Bloch Point Quadrupole Constituting Hybrid Topological Strings Revealed with Electron Holographic Vector Field Tomography.

Topological magnetic (anti)skyrmions are robust string-like objects heralded as potential components in next-generation topological spintronics devices due to their low-energy manipulability via stimuli such as magnetic fields, heat, and electric/thermal current. While these 2D topological objects are widely studied, intrinsically 3D electron-spin real-space topology remains less explored despite its prevalence in bulky magnets. 2D-imaging studies reveal peculiar vortex-like contrast in the core regions of spin textures present in antiskyrmion-hosting thin plate magnets with S4 crystal symmetry, suggesting a more complex 3D real-space structure than the 2D model suggests. Here, holographic vector field electron tomography captures the 3D structure of antiskyrmions in a single-crystal, precision-doped (Fe0.63Ni0.3Pd0.07)3P (FNPP) lamellae at room temperature and zero field. These measurements reveal hybrid string-like solitons composed of skyrmions with topological number W = -1 on the lamellae's surfaces and an antiskyrmion (W = + 1) connecting them. High-resolution images uncover a Bloch point quadrupole (four magnetic (anti)monopoles that are undetectable in 2D imaging) which enables the observed lengthwise topological transitions. Numerical calculations corroborate the stability of hybrid strings over their conventional (anti)skyrmion counterparts. Hybrid strings result in topological tuning, a tunable topological Hall effect, and the suppression of skyrmion Hall motion, disrupting existing paradigms within spintronics.

Semiautomatic contour tracking method for biological object segmentation in thin-section electron microscope images with modified zero DC component-type Gabor wavelets.

In electron microscopic image processing, artificial intelligence (AI) is a powerful method for segmentation. Because creating training data remains time-consuming and burdensome, a simple and accurate segmentation tool, which is effective and does not rely on manual drawings, is necessary to create training data for AI and to support immediate image analysis. A Gabor wavelet-based contour tracking method has been devised as a step toward realizing such a tool. Although many papers on Gabor filter-based and Gabor filter bank-based texture segmentations have been published, previous studies did not apply the Gabor wavelet-based method to straightforwardly detect membrane-like ridges and step edges for segmentation because earlier works used a nonzero DC component-type Gabor wavelets. The DC component has a serious flaw in such detection. Although the DC component can be removed by a formula that satisfies the wavelet theory or by a log-Gabor function, this is not practical for the proposed scheme. Herein, we devised modified zero DC component-type Gabor wavelets. The proposed method can practically confine a wavelet within a small image area. This type of Gabor wavelet can appropriately track various contours of organelles appearing in thin-section transmission electron microscope images prepared by the freeze-substitution fixation method. The proposed method not only more accurately tracks ridge and step edge contours but also tracks pattern boundary contours consisting of slightly different image patterns. Simulations verified these results.

Three-dimensional electron tomography and recent expansion of its applications in materials science.

Electron tomography (ET) is a powerful tool for elucidating the properties and functionalities of materials. The innovative development of aberration-corrected electron microscopy in the early 21st century and the remarkable progress in the development of detectors, equipment and devices related to ET have resulted in substantial improvements in resolution. However, not only advances in hardware but also remarkable developments in reconstruction algorithms and related three-dimensional (3D) analysis methods have contributed to the resolution improvements. ET has its own problems, including the missing-wedge problem due to the limited tilt-angle range and the need to acquire numerous specimen-tilt images, the latter of which is time-consuming and can potentially damage the specimen. This review paper aims to (i) describe the established basic theories and definitions regarding 3D resolution of ET and practical 3D resolution measurement methods, (ii) discuss various reconstruction algorithms that effectively overcome the aforementioned problems and (iii) describe recent progress in the core of ET applications in materials science with respect to atomic ET, analytical ET and in-situ ET. The aforementioned ET problems have been addressed with each method developed in each field of application. Notably, in terms of aim (ii), recently developed reconstruction algorithms can reduce the number of projection images (specimen-tilt images) needed to attain a certain resolution without violating the Nyquist criterion. This approach is interpreted as a novel non-linear sampling theorem.

Novel nonlinear reconstruction method with grey-level quantisation units for electron tomography.

