High-precision correlative fluorescence and electron cryo microscopy using two independent alignment markers.
High-precision correlative fluorescence and electron cryo microscopy using two independent alignment markers.
复制标题
DOI：
10.1016/j.ultramic.2013.10.011
复制
发表时间：
2014-08
期刊：
影响因子：
2.2
通讯作者：
Gruenewald, Kay
中科院分区：
文献类型：
--
作者：
Schellenberger, Pascale;Kaufmann, Rainer;Siebert, C. Alistair;Hagen, Christoph;Wodrich, Harald;Gruenewald, Kay
关键词：
相关光电子显微镜（CLEM）是一种新兴的技术，它将荧光显微镜（FM）提供的功能信息与电子显微镜（EM）提供的高分辨率结构信息相结合。到目前为止，冷冻水化样品的相关低温显微术还没有达到微米级的精度。在这里，提出了一种方法，使荧光标记的蛋白质和电子冷冻断层扫描（cryoET）数据之间的相关性与纳米范围的精度。具体而言，通过相关荧光冷冻显微镜（cryoFM）和cryoET检查玻璃化全细胞的薄区域。所提出的cryoCLEM工作流程的新颖方面不仅包括实现两个独立的电子致密荧光标记以提高对准的精度，而且还包括获得每个感兴趣的个体对象的相关精度的估计的能力。以定位荧光标记的腺病毒颗粒在细胞内运输为例，详细介绍了从急冻到冷冻ET的相关工作流程。玻璃化的哺乳动物细胞通过荧光和电子冷冻显微镜成像。添加TetraSpeck荧光标记物以校正低温荧光通道之间的偏移。FluoSpheres基准点用作参考点，为cryoEM图像分配新坐标。以63 nm的平均相关精度定位腺病毒颗粒。
Correlative light and electron microscopy (CLEM) is an emerging technique which combines functional information provided by fluorescence microscopy (FM) with the high-resolution structural information of electron microscopy (EM). So far, correlative cryo microscopy of frozen-hydrated samples has not reached better than micrometre range accuracy. Here, a method is presented that enables the correlation between fluorescently tagged proteins and electron cryo tomography (cryoET) data with nanometre range precision. Specifically, thin areas of vitrified whole cells are examined by correlative fluorescence cryo microscopy (cryoFM) and cryoET. Novel aspects of the presented cryoCLEM workflow not only include the implementation of two independent electron dense fluorescent markers to improve the precision of the alignment, but also the ability of obtaining an estimate of the correlation accuracy for each individual object of interest. The correlative workflow from plunge-freezing to cryoET is detailed step-by-step for the example of locating fluorescence-labelled adenovirus particles trafficking inside a cell. Vitrified mammalian cell were imaged by fluorescence and electron cryo microscopy. TetraSpeck fluorescence markers were added to correct shifts between cryo fluorescence channels. FluoSpheres fiducials were used as reference points to assign new coordinates to cryoEM images. Adenovirus particles were localised with an average correlation precision of 63 nm.
登录
查看更多内容
影响因子：
3
作者：
Faas, F. G. A.;Barcena, M.;Koster, A. J.
通讯作者：
Koster, A. J.
影响因子：
3
作者：
Kremer, JR;Mastronarde, DN;McIntosh, JR
通讯作者：
McIntosh, JR
影响因子：
3
作者：
Agronskaia, Alexandra V.;Valentijn, Jack A.;Gerritsen, Hans C.
通讯作者：
Gerritsen, Hans C.
使用相关的活细胞显微镜和冷冻电子断层扫描直接可视化HIV-1。
影响因子：
5.7
作者：
Jun, Sangmi;Ke, Danxia;Debiec, Karl;Zhao, Gongpu;Meng, Xin;Ambrose, Zandrea;Gibson, Gregory A.;Watkins, Simon C.;Zhang, Peijun
通讯作者：
Zhang, Peijun
影响因子：
3
作者：
Mastronarde, DN
通讯作者：
Mastronarde, DN