Microscopy (Oxford, England)最新文献

Bone dynamically changes its shape and structure in response to extra-tissue environments, so that bone morphometry has been a substantial method to evaluate pathophysiology of bone. Osteocytes embedded in mineralized bone matrix play key roles in systemic bone metabolism and characterize distinct bone sites. The jawbone has been described as a unique bone in the context of vertebrate evolution and function. Bone loss in the mandibular bone is less obvious in osteoporotic conditions than in other bones, such as vertebral and limb long bones, both in animal models and in clinical studies. Since osteocyte lacunae are complex and small (-10µm in length) in shape and size, respectively, comprehensive and unbiased morphometrical analysis of changes in the size of osteocyte lacunae was still an obstacle. This study established an artificial intelligence-driven morphometry with wide-field microscopy-based imaging of osteocyte lacunae. Successive comparative analyses demonstrated active perilacunar bone remodeling in the mandibular bone than in the parietal bone. This approach enabled us to statistically compare morphometric parameters in a more comprehensive and unbiased manner. We further discuss the possible unique contribution of the mandibular bone to the pathophysiology of osteoporosis. This study established an artificial intelligence-driven morphometry with wide-field microscopy-based imaging of osteocyte lacunae. Successive comparative analyses demonstrated active perilacunar bone remodeling in the mandibular bone than in the parietal bone. This approach enabled us to statistically compare morphometric parameters in a more comprehensive and unbiased manner.

Cryoelectron microscopy is a powerful technique for high-resolution imaging of nonaqueous liquids, but challenges remain regarding imaging and data interpretation. Recent advancements in estimating the physicochemical properties of pure organic liquids at cryogenic temperatures have enhanced the selection of imaging and pre-treatment conditions. However, whether binary mixtures behave similarly to pure substances is still unclear. Furthermore, focused ion beam (FIB) milling facilitates site-specific cross-sectioning, but its effects on the microscopic morphology of frozen organic liquids are not well understood. In this study, we investigated water-tetrahydrofuran (THF) binary mixtures as a model to explore their phase behavior and radiation damage under cryogenic conditions. Spectroscopic analyses indicated microscopic phase separation within the seemingly miscible water-THF mixtures, but their detailed structure has been a subject of ongoing debate. Using cryo-scanning electron microscopy with FIB (cryo-FIB-SEM), we visualized bicontinuous phase-separation. The domain sizes were consistent across spectroscopic data, thermally sublimed surfaces, and FIB cross-sections. Notably, FIB milling caused a significant loss of THF-rich regions, likely due to localized temperature increases of approximately 178 K, which is an order of magnitude greater than that in water-rich domains. We also noted the nanoparticles of electron-resistant carbazole-terminated carbon-bridged oligo(para-phenylenevinylene) (COPV2-G1) formed within the THF-rich phase. Extended electron irradiation led to morphological changes and shrinkage, suggesting THF was incorporated into COPV2-G1 aggregates along with THF decomposition induced by the electron beam. These findings underscore critical considerations in cryo-FIB-SEM imaging of binary organic liquids and solvated particles, providing practical insights for reducing or leveraging ion/electron beam-induced artifacts.

Isotope microscopic imaging and atmospheric pressure matrix-assisted laser desorption/ionization mass spectrometry imaging (AP-MALDI-MSI) provide powerful, complementary approaches for visualizing metabolic dynamics in biological tissues. This study applied these techniques to termite workers fed with 13C-labeled cellulose for one week. Termites are classified as eusocial insects because of their colonies' clear division of labor. The two primary castes in their life cycle are reproductive (king and queen), responsible for reproduction, and non-reproductive (workers and soldiers), who handle tasks such as defense, brood care, and foraging. Although various techniques have been developed to detect 13C-labeled biomolecules in samples, it remains unclear whether the iMScopeTM prototype can visualize these molecules with high spatial resolution. Advanced isotope microscopic imaging technique with high spatial resolution (200-300 nm) offered ultra-high-resolution visualization of the relative abundance of the 13C/12C distribution, suggesting precise localization of isotope enrichment in the abdomen. AP-MALDI-MSI performed in the iMScopeTM prototype enabled spatial mapping of 13C-labeled and unlabeled metabolites, such as acetyl-L-carnitine (ALC) and phosphatidylethanolamine (PE), by detecting characteristic mass shifts due to 13C incorporation. The accumulation of PE in the termite abdomen represents an adaptive strategy to optimize nutrient allocation and promote social cohesion, thereby highlighting its potential role in maintaining colony fitness. Our study shows that the iMScopeTM prototype is a novel AP-MALDI-MSI technique to detect 13C-integrated metabolites in the 13C-labeled sample. This study also demonstrated that this technique can detect 13C-integrated PE, which is abundant mainly in termite abdomen.