We report a new computed tomography reconstruction method, named quantisation units reconstruction technique (QURT), applicable to electron and other fields of tomography. Conventional electron tomography methods such as filtered back projection, weighted back projection, simultaneous iterative reconstructed technique, etc. suffer from the 'missing wedge' problem due to the limited tilt-angle range. QURT demonstrates improvements to solve this problem by recovering a structural image blurred due to the missing wedge and substantially reconstructs the structure even if the number of projection images is small. QURT reconstructs a cross-section image by arranging grey-level quantisation units (QU pieces) in three-dimensional image space via unique discrete processing. Its viability is confirmed by model simulations and experimental results. An important difference from recently developed methods such as discrete algebraic reconstruction technique (DART), total variation regularisation-DART, and compressed sensing is that prior knowledge of the conditions regarding the specimen or the expected cross-section image is not necessary.

Generalized spot auto-focusing method with a high-definition auto-correlation function in transmission electron microscopy.

The spot auto-focusing (AF) method with a unique high-definition auto-correlation function (HD-ACF) proposed in the previous paper is improved and is now applicable to general specimens at a wide range of magnifications. According to the definition where the AF is defocused to obtain the highest resolution, the proposed method achieves the sharpest HD-ACF profile in the AF spot image. The relationship where the sharpest HD-ACF profile gives the highest resolution is theoretically explained, and practical AF examples for different specimens and magnifications are experimentally demonstrated. Specimens include a yeast cell thin section at 10-k magnification, a standard grating replica used as a ruler at 50-k, a crystal lattice of graphitized carbon at 400-k and a 60°-tilted thin section (yeast cell) at 10-k. Different procedures are prepared to actively identify the defocus position that gives the sharpest HD-ACF profile. Every AF result demonstrates the highest-resolution image.

A nuclear membrane-derived structure associated with Atg8 is involved in the sequestration of selective cargo, the Cvt complex, during autophagosome formation in yeast.

Macroautophagy (hereafter autophagy) is a conserved intracellular degradation mechanism required for cell survival. A double-membrane structure, the phagophore, is generated to sequester cytosolic cargos destined for degradation in the vacuole. The mechanism involved in the biogenesis of the phagophore is still an open question. We focused on 4 autophagy-related (Atg) proteins (Atg2, Atg9, Atg14, and Atg18), which are involved in the formation of the phagophore in order to gain a more complete understanding of the membrane dynamics that occur during formation of the autophagosome. The corresponding mutants, while defective in autophagy, nonetheless generate the membrane-bound form of Atg8, allowing us to use this protein as a marker for the nascent autophagosome precursor membrane. Using electron microscopy (EM), we discovered in these atg mutants a novel single-membrane structure (~120 to 150 nm in size). Electron tomography revealed that this structure originates from a part of the nuclear membrane, and we have named it the alphasome. Our data suggest that the alphasome is associated with Atg8, and sequesters selective cargo, the Cvt complex, during autophagy. Abbreviations: 3D: three-dimensional; AB: autophagic body; AP: autophagosome; Atg: autophagy-related; Cvt: cytoplasm-to-vacuole targeting; EM: electron microscopy; IEM: immunoelectron microscopy; L: lipid droplet; N: nucleus; NM: nuclear membrane; PAS: phagophore assembly site; PE: phosphatidylethanolamine; prApe1: precursor aminopeptidase I; rER: rough endoplasmic reticulum; TEM: transmission electron microscopy; V: vacuole; VLP: virus-like particle.

Spot auto-focusing and spot auto-stigmation methods with high-definition auto-correlation function in high-resolution TEM.

As alternatives to the diffractogram-based method in high-resolution transmission electron microscopy, a spot auto-focusing (AF) method and a spot auto-stigmation (AS) method are presented with a unique high-definition auto-correlation function (HD-ACF). The HD-ACF clearly resolves the ACF central peak region in small amorphous-thin-film images, reflecting the phase contrast transfer function. At a 300-k magnification for a 120-kV transmission electron microscope, the smallest areas used are 64 × 64 pixels (~3 nm2) for the AF and 256 × 256 pixels for the AS. A useful advantage of these methods is that the AF function has an allowable accuracy even for a low s/n (~1.0) image. A reference database on the defocus dependency of the HD-ACF by the pre-acquisition of through-focus amorphous-thin-film images must be prepared to use these methods. This can be very beneficial because the specimens are not limited to approximations of weak phase objects but can be extended to objects outside such approximations.

Design of a 300-kV gas environmental transmission electron microscope equipped with a cold field emission gun.

