Higher resolution in localization microscopy by slower switching of a photochromic protein

Photochem Photobiol Sci. 2010 Feb;9(2):239-48. doi: 10.1039/b9pp00124g. Epub 2010 Jan 18.

Abstract

Photoswitchable fluorophores play an essential role in super-resolution fluorescence microscopy, including techniques such as photoactivated localization microscopy (PALM). A determining factor in the precision of the images generated by PALM measurements is the photon numbers that can be detected from the fluorophores. Dronpa is a reversibly photoswitchable fluorescent protein that has been successfully used in PALM experiments. The number of photons per switching cycle that can be acquired for Dronpa depends on its off-switching rate, limiting the number of photons that can be recorded. In this study we report our discovery that the tetrameric ancestor of Dronpa, 22G, shows slower switching, and develop a mutant that displays switching kinetics between those of Dronpa and 22G. We show that the kinetics of the photoswitching are strongly related to self-association of the protein, supporting our view of dynamic flexibility as determining in the photoswitching. Similarly we find that higher-resolution PALM images can be acquired with slower-switching proteins due to their higher number of emitted photons per switching cycle.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Actins / chemistry
  • Actins / genetics
  • Kinetics
  • Luminescent Proteins / chemistry*
  • Luminescent Proteins / genetics
  • Microscopy, Fluorescence
  • Photons*
  • Protein Structure, Quaternary
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / genetics

Substances

  • Actins
  • Luminescent Proteins
  • Recombinant Fusion Proteins