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Sci Rep. 2015 Oct 14;5:13017. doi: 10.1038/srep13017.

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

Liu B1,2, Xue Y1, Zhao W1,3, Chen Y1,3, Fan C1,3, Gu L1,2, Zhang Y1,2, Zhang X1, Sun L1,4, Huang X1,4, Ding W1,4, Sun F1,3,4, Ji W1,3,4, Xu T1,2,3,4.

Author information

1
National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101, China.
2
College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, Hubei, 430074, China.
3
College of Life Sciences, University of the Chinese Academy of Sciences, Beijing, 100049, China.
4
Center for Biological Imaging, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101, China.

Abstract

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.

PMID:
26462878
PMCID:
PMC4604464
DOI:
10.1038/srep13017
[Indexed for MEDLINE]
Free PMC Article

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