In this study, we investigate the feasibility of evaluating the chemical bonding states using an electron-beam-based spectroscopic method implemented on a conventional scanning electron microscope and energy dispersive X-ray spectroscopy (SEM-EDS) system. The widely used surface-sensitive techniques such as X-ray absorption spectroscopy (XAS) often require synchrotron facilities and are not suitable for localized measurements. The critical excitation energies of silicon and silicon oxide featuring distinct absorption edge energies in XAS were determined by measuring the onset of characteristic X-ray emission as a function of incident electron energy. The observed difference in the critical excitation energy is consistent with the reported chemical shifts in the Si 1s binding energies and Si-K absorption edge energies. Systematic evaluations confirmed that variations in the beam current, instrumental instability, and surface charge-up did not significantly affect the measured thresholds. These results indicate that the proposed method captures chemically sensitive information related to the core-level electronic structure. The proposed approach provides a simple and accessible alternative to probe chemical bonding states and offers potential advantages for localized analysis because of the small probe size achievable with electron beams.
We theoretically investigated how the arrangements of tilt points affect the suppression of diffraction contrast in tilt-scan-averaged differential phase contrast scanning transmission electron microscopy (tDPC STEM). To examine the effectiveness of tilt-point arrangement, we compared three sampling patterns: conventional 4-fold, 6-fold symmetric concentric patterns and the Vogel spiral pattern based on Fermat's spiral with the golden angle. Multislice simulations were performed for GaN, GaAs, Si, and SrTiO3 crystals. Although all patterns approached similar converged values with sufficiently high tilt numbers, their convergence behaviors differed markedly. The concentric patterns exhibited non-monotonic decay and showed discrete fluctuations in residual diffraction contrast, whereas the Vogel spiral showed a smoother and more monotonic decrease over a wide range of tilt numbers. This behavior was consistently observed across the crystals with different crystal symmetries. These results show that rotationally asymmetric and uniformly distributed tilt sampling is advantageous for the robust diffraction-contrast suppression in DPC STEM, and identify the Vogel spiral as an effective tilt pattern for this purpose.
In situ tensile testing in a transmission electron microscope (TEM) is a powerful tool for investigating polymer fracture at the nanoscale, but the stochastic nature of crack initiation has long hindered systematic observation. We report a specialized diamond knife, co-developed with Syntek Co., Ltd., that introduces a controlled V-shaped notch into a trimmed specimen block prior to cryo-ultramicrotomy. Ultrathin films (∼100 nm) of a copolymer film containing a well-defined notch were prepared and observed using in situ tensile TEM. Cracks initiated reproducibly from the notch tip and propagated predictably, providing new experimental control for nanoscale fracture studies.
Coherence of electron waves is central to transmission electron microscopy. However, the mechanism underlying partial loss of coherence during inelastic scattering has been a perplexing problem for many decades. Here it is shown that the inelastic collision time is the key parameter governing coherence. Large energy transfers have short collision times and an inversely decaying coherence length. It is also shown that the inelastic coherence volume is highly elongated along the electron beam direction. Plasmon excitation and inter-band transition events, therefore, have only a minor effect on dynamical diffraction, a fact confirmed by energy-filtered measurements on [001]-oriented SrTiO3.
Polyvinylidene fluoride (PVDF) is a polymer with excellent piezoelectric properties. The close relationship between structure and properties necessitates structural investigation. The PVDF structure has been investigated using techniques such as X-ray diffraction, Fourier transform infrared spectroscopy, and polarized light microscopy. However, these methods only provide average structure information about the sample as a whole. To obtain local structure information, transmission electron microscopy should be used. Herein, the nanocrystals constituting the lamellar structure of heat-elongated PVDF were investigated using phase plate scanning transmission electron microscopy. Notably, the length of the nanocrystals increased with the increase in the elongation ratio, with no substantial change in its periodicity, and the nanocrystals exhibited a tendency to align with the lamellar structure orientation. This suggests that (1) the molecular chains within the nanocrystals elongate and tilt owing to the shear stress applied on them during elongation and (2) the entire lamellar structure rotates. To our knowledge, this is the first detailed study of nanocrystals in PVDF, which may further develop functional polymers.