A new in situ environmental transmission electron microscope (ETEM) was developed based on a 300 kV TEM with a cold field emission gun (CFEG). Particular caution was taken in the ETEM design to assure uncompromised imaging and analytical performance of the TEM. Because of the improved pumping system between the gun and column, the vacuum of CFEG was largely improved and the probe current was sufficiently stabilized to operate without tip flashing for 2-3 h or longer. A high brightness of 2.5 × 10(9) A/cm(2) sr was measured at 300 kV, verifying the high quality of the CFEG electron beam. A specially designed gas injection-heating holder was used in the in situ TEM study at elevated temperatures with or without gas around the TEM specimen. Using this holder in a 10 Pa gas atmosphere and specimen temperatures up to 1000°C, high-resolution ETEM performance and analysis were achieved.

A role for the ancient SNARE syntaxin 17 in regulating mitochondrial division.

Recent evidence suggests that endoplasmic reticulum (ER) tubules mark the sites where the GTPase Drp1 promotes mitochondrial fission via a largely unknown mechanism. Here, we show that the SNARE protein syntaxin 17 (Syn17) is present on raft-like structures of ER-mitochondria contact sites and promotes mitochondrial fission by determining Drp1 localization and activity. The hairpin-like C-terminal hydrophobic domain, including Lys-254, but not the SNARE domain, is important for this regulation. Syn17 also regulates ER Ca(2+) homeostasis and interferes with Rab32-mediated regulation of mitochondrial dynamics. Starvation disrupts the Syn17-Drp1 interaction, thus favoring mitochondrial elongation during autophagy. Because we also demonstrate that Syn17 is an ancient SNARE, our findings suggest that Syn17 is one of the original key regulators for ER-mitochondria contact sites present in the last eukaryotic common ancestor. As such, Syn17 acts as a switch that responds to nutrient conditions and integrates functions for the ER and autophagosomes with mitochondrial dynamics.

A new field-of-view autotracking method based on back-projected ray image cross-correlation for online tomography reconstruction.

In general, a tomogram cannot be observed immediately after the acquisition of a series of specimen tilt images, but is instead observed after the post-processing of the tilt series alignment, which often requires a substantial amount of time. Moreover, for general specimens, the automatic acquisition of the tilt series is difficult because field-of-view tracking frequently fails as the tilt angle or specimen thickness increases.In this study, we focus on the improvement of the field-of-view autotracking technique for the purpose of online tomography reconstruction and propose a new alternative technique [1,2]. The method we proposed uses a so-called 'back-projected ray image' instead of a specimen tilt image. The back-projected ray image is a cross-section image calculated from each projection image only during reconstruction. As a result of a study on 'ray images', the quality and accuracy of the cross-correlation between a pair of neighboring ray images among the tilt series were observed to be very high compared with those between a pair of projection images. We observed that a back projected ray image reliably cross-correlates with other neighboring ray images at the position of an existing three-dimensional object. The proposed method can therefore consistently track the field-of-view, overcoming the weakness of a conventional image-matching-based method. In addition, the present method is simple, and high speed processing is expected to be achieved because fast Fourier transform (FFT) and inverse fast Fourier transform (IFFT) algorithms can be used.We applied this method to real specimens in online experiments using a TEM and thereby demonstrated its successful performance. Online autotracking experiments with thin-section samples were used to demonstrate the effectiveness of the proposed method. The field-of-view was automatically tracked with high accuracy through a tilt angle range. Furthermore, online tomograms were obtained immediately after the last specimen tilting. With increases in the tracking speed, in situ tomographic observations for analyzing the dynamic behavior might become feasible in the future.jmicro;63/suppl_1/i23-a/DFU058F1F1DFU058F1Fig. 1.Comparison of the proposed autotracking method with the conventional PCF based alignment method using the yeast cell thin-section. a and b: Reconstructed X-Y cross-section images from tracking results at 8° increment angle with the PCF method and with the proposed method. N, nucleus; V, vacuole; NVJ, nucleus-vacuole junction. c: A reconstructed cross-section image from autotracking result at 1° increment angle with the proposed method. (scale bar: 100 nm).

A new field-of-view autotracking method for online tomography reconstruction based on back-projected ray image cross-correlation.

