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Three-dimensional Imaging of Bacterial Cells for Accurate Cellular Representations and Precise Protein Localization
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Three-dimensional super-resolution protein localization correlated with vitrified cellular context.

Bei Liu1,2, Yanhong Xue1, Wei Zhao1,3

  • 1National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101, China.

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|October 15, 2015
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Summary

Cryogenic super-resolution correlative light and electron microscopy (csCLEM) achieves high-resolution imaging of proteins within native cellular structures. This technique offers improved localization precision and resolution, even in thin sections of vitrified specimens.

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Area of Science:

  • Cell Biology
  • Microscopy
  • Biophysics

Background:

  • Precisely determining the spatial relationship between proteins and cellular structures is crucial for understanding cellular function.
  • Existing super-resolution microscopy techniques face challenges in preserving native cellular states and achieving optimal photon budgets for localization.
  • Correlative light and electron microscopy (CLEM) offers a pathway to bridge light and electron microscopy, but cryogenic methods are needed for optimal preservation.

Purpose of the Study:

  • To demonstrate the utility of cryogenic super-resolution correlative light and electron microscopy (csCLEM) for precise spatial protein localization within native cellular environments.
  • To evaluate the performance of photoswitchable fluorescent proteins under cryogenic conditions for enhanced imaging.
  • To achieve nanometer-scale 3D resolution of protein-membrane interactions in mammalian cells.

Main Methods:

  • Vitrification of specimens using high-pressure freezing followed by cryo-sectioning to preserve native cellular structures and fluorescence.
  • Screening of photoswitchable fluorescent proteins (FPs) and optimization of cryogenic imaging conditions to maximize photon emission and localization precision.
  • Application of csCLEM to mammalian cells, including cryo-sectioning and 3D reconstruction for correlative analysis.

Main Results:

  • Several fluorescent proteins exhibited enhanced photoswitching and photon emission under cryogenic conditions, leading to high localization precision comparable to ambient super-resolution imaging.
  • A 2-3 fold improvement in resolution was achieved compared to previous reports, attributed to optimized imaging and fluorescent protein selection.
  • Successful application of csCLEM to mammalian cells, demonstrating the correlation of a mitochondrial protein with the mitochondrial outer membrane at nanometer resolution in 3D.

Conclusions:

  • Cryogenic super-resolution correlative light and electron microscopy (csCLEM) is a powerful technique for precisely mapping protein localization within native cellular structures.
  • The optimized cryogenic imaging conditions and fluorescent protein selection significantly enhance resolution and localization accuracy.
  • csCLEM provides unprecedented 3D nanoscale insights into molecular organization within mammalian cells, exemplified by mitochondrial protein localization.