The angle-dependences of N-K emission intensity profile of hBN on polar angle θ and azimuth angle ϕ were examined. The θ-dependent N-K emission intensity profiles show different angle dependences in the top and the bottom region of valence band (VB). From the comparison with the theoretically calculated charge distributions, there was a clear relation between the θ-dependence and the corresponding charge distribution was confirmed. No ϕ-dependence of N-K profile was observed and confirmed by a simple calculation, which shows no ϕ-dependence is reasonable for three, four, six-fold symmetrical charge distribution with the same energy. Important factor is the angle θ between a specified axis of the extend/reduce of charge distribution and the detection direction. From the angle dependent B-K emission profile of Fe2B, the presence of the interlayer one-dimensional B-B bonding of pz orbital was assigned at the bottom of VB. For discussing the anisotropic charge distribution in materials other than graphite and hBN, an anisotropy index α(E) was proposed and evaluated for the present experimental data.
Cryogenic transmission electron microscopy (cryo-TEM) is a powerful method that enables the observation of nanomaterials while preserving the specimen in a vitrified state, and minimizing damage caused by the electron-beam irradiation. Although cryo-TEM has been widely applied to aqueous specimens, its application to organic solvent systems remains limited. Because the physicochemical properties of organic solvents differ markedly from those of water, efficient vitrification methods have not yet been well established to date. More critically, organic solvents are highly susceptible to electron-beam irradiation. In this study, we selected methanol-the smallest polar alcohol molecule -and optimized the vitrification protocol for the observation of samples containing organic solvents. This resulted in a reproducible blotting method for forming thin solvent films, along with suitable freezing conditions for producing amorphous methanol. We further aimed to expand the application of cryo-TEM to materials in organic solvent systems by performing cryogenic electron energy-loss spectroscopy (cryo-EELS) elemental mapping. The technique, recently developed by our group for frozen aqueous solutions, enabled the detection of elemental signals even from frozen methanol and provided insights into their spatial distributions. Furthermore, silicon signals from mesoporous silica nanoparticles (MSNs) dispersed in methanol were clearly observed. These results demonstrate that both solvent components and nanomaterials can be visualized and analyzed within frozen organic solvents, thereby expanding the potential of cryo-TEM for advanced materials research involving organic solvent systems.
In-situ biasing differential phase contrast (DPC) scanning transmission electron microscopy (STEM) offers a powerful tool for visualizing local electric fields in devices under operating conditions. However, robust protocols for quantitative operando analysis remain underdeveloped. In this study, we established a methodology for quantitative electric field mapping of a GaAs p-n junction using in-situ biasing DPC-STEM, thereby extending previous quantitative DPC studies to device characterization under applied bias. We successfully visualized the modulation of the depletion layer under forward and reverse biases. By systematically comparing the experimental projected electric field profiles with two-dimensional Poisson simulations, we further revealed that focused ion beam induced electrically inactive layers were formed asymmetrically across the p-n junction, depending on the dopant concentration difference between the n- and p-type regions. These inactive layers significantly modify not only the field intensity but also its spatial distribution compared with the ideal case. Our results demonstrate that quantitative interpretation of in-situ DPC-STEM measurements requires simulation models incorporating realistic specimen structures, particularly asymmetric inactive layers, and provide practical guidance for quantitative characterization of functional semiconductor devices.
Scanning transmission electron microscopy (STEM) has a number of benefits over conventional parallel illumination transmission electron microscopy, such as the ability to simultaneously perform a range of imaging and spectroscopy techniques. However, one distinct disadvantage is the slow imaging speeds, a consequence of the sequential nature of the pixel acquisition, limiting imaging speeds to a few frames per second (fps). This reduces dose-rate control, increases the effect of distortions, and hinder the ability to capture dynamic events for in-situ experiments. The main obstacle to faster framerates is the inductance and hysteresis of the scanning coils that has previously only been addressable with the addition of entirely new hardware coils. Here we demonstrate a predictive scan shaping approach using conventional scanning systems, where scan input is determined based on where the beam will be instead of where the beam should be. We use this new approach to acquire fully sampled 512x512 images at 60 ns per pixel, giving a framerate of 41 fps.
Observation of beam-sensitive materials in scanning transmission electron microscopy (STEM) requires not only the development of highly dose-efficient imaging techniques but also the selection of appropriate imaging conditions such as the raster-scan sampling rate and optical parameters to realise a low-dose condition effectively. In this study, the convergence angle, sampling rates, and electron dose dependencies of the signal-to-noise ratio in optimum bright-field (OBF) STEM, a recently developed dose-efficient STEM imaging method, are investigated, which guides us in choosing optimal conditions to observe beam-sensitive porous materials such as zeolites. The parameters necessary for real-time OBF imaging are also considered towards practical live imaging at low dose. Experimental atomic-resolution OBF STEM observations of zeolites are then demonstrated using the strategy investigated above.
