Abstract Focused ion beam–scanning electron microscopy (FIB–SEM) was used to investigate the effects of acceleration voltage, beam current, and cryogenic conditions on the cross-sectional preparation of proton exchange membrane fuel cell (PEMFC) cathode catalyst layers. Milling at 30 kV and 0.26 nA effectively minimized curtaining artifacts and produced an apparent cross-sectional pore morphology similar to that obtained by cryogenic milling, indicating that optimized room-temperature milling can provide cross sections suitable for consistent two-dimensional (2D) pore-area analysis under the conditions examined. Representative FIB–SEM cross-sectional images of PEMFC cathode catalyst layers prepared under different FIB milling conditions: (a–c) 5 kV at room temperature, (d–f) 30 kV at room temperature, and (g–i) 30 kV under cryogenic conditions (− 150 °C). The indicated voltage and current values represent the FIB acceleration voltage and ion beam current used for milling, respectively. The red dashed box in (f) highlights the optimized room-temperature milling condition (30 kV, 0.26 nA) used for comparison with the corresponding cryogenic condition in (i).
Abstract Optoelectronic characterization of local bandgap, dielectric response, and plasmon at the nanometer scale has become increasingly important for understanding and optimizing modern electronic, optoelectronic, and quantum materials. Conventional optical techniques generally provide spatially averaged information and are therefore limited in their ability to resolve localized electronic variations arising from defects, interfaces, strain fields, and quantum confinement. To overcome these limits, monochromated scanning transmission electron microscopy–valence electron energy loss spectroscopy (STEM-VEELS) has evolved from a qualitative low-loss spectroscopy technique into a quantitative nanoscale bandgap metrology platform. By analyzing low-loss inelastic scattering signals with a sub-nanometer electron probe and sub-100 meV energy resolution, STEM-VEELS enables the direct investigation of bandgap onsets, local density of states (LDOS), and interband transitions at the nanoscale, although the effective spatial resolution of these measurements can be substantially broader than the probe size because of inelastic-scattering delocalization. This review summarizes the fundamental principles and recent advances in monochromated STEM-VEELS for local bandgap measurement. Particular emphasis is placed on the physical origin of low-loss excitations, addressing critical challenges such as momentum transfer and spatial delocalization. Practical experimental strategies for rigorous zero-loss peak (ZLP) subtraction and bandgap onset determination are systematically discussed. Furthermore, major analytical artifacts—such as Cherenkov radiation, guided optical modes, and beam-induced damage—are critically reviewed alongside state-of-the-art mitigation strategies. Recent developments in off-axis and Bessel-aperture STEM-VEELS geometries, machine-learning-assisted spectral analysis, automated onset extraction, and uncertainty-aware bandgap mapping are highlighted. Representative applications involving quantum dots, wide-bandgap oxides, heterointerfaces, and two-dimensional materials demonstrate the unparalleled capability of STEM-VEELS to resolve spatially varying electronic structures. Finally, future perspectives, including operando spectroscopy, ultrafast VEELS, artificial intelligence-driven analysis, and SEM-REELS, are discussed, providing a robust framework for standardization and quantitative nanoscale bandgap metrology.
Abstract Cryogenic-temperature friction stir processing (CT-FSP) was performed on AA6xxx sheets at 600 RPM and 500 mm/min using in-process liquid N2 cooling to investigate microstructure, precipitate evolution, and the resulting mechanical response. Multiscale characterization EBSD, SEM/EDS and microhardness tests were conducted. CT-FSP produced a highly refined predominantly equiaxed stir zone (SZ) with average grain sizes of approximately 1.6–1.8 µm at the surface and mid-thickness, and slightly coarser grains (~ 4.4 µm) near the bottom and within the thermomechanically affected zone (TMAZ). Dynamic recrystallization, evidenced by a high fraction of high-angle grain boundaries and low KAM, dominates the SZ. The recrystallization fraction varies laterally and through the thickness due to heterogeneous strain and thermal fields. Second phase particles experience mechanical fragmentation, partial solvus dissolution, and rapid reprecipitation during CT-FSP, yielding a finer and more uniformly dispersed distribution in the upper SZ, while coarser Al–Fe–Si and Mg2Si particles persist near the bottom/TMAZ. Deep-learning-based image segmentation reveals that the mean equivalent-circle diameters of Al–Fe–Si and Mg2Si phases decrease from ~ 0.97 µm and 0.80 µm in the as-received sheet to ~ 0.6–0.8 µm locally after CT-FSP, accompanied by reduced nearest-neighbor spacing in the SZ. This localized particles refinement and redistribution, together with extensive DRX, accounts for the combined SZ softening and TMAZ hardening, establishing a clear microstructure-property linkage in cryogenic FSPed AA6xxx sheet.
