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Absolute quantum yield measurements of fluorescent proteins using a plasmonic nanocavity.

Daja RuhlandtMartin AndresenNickels JensenIngo GregorStefan JakobsJörg EnderleinAlexey I Chizhik
Published in: Communications biology (2020)
One of the key photophysical properties of fluorescent proteins that is most difficult to measure is the quantum yield. It describes how efficiently a fluorophore converts absorbed light into fluorescence. Its measurement using conventional methods become particularly problematic when it is unknown how many of the proposedly fluorescent molecules of a sample are indeed fluorescent (for example due to incomplete maturation, or the presence of photophysical dark states). Here, we use a plasmonic nanocavity-based method to measure absolute quantum yield values of commonly used fluorescent proteins. The method is calibration-free, does not require knowledge about maturation or potential dark states, and works on minute amounts of sample. The insensitivity of the nanocavity-based method to the presence of non-luminescent species allowed us to measure precisely the quantum yield of photo-switchable proteins in their on-state and to analyze the origin of the residual fluorescence of protein ensembles switched to the dark state.
Keyphrases
  • energy transfer
  • quantum dots
  • living cells
  • label free
  • single molecule
  • molecular dynamics
  • sensitive detection
  • risk assessment
  • monte carlo
  • small molecule