Nanoscale analysis of unstained biological specimens in water without radiation damage using high-resolution frequency transmission electric-field system based on FE-SEM

Ogura, Toshihiko

doi:10.1016/j.bbrc.2015.02.140

Cited by 43 publications

(73 citation statements)

References 34 publications

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“…We recently developed a high-resolution frequency transmission electric-field (FTE) imaging system based on field emission scanning electron microscopy (SEM) of JSM-7000F (JEOL, Tokyo, Japan, http://www.jeol.co.jp), which enables observation of the biological specimens in water without metal staining [39]. Exosomes were imaged by using the FTE imaging system.…”

Section: The High-resolution Frequency Transmission Electric-field Symentioning

confidence: 99%

Mesenchymal Stem Cell-Derived Exosomes Promote Fracture Healing in a Mouse Model

Furuta

Miyaki

Ishitobi

et al. 2016

Stem Cells Translational Medicine

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Paracrine signaling by bone-marrow-derived mesenchymal stem cells (MSCs) plays a major role in tissue repair. Although the production of regulatory cytokines by MSC transplantation is a critical modulator of tissue regeneration, we focused on exosomes, which are extracellular vesicles that contain proteins and nucleic acids, as a novel additional modulator of cell-to-cell communication and tissue regeneration. To address this, we used radiologic imaging, histological examination, and immunohistochemical analysis to evaluate the role of exosomes isolated from MSC-conditioned medium (CM) in the healing process in a femur fracture model of CD9 2/2 mice, a strain that is known to produce reduced levels of exosomes. We found that the bone union rate in CD9 2/2 mice was significantly lower than wild-type mice because of the retardation of callus formation. The retardation of fracture healing in CD9 2/2 mice was rescued by the injection of exosomes, but this was not the case after the injection of exosomes-free conditioned medium (CM-Exo). The levels of the bone repairrelated cytokines, monocyte chemotactic protein-1 (MCP-1), MCP-3, and stromal cell-derived factor-1 in exosomes were low compared with levels in CM and CM-Exo, suggesting that bone repair may be in part mediated by other exosome components, such as microRNAs. These results suggest that exosomes in CM facilitate the acceleration of fracture healing, and we conclude that exosomes are a novel factor of MSC paracrine signaling with an important role in the tissue repair process. STEM CELLS TRANSLATIONAL MEDICINE 2016;5:1620-1630 SIGNIFICANCEThis work focuses on exosomes, which are extracellular vesicles, as a novel additional modulator of cell-to-cell communication. This study evaluated the role of exosomes isolated from mesenchymal stem cell (MSC)-conditioned medium (MSC-CM) in the fracture-healing process of CD9 2/2 mice, a strain that is known to produce reduced levels of exosomes. Retardation of fracture healing in CD9 2/2 mice was rescued by the injection of MSC exosomes, but this was not the case after the injection of exosome-free CM. This study finds that MSC exosomes are a novel factor of MSC paracrine signaling, with an important role in the tissue repair process.

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Section: The High-resolution Frequency Transmission Electric-field Symentioning

confidence: 99%

Mesenchymal Stem Cell-Derived Exosomes Promote Fracture Healing in a Mouse Model

Furuta

Miyaki

Ishitobi

et al. 2016

Stem Cells Translational Medicine

Self Cite

371

362

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“…In a recent study, we developed a novel imaging technology named scanning electron-assisted dielectric microscopy (SE-ADM), which enables the observation of intact cells, bacteria and protein particles in water with extremely low radiation damage and without requiring staining and fixation [18,19]. Our system can produce high-contrast images of untreated biological specimens in aqueous conditions [19][20][21][22]. The biological samples are enclosed in a liquid holder composed of tungsten (W)-coated silicon nitride (SiN) film.…”

Section: Introductionmentioning

confidence: 99%

High-resolution imaging of autophagosome formation by LC3 and ATG12 in cells using scanning electron-assisted dielectric microscopy

Okada

Ogura

2020

Preprint

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Background Autophagy is an intracellular self-eating system that plays a central role in cellular recycling. The formation of functional autophagosomes depends on several autophagy- related proteins, including LC3 and Atg12. To analyse an autophagy system, it is essential to precisely examine the cellular functions of cells in liquid condition. For this purpose, we have developed a novel scanning electron-assisted dielectric microscope (SE-ADM) for nanoscale observations of intact cells in aqueous conditions. Results Here, we first show that our SE-ADM system observes LC3- and Atg12-containing autophagosomes in cells labelled with antibody-binding gold nano-colloids in medium. We observe that during autophagosome formation, Atg12 localises along the actin mesh structure, whereas LC3 forms arcuate or circular alignments. Our system also detects the different distribution properties of LC3 and Atg12. Atg12 is broadly distributed, whereas LC3 is localised over a smaller range. These differences in spatial-distribution manifest as differently sized spots on fluorescence optical microscopy. Conclusion The direct SE-ADM observations of living cells incubated with nanoparticles in a medium may be useful for analysing various cell functions.