In this study, urchin-like CuO/Cu₂O mixed-phase nanostructures were synthesized via a simple surfactant-assisted approach, where the surfactant regulated the nucleation and anisotropic growth, leading to a hierarchical morphology with radially arranged nanoneedles. X-ray diffraction (XRD) and X-ray photoelectron spectroscopy (XPS) analyses verified the coexistence of CuO and Cu₂O phases within individual microspheres. High-resolution transmission electron microscopy (HRTEM) revealed well-defined lattice fringes assigned to Cu₂O, while XRD, XPS, SAED, and EDS analyses collectively suggest the coexistence of CuO and Cu₂O phases within the hierarchical microspheres. Energy-dispersive spectroscopy (EDS) mapping and line-scan profiles indicated a gradual compositional variation of copper and oxygen from the core to the surface region. Furthermore, in situ electron-beam irradiation demonstrated structural densification of the urchin-like architecture, providing insight into its structural stability. These results offer fundamental understanding of mixed-phase copper oxide formation and highlight their potential for high-surface-area functional materials.
This paper reviews a recent microstructure control strategy for steels based on intentional chemical heterogeneity, referred to as “chemically heterogeneous steels” or “chemically patterned steels.” This approach introduces controlled chemical inhomogeneity, which has traditionally been avoided, in order to diversify microstructural design and enhance mechanical properties. The strategy involves a three-step process: (i) introducing compositional variations in a multiphase region, (ii) preserving these variations through rapid austenitization, and (iii) controlling microstructural evolution during subsequent cooling. Chemical heterogeneity is introduced via austenite reversion, carbide precipitation, or pearlitic microstructures, enabling precise control of concentration gradients and characteristic length scales. Building on this processing route, the review emphasizes two primary mechanisms for property improvement. First, localized elemental partitioning stabilizes a higher fraction of retained austenite with a broad stability spectrum, inducing a progressive transformation-induced plasticity (TRIP) effect that enhances ductility. Second, the resulting “chemical boundaries” act as physical barriers to dislocation motion and crack propagation, increasing strength and resistance to hydrogen embrittlement. Finally, the paper suggests extending this concept beyond Mn to other alloying elements such as Cr and Ni, as well as its potential application to titanium alloys and implementation in industrial-scale production lines.
Public biological data repositories not only ensure research reproducibility and transparency but also enable new scientific discoveries that extend beyond the scope of original studies. A previously published study on umbelliferone (UMB) demonstrated recovery of synaptic vesicle architecture under scopolamine (SCO)-induced pathological conditions, and the corresponding high-resolution electron microscopy dataset was deposited in the Korea BioData Station (K-BDS, https://kbds.re.kr ), a national biological data platform. In this study, we reanalyzed this open dataset to investigate whether UMB also affects the postsynaptic density (PSD), a previously unexplored structural determinant of synaptic efficacy and plasticity. Using the original high-resolution transmission electron microscopy images, we performed secondary quantitative analyses focusing on PSD length, width, and ultrastructural features across experimental groups. Our reanalysis revealed that UMB treatment significantly increased PSD length and width compared with the SCO-treated group, indicating a structural restoration of impaired postsynaptic architecture. Notably, these PSD-level alterations were not described in the original publication, demonstrating that novel biological insights can be derived from existing data through purposeful reuse. Collectively, this study underscores the value of K-BDS as a national biological data platform that facilitates secondary discovery and advances data-driven neuroscience research without additional animal experimentation.
