title: Three-dimensional super-resolution protein localization correlated with vitrified cellular context.
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.
authors.affiliation: National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101, China.|College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, Hubei, 430074, China.
authors.forename: Bei
authors.initials: B
authors.lastname: Liu
authors.affiliation: National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101, China.
authors.forename: Yanhong
authors.initials: Y
authors.lastname: Xue
authors.affiliation: National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101, China.|College of Life Sciences, University of the Chinese Academy of Sciences, Beijing, 100049, China.
authors.forename: Wei
authors.initials: W
authors.lastname: Zhao
authors.affiliation: National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101, China.|College of Life Sciences, University of the Chinese Academy of Sciences, Beijing, 100049, China.
authors.forename: Yan
authors.initials: Y
authors.lastname: Chen
authors.affiliation: National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101, China.|College of Life Sciences, University of the Chinese Academy of Sciences, Beijing, 100049, China.
authors.forename: Chunyan
authors.initials: C
authors.lastname: Fan
authors.affiliation: National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101, China.|College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, Hubei, 430074, China.
authors.forename: Lusheng
authors.initials: L
authors.lastname: Gu
authors.affiliation: National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101, China.|College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, Hubei, 430074, China.
authors.forename: Yongdeng
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101, China.
authors.forename: Xiang
authors.initials: X
authors.lastname: Zhang
authors.affiliation: National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101, China.|Center for Biological Imaging, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101, China.
authors.forename: Lei
authors.initials: L
authors.lastname: Sun
authors.affiliation: National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101, China.|Center for Biological Imaging, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101, China.
authors.forename: Xiaojun
authors.initials: X
authors.lastname: Huang
authors.affiliation: National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101, China.|Center for Biological Imaging, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101, China.
authors.forename: Wei
authors.initials: W
authors.lastname: Ding
authors.affiliation: National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101, China.|College of Life Sciences, University of the Chinese Academy of Sciences, Beijing, 100049, China.|Center for Biological Imaging, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101, China.
authors.forename: Fei
authors.initials: F
authors.lastname: Sun
authors.affiliation: National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101, China.|College of Life Sciences, University of the Chinese Academy of Sciences, Beijing, 100049, China.|Center for Biological Imaging, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101, China.
authors.forename: Wei
authors.initials: W
authors.lastname: Ji
authors.affiliation: National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101, China.|College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, Hubei, 430074, China.|College of Life Sciences, University of the Chinese Academy of Sciences, Beijing, 100049, China.|Center for Biological Imaging, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101, China.
authors.forename: Tao
authors.initials: T
authors.lastname: Xu
mesh_terms: D015925:Cryopreservation / Q000295:instrumentation* / Q000379:methods; D004867:Equipment Design; D019544:Equipment Failure Analysis; D057809:HEK293 Cells; D006801:Humans; D007089:Image Enhancement / Q000295:instrumentation / Q000379:methods; D021621:Imaging, Three-Dimensional / Q000295:instrumentation* / Q000379:methods; D055617:Micro-Electrical-Mechanical Systems / Q000295:instrumentation / Q000379:methods; D008854:Microscopy, Electron / Q000295:instrumentation*; D008856:Microscopy, Fluorescence / Q000295:instrumentation* / Q000379:methods; D057054:Molecular Imaging / Q000295:instrumentation / Q000379:methods; D064847:Multimodal Imaging / Q000295:instrumentation / Q000379:methods; D011506:Proteins / Q000378:metabolism*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013223:Statistics as Topic; D013347:Subcellular Fractions / Q000378:metabolism*; D014018:Tissue Distribution; D058989:Vitrification
chemical_list: D011506:Proteins