Electron microscopy of primary cell cultures in solution and correlative optical microscopy using ASEM
Electron microscopy of primary cell cultures in solution and correlative optical microscopy using ASEM
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DOI：
10.1016/j.ultramic.2013.10.010
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发表时间：
2014-08-01
期刊：
影响因子：
2.2
通讯作者：
Sato, Chikara
中科院分区：
文献类型：
--
作者：
Hirano, Kazumi;Kinoshita, Takaaki;Sato, Chikara
细胞在水溶液自然环境中的相关光电子显微镜有助于高通量观察蛋白质复合体的形成。ASEM允许倒置的扫描电子显微镜从下面观察潮湿的样品，而光学显微镜几乎同时从上面观察它。一种带有氮化硅(SiN)薄膜窗口的一次性ASEM培养皿，可以在稳定的培养箱环境中，在几毫升的培养介质中通过不同的涂层实现对底物敏感细胞的原代培养。在光学显微镜或荧光显微镜下观察神经元分化、神经网络、原血小板形成和吞噬作用，并用金标记免疫组织化学方法进行高分辨率成像。Fas在细胞表面的表达与F-肌动蛋白的空间分布相关。用原代培养的神经元进行轴突划分，GluR82-N末端连接的荧光磁珠诱导突触前与突触前标志物Bason相关。此外，还捕获了分泌前血小板的巨核细胞，并将具有黏附活性的P-选择素定位于免疫-ASEM存在的一些颗粒上。此外，还观察了去硫细胞对乳酸菌的吞噬作用。基于这些研究，亚欧会议相关显微镜有望在不久的将来研究各种介观尺度的动力学。(C)2013爱思唯尔B.V.保留所有权利。
Correlative light-electron microscopy of cells in a natural environment of aqueous liquid facilitates highthroughput observation of protein complex formation. ASEM allows the inverted SEM to observe the wet sample from below, while an optical microscope observes it from above quasi-simultaneously. The disposable ASEM dish with a silicon nitride (SiN) film window can be coated variously to realize the primary-culture of substrate-sensitive cells in a few milliliters of culture medium in a stable incubator environment. Neuron differentiation, neural networking, proplatelet-formation and phagocytosis were captured by optical or fluorescence microscopy, and imaged at high resolution by gold-labeled immunoASEM with/without metal staining. Fas expression on the cell surface was visualized, correlated to the spatial distribution of F-actin. Axonal partitioning was studied using primary-culture neurons, and presynaptic induction by GluR82-N-terminus-linked fluorescent magnetic beads was correlated to the presynaptic-marker Bassoon. Further, megakaryocytes secreting proplatelets were captured, and P-selectins with adherence activity were localized to some of the granules present by immuno-ASEM. The phagocytosis of lactic acid bacteria by denthitic cells was also imaged. Based on these studies, ASEM correlative microscopy promises to allow the study of various mesoscopic-scale dynamics in the near future. (C) 2013 Elsevier B.V. All rights reserved.