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Fixation-resistant photoactivatable fluorescent proteins for CLEM.

Maria G Paez-Segala1, Mei G Sun1, Gleb Shtengel1

  • 1Howard Hughes Medical Institute, Janelia Farm Research Campus, Ashburn, Virginia, USA.

Nature Methods
|January 13, 2015
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Summary

Engineered fluorescent proteins maintain fluorescence after heavy fixation. This breakthrough enables correlative super-resolution fluorescence and electron microscopy for detailed biological imaging.

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Area of Science:

  • Biochemistry
  • Microscopy
  • Molecular Biology

Background:

  • Fluorescent proteins are crucial for live and fixed sample imaging.
  • Heavy fixation methods, like osmium tetroxide (OsO4), destroy fluorescence.
  • This loss of fluorescence limits correlative light and electron microscopy (CLEM) applications.

Purpose of the Study:

  • To develop fluorescent protein variants that withstand heavy fixation.
  • To enable super-resolution fluorescence imaging in fixed samples for CLEM.
  • To combine high-resolution fluorescence microscopy with electron microscopy.

Main Methods:

  • Engineering variants of the Eos fluorescent protein.
  • Testing fluorescence and photoconversion in heavily fixed samples (0.5-1% OsO4).
  • Embedding fixed samples in plastic resin for microscopy.
  • Performing correlative super-resolution fluorescence and electron microscopy.

Main Results:

  • Engineered Eos variants retained fluorescence and photoconversion capabilities after heavy fixation.
  • Successful integration of fixed samples into plastic resin.
  • Demonstrated correlative super-resolution fluorescence and electron microscopy on these samples.

Conclusions:

  • Novel fluorescent protein variants overcome fixation-induced fluorescence loss.
  • This enables advanced correlative imaging techniques.
  • Opens new avenues for high-resolution structural and functional analysis in cell biology.