Three-dimensional super-resolution protein localization correlated with vitrified cellular context

分类： 生物学 >> 生物物理学 提交时间： 2016-05-12

Liu, Bei Xue, Yanhong Zhao, Wei Chen, Yan Fan, Chunyan Gu, Lusheng Zhang, Yongdeng Zhang, Xiang Sun, Lei Huang, Xiaojun Ding, Wei Sun, Fei Ji, Wei Xu, Tao Liu, Bei Gu, Lusheng Zhang, Yongdeng Xu, Tao Zhao, Wei Chen, Yan

摘要： We demonstrate the use of cryogenic super-resolution correlative light and electron microscopy (csCLEM) to precisely determine the spatial relationship between proteins and their native cellular structures. Several fluorescent proteins (FPs) were found to be photoswitchable and emitted far more photons under our cryogenic imaging condition, resulting in higher localization precision which is comparable to ambient super-resolution imaging. Vitrified specimens were prepared by high pressure freezing and cryo-sectioning to maintain a near-native state with better fluorescence preservation. A 2-3-fold improvement of resolution over the recent reports was achieved due to the photon budget performance of screening out Dronpa and optimized imaging conditions, even with thin sections which is at a disadvantage when calculate the structure resolution from label density. We extended csCLEM to mammalian cells by introducing cryo-sectioning and observed good correlation of a mitochondrial protein with the mitochondrial outer membrane at nanometer resolution in three dimensions.

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