Standard fluorescent proteins as dual-modality probes for correlative experiments in an integrated light and electron microscope

Elisabeth Brama, Christopher J. Peddie, Martin L. Jones, Marie Charlotte Domart, Xenia Snetkov, Michael Way, Banafshe Larijani, Lucy M. Collinson

Research output: Contribution to journalArticlepeer-review

13 Citations (SciVal)


Integrated light and electron microscopes (ILEMs) will enable a new generation of high-precision correlative imaging experiments. To fully exploit these systems, samples must contain dual-modality probes that highlight the position of macromolecules in the context of cell ultrastructure. We demonstrate that the fluorescent proteins (FPs) GFP (green), YFP (yellow) and mCherry can be used as dual-modality probes for ILEM when preserved using the in-resin fluorescence (IRF) technique, which delivers stable active fluorophores in lightly stained, resin-embedded cells and tissues. However, we found that vacuum pressure in the ILEM affects the photophysics of FPs in IRF sections. Here, we show that reducing the vacuum pressure reduces fluorescence intensity of GFP and YFP, which is a consequence of water extraction from the sample and is reversible on re-creation of partial pressure with water vapour (but not oxygen or nitrogen gas). We also find that, although fluorescence intensity is reduced at a partial pressure of 200 Pa (created using water vapour), the FP intensity is remarkably stable over time in vacuum and resistant to photobleaching during imaging. We are thus able to define imaging strategies for standard FPs acting as dual-modality probes in a single ‘multi-colour’ integrated microscope system.

Original languageEnglish
Pages (from-to)179-188
Number of pages10
JournalJournal of Chemical Biology
Issue number4
Publication statusPublished - 1 Oct 2015

Bibliographical note

Funding Information:
The authors would like to acknowledge funding from Cancer Research UK, and from the MRC, BBSRC and EPSRC under grant award MR/K01580X/1 to LC and Peter O’Toole (York University). We would like to thank Peter O’Toole and Sander den Hoedt (DELMIC B.V.) for useful discussions.

Publisher Copyright:
© 2015, The Author(s).


  • Fluorescent protein
  • GFP
  • In-resin fluorescence
  • Integrated light and electron microscopy
  • Vacuum
  • YFP

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Cell Biology


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