Atmospheric scanning electron microscope observes cells and tissues in open medium through silicon nitride film
Atmospheric scanning electron microscope observes cells and tissues in open medium through silicon nitride film
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DOI：
10.1016/j.jsb.2010.01.005
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发表时间：
2010-03-01
影响因子：
3
通讯作者：
Sato, Chikara
中科院分区：
文献类型：
--
作者：
Nishiyama, Hidetoshi;Suga, Mitsuo;Sato, Chikara
直接观察亚细胞结构及其表征对于了解其生理功能至关重要。为了在开放环境中观察它们，我们开发了一种倒置扫描电子显微镜，带有可拆卸的开放式培养皿，分辨率为 8 nm，并与荧光显微镜结合，从顶部准同时观察同一区域。对于从底部进行扫描电子显微镜，皿底部的氮化硅薄膜窗口在电子枪和打开的样品皿之间保持真空，同时允许电子通过。电子从样品中反向散射，并被培养皿下方的检测器捕获。在开放培养皿上培养的细胞可以在光学显微镜下进行外部操作、固定并使用扫描电子显微镜进行观察。一旦通过扫描电子显微镜揭示了精细结构，就可以通过与单独制备的候选蛋白质的荧光标记光学显微图像及其重金属标记或染色的 ASEM 图像进行比较来鉴定其组成蛋白质。此外，无需薄切片即可成功观察到铂蓝染色组织块中的细胞核，这表明该倒置扫描电子显微镜在癌症诊断中的适用性。该显微镜可实现介观尺度结构的可视化，也适用于包括高分子化学在内的非生物科学领域。 (C) 2010 Elsevier Inc. 保留所有权利。
Direct observation of subcellular structures and their characterization is essential for understanding their physiological functions. To observe them in open environment, we have developed an inverted scanning electron microscope with a detachable, open-culture dish, capable of 8 nm resolution, and combined with a fluorescence microscope quasi-simultaneously observing the same area from the top. For scanning electron microscopy from the bottom, a silicon nitride film window in the base of the dish maintains a vacuum between electron gun and open sample dish while allowing electrons to pass through. Electrons are backscattered from the sample and captured by a detector under the dish. Cells cultured on the open dish can be externally manipulated under optical microscopy, fixed, and observed using scanning electron microscopy. Once fine structures have been revealed by scanning electron microscopy, their component proteins may be identified by comparison with separately prepared fluorescence-labeled optical microscopic images of the candidate proteins, with their heavy-metal-labeled or stained ASEM images. Furthermore, cell nuclei in a tissue block stained with platinum-blue were successfully observed without thin-sectioning, which suggests the applicability of this inverted scanning electron microscope to cancer diagnosis. This microscope visualizes mesoscopic-scale structures, and is also applicable to non-bioscience fields including polymer chemistry. (C) 2010 Elsevier Inc. All rights reserved.