Single-molecule evaluation of fluorescent protein photoactivation efficiency using an in vivo nanotemplate

Nat Methods. 2014 Feb;11(2):156-62. doi: 10.1038/nmeth.2784. Epub 2014 Jan 5.


Photoswitchable fluorescent probes are central to localization-based super-resolution microscopy. Among these probes, fluorescent proteins are appealing because they are genetically encoded. Moreover, the ability to achieve a 1:1 labeling ratio between the fluorescent protein and the protein of interest makes these probes attractive for quantitative single-molecule counting. The percentage of fluorescent protein that is photoactivated into a fluorescently detectable form (i.e., the photoactivation efficiency) plays a crucial part in properly interpreting the quantitative information. It is important to characterize the photoactivation efficiency at the single-molecule level under the conditions used in super-resolution imaging. Here, we used the human glycine receptor expressed in Xenopus oocytes and stepwise photobleaching or single-molecule counting photoactivated localization microcopy (PALM) to determine the photoactivation efficiency of fluorescent proteins mEos2, mEos3.1, mEos3.2, Dendra2, mClavGR2, mMaple, PA-GFP and PA-mCherry. This analysis provides important information that must be considered when using these fluorescent proteins in quantitative super-resolution microscopy.

Publication types

  • Evaluation Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cells, Cultured
  • Female
  • Fluorescent Dyes / chemistry*
  • Green Fluorescent Proteins / analysis*
  • Humans
  • Light
  • Luminescent Proteins / analysis*
  • Microscopy, Fluorescence / methods*
  • Nanotechnology / methods*
  • Oocytes / cytology
  • Oocytes / metabolism*
  • Photobleaching / radiation effects
  • Receptors, Glycine / metabolism*
  • Xenopus laevis / growth & development
  • Xenopus laevis / metabolism


  • Fluorescent Dyes
  • Luminescent Proteins
  • Receptors, Glycine
  • Green Fluorescent Proteins