This review examines the effects of thermal vibrations on core-level excitation spectra, with a particular emphasis on the Ti L  2,3-edge spectra of cubic perovskite-type titanium oxides (SrTiO3 and PbTiO3). Based on combining scanning transmission electron microscopy energy-loss near-edge structure analyses with cluster-type crystal-field multiplet calculations, the influence of atomic thermal vibrations on the fine structure of the Ti L  2,3-edge is investigated, and it is demonstrated that the thermal vibration of oxygen atoms in cubic SrTiO3 can be estimated from the spectrum by fitting experimental and theoretical results. The same approach was extended to cubic PbTiO3 such that isotropic thermal vibrations were identified that relate to the difference in the transition to a low-temperature tetragonal phase. Although the present technique does not directly resolve phonon modes, it treats thermal factors as adjustable parameters, enabling the identification of subtle vibrational features even in materials already widely studied. Further investigation of the relationship between thermal vibrations and the fine structure of core-loss spectra could assist in elucidating certain material properties. This review explores the effects of thermal vibrations on Ti L  2,3-edge spectra of cubic perovskite oxides (SrTiO3, PbTiO3). Combining STEM-ELNES with crystal-field multiplet calculations, it shows that oxygen thermal vibrations can be estimated from spectral fitting, revealing subtle vibrational features and their relation to phase transitions.

In this review, we focus on the ultrastructural characteristics of the Golgi membrane-associated degradation (GOMED) pathway, which have been clarified by electron microscopy, and highlight recent advances in the elucidation of its molecular mechanism and physiological roles. The discovery of GOMED, an Atg5/Atg7-independent degradation pathway that differs from canonical autophagy in membrane origin, stimuli and substrate specificity, has substantially expanded our understanding of intracellular degradation systems. In 2009, we identified GOMED as a novel, evolutionarily conserved autophagic pathway and demonstrated its role in intracellular degradation across eukaryotes, from yeast to mammals. We identified the conserved protein Hsv2/Wipi3 as an essential GOMED protein, which translocates to the trans-Golgi upon induction and remodels Golgi membranes into cup-shaped structures that engulf cytoplasmic components for lysosomal degradation. These processes contribute to organelle and secretory granule turnover, as well as mitochondrial clearance during erythroid differentiation. Moreover, neuronal-specific ablation of Wipi3 in mice causes severe cerebellar degeneration, implicating GOMED in tissue development and homeostasis. As these mechanisms are associated with diseases, such as neurodegenerative disorders and cancer, GOMED mechanisms should also be considered when establishing therapeutic strategies for these diseases.

Collagen fibrils in the dermis are bundled by glycosaminoglycan (GAG) chains of decorin, which contribute to its strength. The three-dimensional structure of collagen fibrils and GAG chains has been discussed on the basis of observations and experiments. This study uses scanning transmission electron microscope (STEM) tomography with high Z-axis resolution to analyze the three-dimensional structure of GAG chains in the dermis from a healthy individual and a patient with musculocontractural Ehlers-Danlos syndrome caused by pathogenic variants in CHST14 (mcEDS-CHST14). This observation revealed that the dermis from a healthy individual featured multiple GAG chains that wrapped around collagen fibrils and formed incomplete ring structures. However, in the dermis from a patient with mcEDS-CHST14, GAG chains were linear and did not form rings. Based on the relationship between collagen fibrils and GAG chains, we suggest the three-dimensional structure of normal GAG chains in a new model named the 'segmented ring-mesh model'. The interactions between collagen fibrils and GAG chains in this model also apply to the dermis of mcEDS-CHST14 patients, in which the GAG chain composition changes to become CS-rich and more linear. This change leads to an increased inter-fibrillar space, which inhibits the dense packing of collagen fibrils. These findings suggest that this phenomenon contributes to the skin fragility observed in mcEDS-CHST14 patients. Our study suggests the 'segmented ring-mesh model' of GAG chains is essential for the dense packing of collagen fibrils in normal dermis. STEM tomography is highly effective in analyzing the three-dimensional structure of collagen fibrils and GAG chains.

