Correlative 3D imaging of whole mammalian cells with light and electron microscopy
Correlative 3D imaging of whole mammalian cells with light and electron microscopy
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DOI：
10.1016/j.jsb.2011.08.013
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发表时间：
2011-12-01
影响因子：
3
通讯作者：
Subramaniam, Sriram
中科院分区：
文献类型：
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作者：
Murphy, Gavin E.;Narayan, Kedar;Subramaniam, Sriram
我们报告的方法的进步，扩展离子磨损扫描电子显微镜（IA-SEM），也被称为聚焦离子束扫描电子显微镜，一个新出现的技术，在3D的大型生物标本的高分辨率成像的当前能力。我们建立的协议，使整个塑料嵌入的哺乳动物细胞的三维图像堆栈的常规生成IA-SEM在类似于10-20 nm的分辨率，高对比度和最小的工件从聚焦离子束。我们建立在这些进展的基础上，通过描述一个详细的方法进行相关的活共聚焦显微镜和IA-SEM相同的细胞。最后，我们证明，通过将相关成像与新开发的自动图像处理工具相结合，可以在整个哺乳动物细胞的SEM图像堆栈中定位100 nm大小的小实体，如HIV-1或金珠。我们预计，这些方法将添加到阿森纳的工具可用于调查机制的宿主-病原体相互作用，更普遍的是，哺乳动物细胞和组织的三维亚细胞结构。(C)2011 Elsevier Inc. All rights reserved.
We report methodological advances that extend the current capabilities of ion-abrasion scanning electron microscopy (IA-SEM), also known as focused ion beam scanning electron microscopy, a newly emerging technology for high resolution imaging of large biological specimens in 3D. We establish protocols that enable the routine generation of 3D image stacks of entire plastic-embedded mammalian cells by IA-SEM at resolutions of similar to 10-20 nm at high contrast and with minimal artifacts from the focused ion beam. We build on these advances by describing a detailed approach for carrying out correlative live confocal microscopy and IA-SEM on the same cells. Finally, we demonstrate that by combining correlative imaging with newly developed tools for automated image processing, small 100 nm-sized entities such as HIV-1 or gold beads can be localized in SEM image stacks of whole mammalian cells. We anticipate that these methods will add to the arsenal of tools available for investigating mechanisms underlying host-pathogen interactions, and more generally, the 3D subcellular architecture of mammalian cells and tissues. (C) 2011 Elsevier Inc. All rights reserved.