We devised a new field-of-view autotracking method for online tomography reconstruction based on a cross-correlation between a pair of neighbours, called back-projected ray images, among a specimen tilt sequence. One ray image is calculated through normal filtered back-projection only in the cross-sectional plane from each projection image. This ray-image matching can reliably track the field-of-view because a pair of neighbouring ray images mostly cross-correlates at the existing three-dimensional object position. Online experiments using real specimens resulted in successful autotracking performance with high accuracy, and online tomograms were obtained immediately after the final tracking at the last tilting angle.

High-precision image-drift-correction method for EM images with a low signal-to-noise ratio.

The phase correlation method (PCM) is well known for high-precision matching between images. However, if the signal-to-noise ratio of an image is low, the method is difficult to apply. To solve this problem, we developed an improved PCM that can match images automatically with sub-pixel matching precision. Using this method, a 0.2-nm crystal lattice spacing was clearly revealed after 10 blurred images were processed in a verification experiment; such a lattice could not be recognized or hardly be recognized in each individual image.

Alternative automatic alignment method for specimen tilt-series images based on back-projected volume data cross-correlations.

We devised a new automatic image alignment method for a specimen tilt series; this method is based on the volume data cross-correlation among 3-D cross-sections reconstructed from different sets of projection images (including a single image) for tilt-series alignment or tilt-axis search purposes. This method requires neither markers nor image feature points traceable through the tilt series, and it was examined through simulations and applied to biological thin sections. The method automatically aligned tilt series centred at the correctly detected tilt axis with a precision sufficient for practical applications.

Novel configuration of a myosin II transient intermediate analogue revealed by quick-freeze deep-etch replica electron microscopy.

In the present paper, we described our attempt to characterize the rough three-dimensional features of the structural analogue of the key intermediate of myosin's cross-bridge cycle. Using quick-freeze deep-etch replica electron microscopy, we observed that actin-attached myosin during in vitro sliding was bent superficially as postulated by the conventional hypothesis, but in the opposite direction of the putative pre-power-stroke configuration, as for ADP·Vi (inorganic vanadate)-bound myosin. We searched for the conformational species with a similar appearance and found that SH1-SH2 (thiols 1 and 2)-cross-linked myosin is a good candidate. To characterize such small asymmetric structures, we employed a new pattern-recognition procedure that accommodates the metal-replicated samples. In this method, the best-matched views of the target microscopic images were selected from a comprehensive set of images simulated from known atomic co-ordinates of relevant proteins. Together with effective morphological filtering, we could define the conformational species and the view angles of the catalytic domain and the lever arm cropped from averaged images of disulfide-cross-linked myosin. Whereas the catalytic domain of the new conformer closely resembled the pPDM (N,N'-p-phenylenedimaleimide)-treated, but SH2 Lys705-cross-linked, structure (PDB code 1L2O), a minor product of the same cross-linking reaction, the lever arm projected differently. Using separately determined view angles of the catalytic domain and the lever arm, we built a model of disulfide-cross-linked myosin. Further combination with the 'displacement-mapping' procedure enabled us to reconstruct the global three-dimensional envelope of the unusual structure whose lever arm orientation is compatible with our reports on the actin-sliding cross-bridge structure. Assuming this conformer as the structural analogue of the transient intermediate during actin sliding, the power stroke of the lever arm might accompany the reversal of the disorganized SH1 helix.

Extended morphological processing: a practical method for automatic spot detection of biological markers from microscopic images.

BACKGROUND: A reliable extraction technique for resolving multiple spots in light or electron microscopic images is essential in investigations of the spatial distribution and dynamics of specific proteins inside cells and tissues. Currently, automatic spot extraction and characterization in complex microscopic images poses many challenges to conventional image processing methods. RESULTS: A new method to extract closely located, small target spots from biological images is proposed. This method starts with a simple but practical operation based on the extended morphological top-hat transformation to subtract an uneven background. The core of our novel approach is the following: first, the original image is rotated in an arbitrary direction and each rotated image is opened with a single straight line-segment structuring element. Second, the opened images are unified and then subtracted from the original image. To evaluate these procedures, model images of simulated spots with closely located targets were created and the efficacy of our method was compared to that of conventional morphological filtering methods. The results showed the better performance of our method. The spots of real microscope images can be quantified to confirm that the method is applicable in a given practice. CONCLUSIONS: Our method achieved effective spot extraction under various image conditions, including aggregated target spots, poor signal-to-noise ratio, and large variations in the background intensity. Furthermore, it has no restrictions with respect to the shape of the extracted spots. The features of our method allow its broad application in biological and biomedical image information analysis.

