Methylene blue- and thiol-based oxygen depletion for super-resolution imaging

Philip Schäfer, Sebastian van de Linde, Julian Lehmann, Markus Sauer, Sören Doose

Research output: Contribution to journalArticlepeer-review

44 Citations (Scopus)


Anaerobic conditions are often required in solution-based bionanotechnological applications. Efficient oxygen depletion is essential for increasing photostability, optimizing fluorescence signals, and adjusting kinetics of fluorescence intermittency in single-molecule fluorescence spectroscopy/microscopy, particularly for super-resolution imaging techniques. We characterized methylene blue (MB)- and thiol-based redox reactions with the aim of designing an oxygen scavenger system as an alternative to the established enzyme-based oxygen scavenging systems or purging procedures. Redox reactions of the chromophore methylene blue in aqueous solution, commonly visualized in the blue bottle experiment, deplete molecular oxygen as long as a sacrificial reduction component is present in excess concentrations. We demonstrate that methylene blue in combination with reducing compounds such as β-mercaptoethylamine (MEA) can serve as fast and efficient oxygen scavenger. Efficient oxygen scavenging in aqueous solution is also possible with mere β-mercaptoethylamine at mM concentrations. We present kinetic parameters of the relevant reactions, pH-stability of the MB/MEA-oxygen scavenging system, and its application in single-molecule based super-resolution imaging.

Original languageEnglish
Pages (from-to)3393-400
Number of pages8
JournalAnalytical Chemistry
Issue number6
Publication statusPublished - 19 Mar 2013


  • HeLa cells
  • spectrometry
  • methylene blue
  • microscopy, fluorescence/methods
  • microtubules/ultrastructure
  • fluorescence


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