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

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

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cryopreservation / instrumentation*
  • Cryopreservation / methods
  • Equipment Design
  • Equipment Failure Analysis
  • HEK293 Cells
  • Humans
  • Image Enhancement / instrumentation
  • Image Enhancement / methods
  • Imaging, Three-Dimensional / instrumentation*
  • Imaging, Three-Dimensional / methods
  • Micro-Electrical-Mechanical Systems / instrumentation
  • Micro-Electrical-Mechanical Systems / methods
  • Microscopy, Electron / instrumentation*
  • Microscopy, Fluorescence / instrumentation*
  • Microscopy, Fluorescence / methods
  • Molecular Imaging / instrumentation
  • Molecular Imaging / methods
  • Multimodal Imaging / instrumentation
  • Multimodal Imaging / methods
  • Proteins / metabolism*
  • Reproducibility of Results
  • Sensitivity and Specificity
  • Statistics as Topic
  • Subcellular Fractions / metabolism*
  • Tissue Distribution
  • Vitrification

Substances

  • Proteins