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Abstract

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.