Abstract We present an FPGA-based reconfigurable scanning and data acquisition system for scanning electron microscopy (SEM). Built on the Xilinx Artix-7 (XC7A35T), the system integrates dual-channel 14-bit DAC raster scan waveform generation, dual-channel 12-bit ADC signal acquisition with on-chip averaging, and real-time USB 2.0 High-Speed data streaming at up to 40 MB/s. Integration with a commercial SEM (ModuleSci PicoEye-100) produced clearly resolved secondary-electron images, demonstrating stable raster operation in the fast-scan mode used for alignment and focusing. Standard data acquisition was performed at a per-frame acquisition time of 10 s, and a quantitative image-quality benchmark against the instrument’s built-in acquisition channel under this condition, using a grid-hole masking protocol and sub-pixel cross-correlation drift correction (Guizar-Sicairos et al. 2008), demonstrates substantial SNR improvements. The FPGA-based system achieves 41–47% higher spatial SNR and near-theoretical temporal SNR scaling, reaching a $$4.98\times$$ improvement over the commercial reference. These results highlight the effectiveness of hardware-level synchronization for improving the practical recoverability of high-frequency spatial detail under reduced acquisition time. The modular architecture is applicable to a broad range of point-scanning instruments beyond electron microscopy.
Abstract Hydrocarbon contamination is a common problem in transmission electron microscopy (TEM), affecting image contrast and the accuracy of quantitative spectroscopic analysis. Although plasma cleaning of specimen holders and samples is widely used, fully removing hydrocarbon contaminants from inside the microscope chamber has mostly involved slow processes like column baking, which can take several days of instrument downtime. In this study, we introduce a holder-type plasma cleaner that can be inserted directly into the TEM chamber through the specimen port. The device produces oxygen-radical plasma powered by radio frequency (RF) at 13.56 MHz, allowing in-situ cleaning of internal chamber surfaces in around three hours. We evaluated the cleaning performance quantitatively using scanning TEM (STEM)-energy-dispersive X-ray spectroscopy (EDX) and electron energy-loss spectroscopy (EELS). After one cleaning cycle, the carbon-K signal increase rate dropped from 2.81 to 0.30%/frame, and the carbon deposition rate decreased from 0.54 to 0.02 nm/frame, roughly a tenfold decrease. Repeated cleaning after three months of routine use further lowered contamination rates. These test results show that holder-type plasma cleaning is an effective, time-saving alternative for regular maintenance of TEM chamber cleanliness.
Abstract The histological structure and morphometric characteristics of the skin of the Korean ice goby Leucopsarion petersii were investigated using light microscopy, two histological staining techniques, and SPSS statistical analysis. Skin samples from adult L. petersii fishes were collected from five body regions, including the head, operculum, dorsal body, lateral body, and ventral body. In all examined regions, the skin of L. petersii exhibited the general structure as in other teleost fishes, consisting of an epidermis and dermis separated by a distinct basement membrane with underlying skeletal muscle. The epidermis was composed of outermost flattened cells, basal cells, club cells, and mucous cells. However, some histological findings differed from the typical teleost fishes: I), the dermis was mainly characterized by a well-developed stratum compactum with an almost complete absence of the stratum laxum; II), Although epithelial thickness showed significant regional variation, it was positively correlated with basement membrane thickness (r = 0.417, p < 0.001; n = 100); III), Mucous cells, suggesting the formation of a protective mucosal barrier on the skin surface, were distributed in the outer epidermal layer; IV), club cells, implying retention of chemical defense mechanisms against environmental stress, were confirmed within the epidermis. Consequently, the skin histology of the skin of L. petersii may reflect adaptive responses to its thin body surface and to the fluctuating environmental conditions encountered in coastal and estuarine habitats.