Focused ion beam (FIB) processing is widely used for preparing transmission electron microscopy (TEM) specimens of polymer materials. However, it often introduces damaged layers that hinder structural analysis. In this study, we demonstrate the effectiveness of gas cluster ion beam (GCIB) irradiation in reducing such damaged layers in polyethylene (PE) extracted from a PE/polyamide (PA) multilayer film, used here as a representative single-component polymer. GCIB treatment successfully removed the FIB-induced damaged layers and enabled access by ruthenium tetroxide (RuO4), which allowed clear visualization of the lamellar structure in PE. Electron tomography confirmed that GCIB not only eliminated damaged layers but also thinned the specimen. These findings suggest that GCIB is a promising technique for preparing ultra-thin, low-damage TEM specimens of polymer materials and can facilitate more accurate structural and chemical analyses.
Transmission electron microscopy (TEM) provides nanometer-scale resolution, which is essential for ultrastructural analysis of biological tissue. However, its application for large tissue areas is limited by a restricted field of view and observer-dependent sampling. In this study, we present a workflow that combines wide-field montage TEM-via "network tele-microscopy"-with correlative light and electron microscopy to enable large-area ultrastructural analysis while preserving synapse-level resolution. We demonstrate this approach in the glomerular layer and tyrosine hydroxylase-positive neurons in mouse olfactory bulb glomeruli, which exhibit dense, heterogeneous synaptic organization. Light and confocal laser scanning microscopy was first used for orientation in regions of interest, allowing the generation of continuous wide-field montage TEM datasets. This approach allowed systematic identification and quantification of synapses across an entire glomerulus while maintaining spatial relationships among ultrastructural elements. This study demonstrates a technically feasible platform for the development of TEM and the future integration of network tele-microscopy with computational methods.
We systematically engineered fluorescent protein-nanobody probes with different coupling rigidities and binding geometries to specific targets and analyzed the diffusion properties of their complexes by polarization-dependent fluorescence correlation spectroscopy (Pol-FCS). The results demonstrate that probe architecture critically affects Pol-FCS readouts, suggesting practical design principles for structure-sensitive probes. Polarization-dependent fluorescence correlation spectroscopy (Pol-FCS) simultaneously measures translational and rotational diffusion of fluorescently labeled molecules and provides information on molecular size and shape. Rotational diffusion is, in principle, expected to provide even higher sensitivity than translational diffusion, which is routinely used in conventional FCS to detect binding of fluorescent protein (FP)-fused binders to their targets. However, when FPs are fused to binders via flexible linkers, local wobbling of the FP can decouple its rotational diffusion from that of the binding complex. Quantitative evaluation of this decoupling effect remains lacking. Here, leveraging our expertise from polarization-dependent orientation probes, POLArIS, we designed and compared two classes of probes, termed "Rigid" and "Flex," that differ in how tightly the FP is linked to the anti-ALFAtag nanobody, to evaluate how probe architecture affects Pol-FCS readouts. Using ALFAtag and the rod-like repeat protein DHR10 as a structurally anisotropic binding target, we varied probe binding geometry while keeping molecular weight nearly constant and measured rotational and translational diffusion. Flex probes exhibited shorter rotational diffusion times than Rigid probes when bound to the same targets, consistent with the expected local reorientation. Notably, translational diffusion times also showed architecture-dependent differences between Rigid and Flex probes. These results demonstrate that probe architecture is a key determinant of diffusion readouts in FP-based Pol-FCS and suggest practical probe design principles for sensitive reporters of intermolecular binding accompanied by changes in complex shape.
High-order transfer maps offer many advantages in the study of both singlepass systems, where they represent optical aberrations, and multipass systems, where they allow the direct computation of relevant properties like high-order dispersions, chromaticities, and amplitude- and parameter-dependent tune shifts. However, one remaining question is always how accurate a map of a given expansion order really is-while in many cases, the sizes of high-order contributions decrease as a function of order, this does not always have to be the case. To address these topics, we present methods to determine mathematically rigorous bounds on all missing orders beyond the one explicitly considered. The computational effort to determine these bounds is small compared to the cost of the underlying high-order transfer map computation. Furthermore, the method allows for rigorous step size control in the Differential Algebra-based integration of high-order transfer maps.
