Absolute quantum yield measurements of fluorescent proteins using a plasmonic nanocavity

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. Ruhlandt et al. report a plasmonic nanocavity-based method to measure absolute values of quantum yield 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. Authors are further able to determine lifetime and quantum yield of several fluorescent proteins, which would be a good resource for researchers working with them.