Array tomography using scanning electron microscopy (SEM) enables large-volume, nanometer-scale reconstruction of neural circuits and allows nanometer resolution of synaptic vesicles and other ultrastructural features. For stable serial imaging, Kapton tape, carbon nanotube (CNT) tape mounted on silicon wafers, and indium tin oxide (ITO) glass have commonly been used. However, each tape shows practical limitations: CNT tape is not consistently available, whereas Kapton tape often requires additional glow discharge or carbon coating steps to reduce charging artifacts and improve imaging stability. Here, we report the development of a lab-made tape using a cellulose/CNT/AgNW composite tape designed for SEM-based array tomography. By integrating intrinsically hydrophilic natural cellulose fibrils with conductive carbon or silver nanotubes, the newly developed tape provides sufficient surface hydrophilicity and conductivity without additional glow discharge or carbon coating treatment. This simplifies workflow while maintaining stable imaging conditions. Notably, the cellulose fibrils containing tape demonstrated reduced charging artifacts, improved section adhesion, and stable high-resolution imaging suitable for nanometer level analysis. Our results establish cellulose-based conductive tape as a practical advancement for array tomography, enhancing reliability and reproducibility in large-scale SEM imaging.
The deformation behavior of a high-Mn austenitic steel was investigated using in-situ straining transmission electron microscopy (TEM), with emphasis on the dynamic interaction between stacking faults and partial dislocations during plastic deformation. Owing to the low stacking-fault energy of the alloy, plastic deformation is primarily governed by Shockley partial dislocations, leading to extensive stacking-fault formation on multiple 111 planes. As deformation proceeds, stacking faults generated on different slip variants frequently intersect within grain interiors. Real-time observations reveal that these intersections act as strong deformation-induced barriers that impede partial dislocation motion, resulting in dislocation pile-up and localized strain concentration. Such interactions are considered to contribute significantly to strain hardening in low stacking-fault energy austenitic alloys. Despite their strong blocking effect, stacking-fault intersections are not strictly impenetrable. Under conditions of significant dislocation accumulation, the separation distance between leading and trailing partial dislocations can locally decrease, allowing temporary recombination into a perfect dislocation segment. The recombined dislocation adopts screw character, enabling cross-slip onto a secondary 111 slip plane, followed by re-dissociation into Shockley partial dislocations.These findings demonstrate that stacking-fault intersections function as dynamic microstructural features that both impede and mediate dislocation motion. The present in-situ TEM observations provide direct mechanistic insight into stacking-fault intersection-controlled dislocation dynamics and their role in strain hardening and local plastic accommodation in low stacking-fault energy austenitic steels.
Electron microscopy (EM) allows ultrastructural analysis of biological tissues and cells, but images frequently contain artifacts because biological samples have to undergo rigorous preparation to be resistant to vacuum conditions and electron beam exposure. Knowledge about the appearance of image artifacts and how they arise is crucial for their recognition and mitigation and for proper image interpretation. How artifacts appear depends strongly on the electron detection modality and the imaging conditions. Optical scanning transmission EM (OSTEM) is a detection technique compatible with single-beam and multibeam electron microscopes, in which tissue samples are directly deposited on a scintillator for imaging in transmission mode. Here, we identified several types of artifacts that may occur in single-beam and multibeam OSTEM. These artifacts arise or appear as a result of combining established sample preparation protocols with solid scintillator substrates and optical transmission detection. Artifacts can be effectively mitigated or minimized to ultimately enable high quality large-scale 2D and 3D acquisitions.
Cryo-electron tomography (cryo-ET) has become a key technique for observing the three-dimensional structures of biomolecular complexes and organelles in cellular and tissue environments in situ. However, for thicker specimens that cannot be directly imaged at an electron-transparent thickness, the practical success of cryo-ET depends largely on workflow design that reproducibly yields high-quality, target-containing lamellae. Cryo-FIB-SEM is the de facto standard platform for precisely machining frozen specimens to an electron-transparent thickness, and it integrates the overall process into a single system, encompassing targeting via cryo-CLEM, the management of major failure modes such as contamination, charging, curtaining, and downstream steps for data reconstruction and interpretation. This review summarizes a cryo-FIB-SEM–centered cryo-ET workflow from the perspectives of specimen preparation, targeting, milling, acquisition, reconstruction, and interpretation. In addition, we discuss extended preparation and imaging strategies for thicker specimens, including high-pressure freezing (HPF), the waffle method, cryo-FIB-SEM slice-and-view imaging, and Serial Lift-Out, highlighting both their expanding capabilities and the operational challenges that remain for robust and scalable implementation.
