Evaluation of fluorophores for optimal performance in localization-based super-resolution imaging

Nat Methods. 2011 Nov 6;8(12):1027-36. doi: 10.1038/nmeth.1768.


One approach to super-resolution fluorescence imaging uses sequential activation and localization of individual fluorophores to achieve high spatial resolution. Essential to this technique is the choice of fluorescent probes; the properties of the probes, including photons per switching event, on-off duty cycle, photostability and number of switching cycles, largely dictate the quality of super-resolution images. Although many probes have been reported, a systematic characterization of the properties of these probes and their impact on super-resolution image quality has been described in only a few cases. Here we quantitatively characterized the switching properties of 26 organic dyes and directly related these properties to the quality of super-resolution images. This analysis provides guidelines for characterization of super-resolution probes and a resource for selecting probes based on performance. Our evaluation identified several photoswitchable dyes with good to excellent performance in four independent spectral ranges, with which we demonstrated low-cross-talk, four-color super-resolution imaging.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Buffers
  • Color
  • Fluorescence
  • Fluorescent Dyes / analysis*
  • Light
  • Luminescent Measurements*
  • Microscopy, Fluorescence
  • Microtubules / metabolism
  • Molecular Imaging / methods*
  • Molecular Probes / analysis
  • Molecular Probes / chemistry
  • Particle Size
  • Photons


  • Buffers
  • Fluorescent Dyes
  • Molecular Probes