Graphene Enclosure of Chemically Fixed Mammalian Cells for Liquid-Phase Electron Microscopy
Graphene Enclosure of Chemically Fixed Mammalian Cells for Liquid-Phase Electron Microscopy
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DOI：
10.3791/61458
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发表时间：
2020-09-01
影响因子：
1.2
通讯作者：
de Jonge, Niels
中科院分区：
文献类型：
--
作者：
Blach, Patricia;Keskin, Sercan;de Jonge, Niels
介绍了一种利用扫描电子显微镜(STEM)研究乳腺癌细胞完整质膜中人表皮生长因子受体2(HER2)的方法。哺乳动物乳腺癌细胞系SKBR3的细胞生长在带有氮化硅(SIN)窗口的硅微芯片上。细胞被化学固定，HER2蛋白被量子点纳米颗粒(Qds)标记，使用两步生物素-链霉亲和素结合方案。这些细胞被多层石墨烯覆盖，以保持水合状态，并在STEM过程中保护它们免受电子束的破坏。为了考察样品在电子束辐照下的稳定性，进行了剂量系列实验。比较了石墨烯包覆和未包覆的样品。随着电子剂量D的增加，一些未镀膜的样品出现了以明亮伪影的形式出现的束致损伤，而镀膜的样品没有出现伪影。
A protocol is described for investigating the human epidermal growth factor receptor 2 (HER2) in the intact plasma membrane of breast cancer cells using scanning transmission electron microscopy (STEM). Cells of the mammalian breast cancer cell line SKBR3 were grown on silicon microchips with silicon nitride (SiN) windows. Cells were chemically fixed, and HER2 proteins were labeled with quantum dot nanoparticles (QDs), using a two-step biotin-streptavidin binding protocol. The cells were coated with multilayer graphene to maintain a hydrated state, and to protect them from electron beam damage during STEM. To examine the stability of the samples under electron beam irradiation, a dose series experiment was performed. Graphene-coated and non-coated samples were compared. Beam induced damage, in the form of bright artifacts, appeared for some non-coated samples at increased electron dose D, while no artifacts appeared on coated samples.