Mechanistic investigation of mEos4b reveals a strategy to reduce track interruptions in sptPALM

Nat Methods. 2019 Aug;16(8):707-710. doi: 10.1038/s41592-019-0462-3. Epub 2019 Jul 8.


Green-to-red photoconvertible fluorescent proteins repeatedly enter dark states, causing interrupted tracks in single-particle-tracking localization microscopy (sptPALM). We identified a long-lived dark state in photoconverted mEos4b that results from isomerization of the chromophore and efficiently absorbs cyan light. Addition of weak 488-nm light swiftly reverts this dark state to the fluorescent state. This strategy largely eliminates slow blinking and enables the recording of longer tracks in sptPALM with minimum effort.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • B7-2 Antigen / analysis*
  • B7-2 Antigen / genetics
  • COS Cells
  • Cell Tracking / methods*
  • Chlorocebus aethiops
  • Crystallography, X-Ray
  • HeLa Cells
  • Humans
  • Luminescent Proteins / analysis*
  • Luminescent Proteins / chemistry
  • Luminescent Proteins / genetics
  • Microscopy, Fluorescence / methods*
  • Mutation
  • Photochemical Processes
  • Protein Conformation


  • B7-2 Antigen
  • Luminescent Proteins