The technology of volume electron microscopy (vEM), which enables three-dimensional (3D) observation of organelles, cells and even tissues at the nanoscale, is advancing and being applied to various fields of life science. As the demand for vEM grows, there is an increasing need for Volume Correlative Light and Electron Microscopy (Volume CLEM) to capture targets without missing the region of interest (ROI) or to observe the localization of molecules (often proteins) at the exact location of the fluorescent signals. This mini review provides an overview of the current state of Volume CLEM and introduces several unique approaches. My aim is to share the diverse fundamental technologies and broad application scope of this technique. vEM provides nanoscale imaging for life science applications. CLEM, is very powerful for linking molecular localization to ultrastructure. This mini review outlines advances, distinctive approaches and its broad technological and application scope.
The Li K-L emission spectra of lithium metal and its binary compounds (Li3N, Li2O, and LiF) were measured by using a soft X-ray emission spectrometer (SXES), which was equipped with a newly developed high-efficiency diffraction grating (JS35BC) and attached to an electron probe microanalyzer (EPMA). The new grating enables the observation of the entire intensity profile of both Li K-L and Mg L2,3-M emission spectra. The systematic energy shift of the Li K-L spectra to the lower energy side with an increasing electronegativity (or the ionization tendency) of elements of N, O, and F bonded with the Li atom was clearly detected. This shift was reproduced by theoretical calculations and was assigned mainly due to the change of the binding energy of the valence band of those materials. The main peak and its distinct shoulder structure observed in Li K-L spectrum of LiF were assigned as 2p dominated and 2 s + 2p mixed states, respectively, from the comparison with theoretical calculations.
Osteoporosis frequently presents with lower back pain, often accompanied by hypersensitivity, even in the absence of vertebral fractures, suggesting the involvement of central neuroinflammatory mechanisms. The activation of spinal glia has been implicated as a key driver of pain. Clinical and preclinical studies have also shown that teriparatide (TPTD), a bone anabolic drug, alleviates osteoporotic pain and raises the possibility of anti-neuroinflammatory effects. We previously reported that TPTD suppresses neuroinflammatory microglial proliferation in the spinal dorsal horn of an ovariectomized (OVX) rat model of postmenopausal osteoporosis. To further substantiate these previous findings, this study morphometrically investigated neuroinflammatory alterations in the same patho-pharmacological setting by establishing an AI-driven morphometric pipeline applied to DAB-stained bright-field images. OVX increased the number of microglia, induced process shortening and higher circularity, and expanded GFAP-positive astrocytic areas. TPTD partially attenuated OVX-induced glial changes. These findings indicate that OVX induces spinal neuroinflammation involving both microglia and astrocytes and that TPTD mitigates these neuroinflammatory responses. Moreover, the combination of 2D bright-field imaging and AI-driven morphometry represents a practical, accessible approach that requires minimal specialized equipment, yet sensitively captures OVX- and TPTD-induced microglial alterations, and enables phenotype-based classification.
We discuss the computational treatment of both singlepass systems like those used in microscopy and spectroscopy where careful consideration and control of aberrations is important, as well as multipass systems where other priorities arise. This is largely due to the fact that many of the aberrations cancel out when traversing a system repeatedly, which is greatly helped by the adjustment of the linear transfer matrix to be non-resonant. However, some other effects have a tendency to build up over time either linearly or exponentially, and a specific form of analysis is necessary to understand and control this behavior. This is achieved using normal form methods which allow a clear separation of multipass effects that are transient and those that are persistent, as well as the use of symplectic integration. The Differential Algebraic (DA) methods employed in COSY INFINITY allow for the computation of aberrations of arbitrary order and also the relevant normal forms. The tools allow the automatic computation of fully Maxwellian 3D fields if only midplane or on-axis field information is available, which for example allows recovering all nonlinear effects arising from increasing or decreasing fields in the fringes of particle optical elements. They also allow the computation of such fields from surface or volume field measurements, leading to a fully Maxwellian representation even in the presence of noise in the data. Utilizing metrics on symplectic spaces, it is possible to construct minimally invasive symplectification schemes for study of multipass systems based on transfer maps.
