Applied Microscopy最新文献

Negative staining electron microscopy remains a valuable tool for structural biology, particularly for initial characterization of large protein complexes. However, the limitations of single staining methods often result in incomplete structural information. Here, we present a novel multi-stain approach for negative staining electron microscopy, applied to the structural analysis of the pyruvate dehydrogenase E2 (PDH E2) complex. By integrating data from three distinct staining agents (uranyl acetate, ammonium phosphotungstate, and ammonium molybdate) we demonstrate significant improvements in structural resolution and detail. Our method improved the resolution from a range of approximately 27–31 Å (observed with individual stains) to about 21.7 Å in the combined dataset. This enhancement facilitated a clearer visualization of the complex’s icosahedral symmetry and allowed for a more precise determination of the overall shape and domain organization of the PDH E2 complex. The multi-stain approach revealed complementary structural information, with each stain highlighting different aspects of the protein complex. Uranyl acetate provided excellent overall contrast, while ammonium phosphotungstate and molybdate offered enhanced visibility of specific structural elements. The integration of these complementary data sets resulted in a more comprehensive structural model. Our findings suggest that this multi-stain negative staining approach can be a powerful tool for enhancing low-resolution structural information of large protein complexes, bridging the gap between initial characterization and high-resolution studies. This method holds promise for improving our understanding of challenging macromolecular assemblies and may serve as a valuable precursor to more advanced structural biology techniques.

Diatoms are microalgae with a significant ecological role as primary producers, contributing approximately 20% of global carbon fixation. They exhibit remarkable evolutionary success and biodiversity, with approximately 18,000 species identified globally, including 2,323 species reported in Korea. In this study, we identified 13 unrecorded diatom species from various freshwater environments in Korea, including rivers, streams, reservoirs, and lagoons. Among these, the genera Brachysira, Gomphonella, and Staurophora were recorded for the first time in Korea, expanding the national species list. The unrecorded species included 12 pennate diatoms (Adlafia multnomahii, Encyonema cespitosum, Placoneis symmetrica, Gomphonema incognitum, Rhoicosphenia baltica, Brachysira vitrea, Gomphonella fogedii, Hantzschia psammicola, Hyalosynedra laevigata, Pseudostaurosira parasitica, Tryblionella calida, and Staurophora amphioxys) and one centric diatom (Discostella lacuskarluki). Morphological and ultrastructural analyses were conducted using scanning electron microscopy to characterize these species. This research highlights the biodiversity of Korean diatoms and their potential applications in environmental monitoring and nanotechnology, contributing to the broader understanding of diatom taxonomy and ecology.

Optical clearing of apple tissues was performed to observe the pre-penetration behavior of Botryosphaeria dothidea. Mature red fruits and two-year-old twigs were artificially inoculated with the fungal conidia. Fruit epidermis and twig cork tissues were excised and immersed overnight in an ethanol-chloroform solution amended with trichloroacetic acid. Lactophenol cotton blue was used to stain the fungus on the host surfaces. The morphology and behavior of the inoculated B. dothidea could be clearly observed in the two types of optically cleared specimens. The conidia showed either monopolar or bipolar germination, leading to the emergence of germ tubes from one or both conidial ends. Conidia formed appressoria at the terminal ends of germ tubes. They appeared round, hook-shaped, and irregular-shaped in two-dimensional light micrographs. Multiple appressoria were observed on the suberized phellem cells in twig lenticels. These results suggest that the optical clearing technique and fungal staining were effective in partially decolorizing apple tissues and revealing the fungal structures on the host surfaces.

X-ray micro-computed tomography (XCT) is an X-ray-based three-dimensional (3D) imaging technique that enables non-destructive imaging of both external and internal structures. It is widely used for studying biological specimens such as animals and plants. In this review, we discuss various specimen preparation methods for the technique, particularly focusing on forest pests, with six representative cases. Specimen preparation methods for forest pests can be broadly categorized into three groups based on mounting types: (i) simple mounting, (ii) liquid-cell mounting, and (iii) dry-cell mounting. The simple mounting method is particularly suitable for adult beetles due to their exoskeleton. The dehydration process minimizes specimen movement during scanning, ensuring better imaging quality. In the case of liquid-cell mounting, the specimen is immersed in a liquid medium for scanning, which effectively preserves the soft tissues of larvae and pupae. The dry-cell mounting does not involve fixation or dehydration and is particularly useful for analyzing immobilized specimens. To enhance the quality of 3D images, selecting an appropriate preparation method is essential. Since forest pests display varying sizes and types, the choice of preparation method should be based on the specific characteristics of the specimens of interest and research objectives. This review provides valuable insights for researchers and practitioners seeking to identify the most suitable and effective mounting method for XCT scanning of forest pests.

Transmission electron microscopy (TEM), which can analyze the shape and crystallinity of materials as well as the chemical bonding of ions and the states of elements, operates at different accelerating voltages depending on the type of specimen analyzed and the analysis area. Electron-beam irradiation can be used to induce structural transitions and crystallization of materials. Therefore, studies on phase transition using electron beams have been frequently conducted. Cobalt oxides, including cobalt hydroxides, have various phases and crystal structures, depending on their stoichiometric compositions. Specific synthesis methods can be used to synthesize these at low dimensions, in addition to large nanosheet structures. In this study, the crystallization and phase transition of amorphous cobalt hydroxide nanosheets induced by continuous electron-beam irradiation were analyzed using high-resolution TEM (HR-TEM). The synthesized cobalt hydroxide nanosheets were partially converted into cobalt oxides, and the transferred area expanded as the irradiation time increased. Under 300 kV of accelerating voltage, the transition to cubic cobalt oxides was dominant, suggesting a frequent transitional behavior of amorphous metal hydroxides upon electron-beam irradiation.