The development of a new windowless XEDS detector.

A new windowless X-ray energy-dispersive spectroscopy (XEDS) detector has been developed for an analytical electron microscope (AEM). Different from the conventional XEDS detectors, the new detector does not contain an ultra-thin window (UTW) and a vacuum gate valve which are the major causes of low X-ray detection sensitivity and vibration problems for AEM imaging, respectively. The performance of the newly designed detector was examined at an AEM column vacuum level of 10⁻5 Pa. The X-ray detectability was improved considerably; in particular, the sensitivity for detecting nitrogen characteristic X-ray signal was three times higher than that of the conventional UTW detectors.

Nanostructural characterization and catalytic analysis of hybridized platinum/phthalocyanine nanocomposites.

Organic crystals, such as phthalocyanine nanocrystal, were successfully hybridized with Pt nanoparticles using a nanohybridization technique. The presence of highly dispersed Pt nanoparticles on the surface of phthalocyanine was confirmed by the combination of transmission electron microscopy and three-dimensional electron tomography. Catalytic activities of hybridized samples with different degrees of dispersions were also examined as oxygen reduction reactivity (ORR) with a linear potential sweep method. It was found that oxygen reduction activity increased with increasing Pt dispersion, and reasonably high ORR was observed on Pt-dispersed phthalocyanine nanocrystal even at 2 wt% Pt loading.

The development and characteristics of a high-speed EELS mapping system for a dedicated STEM.

A new EELS (electron energy loss spectroscopy) real-time elemental mapping system has been developed for a dedicated scanning transmission electron microscope (STEM). The previous two-window-based jump-ratio system has been improved by a three-window-based system. It is shown here that the three-window imaging method has less artificial intensity in elemental maps than the two-window-based method. Using the new three-window system, the dependence of spatial resolution on the energy window width was studied experimentally and also compared with TEM-based EELS. Here it is shown experimentally that the spatial resolution of STEM-based EELS is independent of the energy window width in a range from 10 eV to 60 eV.

A novel "ghost"-free tomographic image reconstruction method applicable to rotary-shadowed replica specimens.

Electron tomography by conventional filtered back-projection is often seriously impaired by anisotropic resolution due to unavoidable limitation in specimen tilt-angles. We propose a new approach to overcome the problem for thin film-like replica-type specimens in which internal density is supposed as homogenous and contiguously distributed, by imposing a reasonable constraint of density-existing region in the reconstruction procedure. The objects were approximated as a distribution of binary voxels and the intensity of the projected images being proportional to the thickness along the projection ray. The new reconstruction algorithm consists of initial determination of approximate constraint region by a topographic analysis by stereo-photogrammetry, followed by iterative computation to find the unique solution of simultaneous equations, so that all the intensity distribution in tilt-series images are included within pre-determined voxel arrangement. During a trial run with a new methodology, we realized its significantly advantageous feature that much less number of projection images than conventional back-projection is required to perform the reconstruction of almost equivalent quality. Here, we show the performance of this novel algorithm by 3-D reconstruction of quick-freeze deep-etch replica specimens without any trace of spurious ghosting caused by missing-wedge problems.

A procedure to analyze surface profiles of the protein molecules visualized by quick-freeze deep-etch replica electron microscopy.

Quick-freeze deep-etch replica electron microscopy gives high contrast snapshots of individual protein molecules under physiological conditions in vitro or in situ. The images show delicate internal pattern, possibly reflecting the rotary-shadowed surface profile of the molecule. As a step to build the new system for the "Structural analysis of single molecules", we propose a procedure to quantitatively characterize the structural property of individual molecules; e.g. conformational type and precise view-angle of the molecules, if the crystallographic structure of the target molecule is available. This paper presents a framework to determine the observed face of the protein molecule by analyzing the surface profile of individual molecules visualized in freeze-replica specimens. A comprehensive set of rotary-shadowed views of the protein molecule was artificially generated from the available atomic coordinates using light-rendering software. Exploiting new mathematical morphology-based image filter, characteristic features were extracted from each image and stored as template. Similar features were extracted from the true replica image and the most likely projection angle and the conformation of the observed particle were determined by quantitative comparison with a set of archived images. The performance and the robustness of the procedure were examined with myosin head structure in defined configuration for actual application.