing and transporting hydrogen in containers and pipes, and fuel cells and water electrolysis using metal catalysts and oxides as electrode materials. All of these processes are important for practical applications of hydrogen. Numerous in situ TEM studies have revealed the microscopic structural changes when hydrogen reacts with the materials, when hydrogen is solidly dissolved in the materials, and during the operation of the material. This review is expected to facilitate further development of TEM operando observations of hydrogen-related materials.

Abstract: During the in-situ TEM observations, the diverse functionalities of different specimen holders play a crucial role. We hereby provide a comprehensive overview of the main types of holders, associated technologies, and case studies pertaining to the widely employed heating and gas heating methods, from their initial developments to the latest advancement. In addition to the conventional approaches, we also discuss the emergence of holders that incorporate a micro-electro-mechanical system (MEMS) chip for in-situ observations. The MEMS technology offers a multitude of functions within a single chip, thereby enhancing the capabilities and versatility of the holders. MEMS chips have been utilized in the environmental-cell designs, enabling customized fabrication of diverse shapes. This innovation has facilitated their application in conducting in-situ observations within gas and liquid environments, particularly in the investigation of catalytic and battery reactions. We summarize recent noteworthy studies conducted using in-situ liquid TEM. These studies highlight significant advancements and provide valuable insights into the utilization of MEMS chips in environmental-cells, as well as the expanding capabilities of in-situ liquid TEM in various research domains.

Abstract: We have developed a method to quantitatively measure image distortion, one of the five Seidel aberrations, in transmission electron microscopes without using a standard sample with a known structure. Displacements of small local segments in an image due to image distortion of the intermediate and projection lens system are first measured by comparing images taken before and after a given shift at the first image plane of the objective lens. Then, the sum of the second partial derivatives, or the Laplacian, of the displacement field is measured, and the radial and azimuthal distortion parameters are determined from the measured results. We confirmed using numerically distorted images that the proposed method can measure the image distortion within a relative error ratio of 0.04 for a wide range of distortion amount from 0.1 to 5.0%. The distortion measurement and correction were confirmed to work correctly by using the experimental images, and the iterative measurement and correction procedure could reduce the distortion to a level where the average image displacement was less than 0.05 pixels.

Abstract: Electrically assisted heat treatment is the process of applying an electric current to a sample during heat treatment. Literature has generally shown there to be a difference in the resulting effects of DC current and highly transient current (i.e. electropulsing). However, these differences are poorly characterized. In situ TEM observation of an AA7075 sample while DC and pulsed current were passed through it was performed herein to explore the effects of an electric current on precipitate development. Numerical simulation results indicate that the thermal response of the samples was very rapid, causing the sample to reach steady-state temperatures almost instantly. There does not appear to be any significant difference between the results of pulsed current application and DC current. Additionally, the failure mechanism of an electrical biasing TEM sample is explored.

Abstract: The two-dimensional observation of ultrathin sections from resin-embedded specimens provides insufficient understanding of the three-dimensional (3D) morphological information of membranous organelles. The osmium maceration method, developed by Professor Tanaka's group over 40 years ago, is the only technique that allows direct observation of the 3D ultrastructure of membrane systems using scanning electron microscopy (SEM), without the need for any reconstruction process. With this method, the soluble cytoplasmic proteins are removed from the freeze-cracked surface of cells while preserving the integrity of membranous organelles, achieved by immersing tissues in a diluted osmium solution for several days. By employing the maceration method, researchers using SEM have revealed the 3D ultrastructure of organelles such as the Golgi apparatus, mitochondria, and endoplasmic reticulum in various cell types. Recently, we have developed new SEM techniques based on the maceration method to explore further possibilities for this method. These include: (1) a rapid osmium maceration method that reduces the reaction duration of the procedure, (2) a combination method that combines agarose embedding with osmium maceration to elucidate the 3D ultrastructure of organelles in free and cultured cells, and (3) a correlative immunofluorescence and SEM technique that combines cryosectioning with the osmium maceration method, enabling the correlation of the immunocytochemical localization of molecules with the 3D ultrastructure of organelles. In this paper, we review the novel osmium maceration methods described above and discuss their potential and future directions in the field of biology and biomedical research.

Abstract: Journal Article In This Issue Get access Microscopy, Volume 72, Issue 1, February 2023, Pages 1–2, https://doi.org/10.1093/jmicro/dfac074 Published: 08 February 2023 Article history Corrected and typeset: 11 January 2023 Published: 08 February 2023

Abstract: Glucose is the most important energy source in all organisms; however, our understanding of the pathways and mechanisms underlying glucose transportation and localization in living cells is incomplete. Here, we prepared two glucose analogs labeled with a dansylamino group at the C-1 (1-Dansyl) or C-2 (2-Dansyl) position; the dansyl group is a highly fluorescent moiety that is characterized by a large Stokes shift between its excitation and emission wavelengths. We then examined the cytotoxicity of the two glucose analogs in mammalian fibroblast cells and in the ciliated protozoan Tetrahymena thermophila. In both cell types, 2-Dansyl had no negative effects on cell growth. The specificity of cellular uptake of glucose analogs was confirmed using inhibitor of glucose transporter in NIH3T3 cells. In NIH3T3 cells and T. thermophila, fluorescence microscopy revealed that the glucose analogs localized throughout the cytoplasm, but especially at the periphery of the nucleus. In T. thermophila, we also found that swimming speed was comparable in media containing non-labeled glucose or one of the glucose analogs, which not only provided more evidence that the analogs were not cytotoxic in these cells, but also that the analogs were not affect the ciliary motion. Together, the present results suggest that the glucose analogs have low toxicity and will be useful for bioimaging of glucose-related systems.

Abstract: Electron holography is a useful tool for analyzing functional properties, such as electromagnetic fields and strains of materials and devices. The performance of electron holography is limited by the "shot noise" inherent in electron micrographs (holograms), which are composed of a finite number of electrons. A promising approach for addressing this issue is to use mathematical and machine learning-based image-processing techniques for hologram denoising. With the advancement of information science, denoising methods have become capable of extracting signals that are completely buried in noise, and they are being applied to electron microscopy, including electron holography. However, these advanced denoising methods are complex and have many parameters to be tuned; therefore, it is necessary to understand their principles in depth and use them carefully. Herein, we present an overview of the principles and usage of sparse coding, wavelet hidden Markov model, and tensor decomposition, which have been applied to electron holography. We also present evaluation results for the denoising performance of these methods obtained through their application to simulated and experimentally recorded holograms. Our analysis, review, and comparison of the methods clarify the impact of denoising on electron-holography research.