The development of bispecific antibodies (BsAbs) represents a significant advancement in therapeutic antibody design, enabling the simultaneous targeting of two different antigens. This dual-targeting capability enhances therapeutic efficacy, particularly in complex diseases like cancer, where tumor heterogeneity presents a significant challenge for traditional treatments. By bridging two distinct pathways, BsAbs can improve specificity and minimize off-target effects, making them invaluable in therapeutic contexts. Integrating advanced imaging techniques, particularly Correlative Light and Electron Microscopy (CLEM), offers a unique opportunity to visualize the dynamic interactions of BsAbs within cellular environments. CLEM combines the strengths of optical and electron microscopy, allowing researchers to observe real-time antibody-antigen interactions at nanoscale resolution. This synergy not only deepens our understanding of BsAbs’ mechanisms of action but also provides critical insights into their spatial distribution, binding kinetics, and functional dynamics in live cells. In this review, the integration of BsAbs and CLEM paves the way for targeted therapeutic strategies, fostering the development of more effective treatments that can adapt to the complexities of disease pathology.

The olfactory organ of Synechogobius hasta was investigated with a focus on its environmental adaptation, using stereo microscopy and light microscopy. This research revealed the following anatomical and histological characteristics: (i) tubular anterior nostril, (ii) one longitudinal lamella, (iii) two accessory nasal sacs, (iv) lymphatic cells in the lower part of the sensory epithelium, (v) four to five villi of olfactory receptor neurons, (vi) abundant blood capillaries beneath the sensory epithelium, and (vii) rod-shaped erythrocytes. These findings hint that the olfactory organ of S. hasta has anatomical and histological adaptations to intertidal pools that undergo periodic hypoxia and increased temperature under stagnant water conditions due to the tidal cycle.

This study investigates the impact of excessive bleaching on the external morphology and internal microstructure of hair, compared to untreated hair. Using scanning electron microscopy (SEM) and transmission electron microscopy (TEM), we observed significant changes in both the surface and internal structures of bleached hair. SEM analysis of normal hair revealed a relatively clean surface with intact cuticle scales, while bleached hair showed brittle, torn scales with a rough appearance. In areas where the cuticle was broken, remnants of endocuticle debris were still attached, contributing to the rough surface. Complete separation of the cuticle layer resulted in numerous longitudinal fissures along the exposed cortical surface of bleached hair. TEM analysis further confirmed distinct differences; in normal hair, the cuticle layer and cortex were well-separated, and a small hole was observed within the endocuticle of the cuticle cells. Conversely, in bleached hair, the cuticle layer was separated from the cortex, with numerous pores formed by the dissolution of melanin granules scattered within the cortex, specifically between the macrofibrils. No melanin granules were detected in the cortex of bleached hair, although the macrofibril structure remained intact. The findings clearly indicate that excessive bleaching leads to the loss of the cuticle layer, exposing the cortex and significantly altering the hair’s structural integrity.

The cosmetic-tattoo industry is evolving every year and the microstructures of the equipment have the great potential for semi-permanent makeup applications. Present paper explores the materials and microparticles of semi-permanent makeup tattoo needles. The surface of the five-round-shader tattoo needle used in semi-permanent makeup process was examined by scanning electron microscopy, and its elemental composition was analyzed by energy-dispersive X-ray spectroscopy. The comparison of five-round-shader needles have undergone thorough observation: original five-round-shader needle and distorted five-round-shader needle. The diameter of the sharp and rounded needle tip was measured at approximately 6.80 μm, while the deformed needle tip was approximately 16 μm thick, about 2.5 times thicker than the rounded needle tip. Many rosette-shaped lead (Pb) particles and irregular clusters adhere to the welded areas and closely adjacent needle shaft surfaces. The lead particles have a diameter ranging from 4 μm to 5 μm and exhibit a grid-like structure with a consistent thickness of plate-like shape. The distorted structure of Pb in rosette-shaped formations is shown to have originated from the grinding and polishing processes during needle manufacturing. To produce sterilized tattoo needles, high-quality tattoo needle inspection processes are necessary to remove any unhygienic substances adhering to the needle surface.

Glass vials are the most widely used primary containers for the packaging of parenteral products due to their optical clarity, general inertness, and hermetic properties, but under certain circumstances, they can pose safety concerns. Most of these issues are related to the potential formation of glass particulates through delamination or precipitation, resulting from the chemical interaction between the drug product and the inner surface of the glass vial. Hence, it is imperative for pharmaceutical companies to conduct product-vial compatibility studies to determine the appropriate packaging/container closure system. To support this development activity, scientists need to develop analytical methods to detect subvisible glass particulates in parenteral products, along with the appropriate positive controls, to facilitate detection and identification. This paper outlines the utilization of coaxial/episcopic and oblique illumination microscopy, combined with spectroscopic techniques, to detect thin glass particulates generated from a modified procedure. It also showcases the importance of angle-dependent lighting in visualizing positive control samples containing thin glass particulates. The analytical microscopy techniques discussed in this paper can assist scientists in selecting suitable container closure systems for developing parenteral products.