The contribution of reactive oxygen species to the photobleaching of organic fluorophores Academic Article uri icon


MeSH Major

  • Fluorescent Dyes
  • Organic Chemicals
  • Photobleaching
  • Reactive Oxygen Species


  • Photoexcitation of fluorophores commonly used for biological imaging applications generates reactive oxygen species (ROS) which can cause bleaching of the fluorophore and damage to the biological system under investigation. In this study, we show that singlet oxygen contributes relatively little to Cy5 and ATTO 647N photobleaching at low concentrations in aqueous solution. We also show that Cy5 generates significantly less ROS when covalently linked to the protective agents, cyclooctatetraene (COT), nitrobenzyl alcohol (NBA) or Trolox. Such fluorophores exhibit enhanced photostability both in bulk solutions and in single-molecule fluorescence measurements. While the fluorophores ATTO 647N and ATTO 655 showed greater photostability than Cy5 and the protective-agent-linked Cy5 derivatives investigated here, both of ATTO 647N and ATTO 655 generated singlet oxygen and hydroxyl radicals at relatively rapid rates, suggesting that they may be substantially more phototoxic than Cy5 and its derivatives.

publication date

  • January 2014



  • Academic Article



  • eng

PubMed Central ID

  • PMC3959289

Digital Object Identifier (DOI)

  • 10.1111/php.12204

PubMed ID

  • 24188468

Additional Document Info

start page

  • 448

end page

  • 54


  • 90


  • 2