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“…These preparations also have positive effects in terms of contrast enhancement. To date, atmospheric and/or wet biological specimens have been examined using atmospheric holders, but they undergo heavy radiation damage caused by electron beam (EB).We recently developed a new imaging technology called a scanning-electron assisted dielectricimpedance microscopy (SE-ADM) system based on SEM [1][2]. Our system provides high-contrast imaging of the intact biological specimens in water introduced in an atmospheric sample holder comprising two silicon nitride (SiN) films (Fig.…”

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“…1b) [1]. In this method, the biological samples are not directly exposed to the EB, which prevents electron radiation damage [1].The intact bacteria (Rhodobacter capsulatus) immersed in water were examined using the highresolution SE-ADM system at 10,000× magnification, 3.6-kV EB acceleration [2]. The intact bacteria were visible with clear-cut white contrast; the inner structure exhibited complex undulation (Fig.…”

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Nanoscale Observation of Intact Biological Specimens in Water with High-contrast Imaging by Scanning Electron Assisted Dielectric-impedance Microscopy

Ogura

Okada

2017

Microsc Microanal

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Scanning electron microscopy (SEM) has been widely used to analyse biological specimens. However, SEM observations of these specimens under high vacuum conditions require specific sample preparation protocols involving glutaraldehyde fixation, negative staining, cryo-techniques and metal coating to avoid electrical radiation damage. These preparations also have positive effects in terms of contrast enhancement. To date, atmospheric and/or wet biological specimens have been examined using atmospheric holders, but they undergo heavy radiation damage caused by electron beam (EB).We recently developed a new imaging technology called a scanning-electron assisted dielectricimpedance microscopy (SE-ADM) system based on SEM [1][2]. Our system provides high-contrast imaging of the intact biological specimens in water introduced in an atmospheric sample holder comprising two silicon nitride (SiN) films (Fig. 1a). The upper SiN film, which is coated with tungsten (W), is irradiated using a focused scanning EB. Irradiated electrons are strongly scattered and absorbed in the 15 nm-thick W layer resulting in a negative electric-field potential at the irradiated position. This negative potential transmitted to the bottom SiN film through the biological specimens in water (Fig. 1b) [1]. In this method, the biological samples are not directly exposed to the EB, which prevents electron radiation damage [1].The intact bacteria (Rhodobacter capsulatus) immersed in water were examined using the highresolution SE-ADM system at 10,000× magnification, 3.6-kV EB acceleration [2]. The intact bacteria were visible with clear-cut white contrast; the inner structure exhibited complex undulation (Fig. 2a). We also used the new method to analyse unstained and unfixed IgM antibody molecules in water. The IgM antibody has molecular weight of 900 kDa and consists of five IgG antibodies. We examined intact IgM protein in water at 50,000× magnification and 4-kV EB acceleration (Fig. 2b). Several small particles of approximately 50-nm diameter were dispersed throughout the whole visual field. Fig. 2c shows three individual IgM molecules indicated in Fig. 2b with white arrows, and the pseudo-colour maps are shown in Fig. 2d. Individual IgM molecules are visible as slightly star shaped with a dome-like centre. Further, we demonstrate the nanoscale observation of living mammalian cells using SE-ADM system with a culture dish holder [3]. Our system clearly visualised unstained and unfixed cancer cells in medium (Fig. 3). Figure 3a clearly shows intracellular structures of the nucleus and endoplasmic reticulum (ER). Moreover, SE-ADM images indicated various vesicles and/or ER near the nucleus (Fig. 3b). Two enlarged vesicles of red-boxed area from Fig. 3b clearly reveal a spherical shape with rough surface membrane (Fig. 3c, d), and the vesicles were found to be interconnected [3].Taken together, we developed a SE-ADM system based on SEM. This system enables high-resolution imaging of intact biological specimens in water without radiation damage. Furth...

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Nanoscale analysis of unstained biological specimens in water without radiation damage using high-resolution frequency transmission electric-field system based on FE-SEM

Cited by 43 publications

References 34 publications

Mesenchymal Stem Cell-Derived Exosomes Promote Fracture Healing in a Mouse Model

Mesenchymal Stem Cell-Derived Exosomes Promote Fracture Healing in a Mouse Model

High-resolution imaging of autophagosome formation by LC3 and ATG12 in cells using scanning electron-assisted dielectric microscopy

Nanoscale Observation of Intact Biological Specimens in Water with High-contrast Imaging by Scanning Electron Assisted Dielectric-impedance Microscopy

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