Electron tomography is a powerful tool for structural studies in cell biology, but specimen thickness remains a significant limitation. Scanning transmission electron microscopy (STEM) tomography offers advantages in this regard. Recent developments in focused ion beam slicing methods for cryo-cell biology have enabled the observation and 3D reconstruction of relatively thick specimens (300-500 nm) using cryo-STEM tomography. Organelles such as mitochondria and the nuclear membrane have been clearly reconstructed, demonstrating the promise of STEM tomography for structural studies in cell biology.

Atomic force microscopy (AFM) allows direct imaging of atomic- or molecular-scale surface structures in liquid. However, such subnanoscale measurements are often sensitive to the AFM tip properties. To overcome this problem, 30 nm Si-sputter coating was proposed, and its effectiveness in improving stability and reproducibility has been demonstrated in atomic-scale imaging of various materials. However, this method involves tip blunting, enhancing the tip-induced dilation effect. As an alternative method, here we investigate atomic layer deposition (ALD) Al2O3-coating, where the film thickness is atomically well-controlled. Our transmission electron microscopy, contact angle and force curve measurements consistently suggest that as-purchased tips are covered with organic contaminants, and the initial 20 cycles gradually remove them, reducing the tip radius (Rt) and hydrophobicity. Further deposition increases Rt and hydrophilicity and forms an intact Al2O3 film over 50 cycles. We compared 50-cycle ALD-coated tips with 30 nm Si-sputter-coated tips in imaging mica and chitin nanocrystals (NCs). On mica, ALD coating gives slightly less stability and reproducibility in hydration force measurements than the Si sputter coating, yet they are sufficient in atomic-scale imaging. In imaging chitin NCs, ALD-coated tips give a less tip-induced dilation effect while maintaining molecular-scale imaging capability. We also found that 10-cycle-ALD coated tips covered with carbon give a better resolution and reproducibility in observing subnanoscale features at chitin NC surfaces. This result and our experience empirically suggest carbon-coated tips' effectiveness in observing carbon-based materials.

We have studied the effects of electron irradiation on Pt/a-SiOx thin films by transmission electron microscopy and electron diffraction. Pt2Si was formed by 75 keV electron irradiation at 298 K and 90 K. Such a low-temperature synthesis of Pt2Si can be attributed to the dissociation of a-SiOx induced by electronic excitation; Si-O bonds dissociate through Auger decay of core-holes generated by electronic excitation, and then, dissociated Si atoms form Pt-Si bonds. The morphology of Pt islands extensively changed during Pt2Si formation, even at 90 K. Coalescence and growth of metallic particles are not due to thermal effects during electron irradiation but to athermal processes accompanied by silicide formation. To maintain the reaction interface between metallic particles and the dissociated Si atoms by electronic excitation, a considerable concomitant morphology change occurs. Similarly, Fe2Si was synthesized by using the same technique. In this way, we have demonstrated a versatile method for selectively forming nanoscale metal silicides in electron-irradiated areas at room temperature. We also propose a new mechanism for the crystallization of amorphous alloys, which is mediated by additional solute atoms produced by electronic excitation. Crystallization of amorphous Pd-Si alloy thin films can be realized by 75 keV electron irradiation at 90 K via the electronic excitation, where both knock-on damage and possible thermal crystallization can be excluded. Supply of dissociated Si to the Pd-Si layer may cause instability of the amorphous phase, which serves as the trigger for the remarkable structural change, i.e. additional solute atom-mediated crystallization.