Over the past few decades, numerous biological therapeutics, such as RNAs and polypeptides, have emerged as promising alternatives to traditional chemotherapy. While their molecular mechanisms of action are well-understood, their clinical application remains hindered by several critical barriers, including inherent intracellular instability, the need for precise target-site delivery, poor cellular uptake, and immune system clearance. To overcome these challenges, the development of selective delivery systems has been raised as an indispensable strategy. Among various drug carriers, cationic lipid-based platforms have garnered significant attention and are increasingly exploited in oncology. This review summarizes the physicochemical characteristics of cationic lipid-based nanoparticles and evaluates various therapeutic cargos based on their biological properties. Furthermore, we explore the current research applications and clinical potential of these nanoparticles in cancer treatment.
The strength of precipitation-hardened Al–Si–Mg alloys primarily derives from the nanoscale coherent β″ precipitates, which are the dominant contributors to strengthening under peak-aged conditions. Observing these nanoscale precipitates via TEM is particularly challenging due to their strict orientation relationship with the matrix and the subtle atomic number differences between Al, Si, and Mg. In this study, we investigate the precipitation behavior of a high-Si Al–Si–Mg alloy through a comprehensive and systematic microstructural analysis. We demonstrate that the key strengthening mechanism in the peak-aged alloy is the high number density of nanoscale β″ precipitates. The atomic structure of the β″ phase is revealed using HR-TEM, HAADF, and LAADF imaging along the < 010 > Al zone axis. HR-TEM provides clear reflections in the FFT, facilitating effective observation and identification of the β″ phase. Additionally, LAADF imaging proves particularly useful in resolving the atomic structure of the β″ phase. These precipitates display an eye-like morphology, with four Mg and Si atoms arranged in a rim and a central Mg/Al atom, forming a long-range ordered structure. This detailed characterization offers valuable insights into the contribution of β″ precipitates to the alloy’s age-hardening behavior.
Abstract In research on cells conducted in vitro, cell viability is determined using staining techniques. However, interference with subsequent observation of live cell growth limits their applicability for real-time or continuous investigation. To address this limitation, we developed a deep learning–based algorithm capable of classifying live and dead cancer cells from microscopic images without staining. In this study, microscopic images were first captured prior to staining, and then the same regions were imaged again after staining to obtain live, dead, and other cell labels using a conventional staining method. The stained images served as ground truth data for supervised training with the corresponding pre-staining images. The proposed model achieved an accuracy of 0.931 after 99 training epochs in distinguishing live and dead cells from unstained images. This framework accurately differentiated live and dead cells directly from pre-staining images, demonstrating performance comparable to conventional stained-image analysis. Moreover, the approach enabled estimation of spatial boundaries between live and dead cell populations. These results demonstrate the potential of this approach as a non-invasive technique for assessing cell viability in in vitro studies.
Gamma prime (γ′) precipitates and grain boundary (GB) carbides govern the high-temperature performance of Ni-based superalloys, and their reliable quantification is essential for microstructural evaluation and alloy development. However, conventional etching procedures are often transferred between alloys without considering composition-dependent changes in γ′ size and fraction or carbide population, which can cause unstable contrast, γ-matrix damage, and unreliable image-based interpretation. Here, we establish composition-tailored etching conditions for Haynes® 282 (γ′ 23 nm) and two model alloys with modified Al–Ti and Nb–Ta contents, and evaluate their suitability for phase-selective Scanning electron microscopy (SEM) imaging. After identical mechanical preparation, γ′ precipitates in Haynes® 282 and Model alloy 1 are clearly revealed using a nitric-acid etchant, whereas the same condition fails in Model alloy 2 with reduced Al and Ti, where much finer γ′ precipitates form. An HF-containing mixed-acid etchant is introduced to obtain stable γ′ contrast in Model alloy 2 without excessive surface relief. GB carbides also show composition-dependent responses. In Haynes® 282, Cr-rich M₂₃C₆-decorated boundaries are revealed by nitric acid, whereas Nb/Ta-containing model alloys require chloride-based etchants to expose both MC and M₂₃C₆ carbides. These protocols provide reproducible, phase-selective SEM contrast for robust image-based quantification.
