A Comparison of the Fluorescence Dynamics of Single Molecules of a Green Fluorescent Protein: One‐ versus Two‐Photon Excitation

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Abstract
We report on the dynamics of fluorescence from individual molecules of a mutant of the wild‐type green fluorescent protein (GFP) from Aequorea victoria, super folder GFP (SFGFP). SFGFP is a novel and robust variant designed for in vivo high‐throughput screening of protein expression levels. It shows increased thermal stability and is able to retain its fluorescence when fused to poorly folding proteins. We use a recently developed single‐molecule technique which combines fluorescence‐fluctuation spectroscopy and time‐correlated single photon counting in order to characterize the photophysical properties of SFGFP under one‐ (OPE) and two‐ (TPE) photon excitation conditions. We use Rhodamine 110 as a model chromophore to validate the methodology and to explain the single‐molecule results of SFGFP. Under OPE, single SFGFP molecules undergo fluorescence flickering on the time scale of μs and tens of μs due to triplet formation and ground‐state protonation–deprotonation, respectively, as demonstrated by excitation intensity‐ and pH‐dependent experiments. OPE single‐molecule fluorescence lifetimes indicate heterogeneity in the population of SFGFP, indicating the presence of the deprotonated I and B forms of the SFGFP chromophore. TPE of single SFGFP molecules results in the photoconversion of the chromophore. TPE of single SFGFP molecules show fluorescence flickering on the time scale of μs due to triplet formation. A flicker connected with protonation–deprotonation of the SFGFP chromophore is detected only at low pH. Our results show that SFGFP is a promising fusion reporter for intracellular applications using OPE and TPE microscopy.