Abstract The internal structures of fractured white charcoal were investigated using a scanning electron microscope. The charcoal was fractured using a razor blade and hammer, gold-coated, and observed under the electron microscope. Both embolized and conductive vessels coexisted across the transverse surfaces of the fractured charcoal. Vessels were predominantly ellipsoidal, with an average diameter of approximately 250 μm. Embolized vessels exhibited membranous tyloses within their lumens. The presence of ring-porous wood, axial parenchyma cells, and xylem fibers within the secondary xylem implies that the white charcoal was produced from a Quercus species. Fungal hyphae were observed to proliferate, branch, and sporulate on the secondary cell walls of vessels. The conidiogenous cells and conidia closely resembled those of a fungal pathogen known to cause oak wilt disease in South Korea. Fungal hyphae were also found within the pits of the secondary cell walls. These observations suggest that the oak tree used for charcoal production in this study may have been diseased. These results indicate that wood structures are preserved through the white charcoal production process, allowing the observation of fungal structures within the host.
Plastic deformation in Mg alloys requires a full understanding of slip activity and intergranular interactions, which determine mechanical behavior and strain localization. Electron backscatter diffraction (EBSD) has emerged as a versatile technique to map crystallographic orientations, slip systems, and lattice rotations, permitting the systematic analysis of deformation mechanisms across polycrystalline aggregates. The coupling of EBSD with metrics including Schmid factors, ingrain misorientation axes, and slip-transfer criteria permits a quantitative assessment of slip compatibility and the role of grain boundaries in strain accommodation. Limitations related to conventional 2D surface characterization create a growing need for novel three-dimensional techniques that can accurately represent grain boundary geometry as well as complex intergranular deformation pathways. A focused review of such methodologies will compile current knowledge on these methods and their capabilities and limitations, guiding future investigations toward a deeper understanding of microstructure-mechanics relationships in Mg alloys.
Antonie van Leeuwenhoek (1632–1723) transformed observation into science through the power of a single handmade lens. His work emerged from the visual culture of seventeenth-century Delft, where craftsmanship, optics, and artistic precision intersected. While Robert Hooke’s compound microscope introduced the idea of microscopic visualization, Leeuwenhoek’s single-lens instruments achieved far superior magnification and resolution by minimizing optical interfaces. Using these deceptively simple devices, he documented the first observations of free-living microorganisms, fungal hyphae, red blood cells, capillary flow, oral bacteria, and spermatozoa in more than two hundred letters to the Royal Society of London. But his investigations reached far beyond microbiology. Leeuwenhoek also examined the barbed structure of the bee sting, the ordered vessels of ash wood, and the geometric microstructure of crystals and salts—demonstrating that hidden organization pervades both living and non-living matter. These studies established microscopy as a universal investigative tool, capable of unifying biology, medicine, botany, and early materials science under a single optical principle. Leeuwenhoek’s work marks one of the earliest examples of how rigorous observation can redefine scientific domains. His use of a home-crafted single lens created an empirical foundation for biological microscopy that persists to this day. The legacy of his minimalist optical design also survives in the digital age: modern clip-on smartphone microscopes and paper-based platforms such as the Foldscope reproduce the same single-lens principle through micro-optics mounted directly onto digital sensors. Three and a half centuries later, his work continues to remind us that new worlds do not emerge from new theories alone, but from new ways of seeing.
