Time and frequency-domain measurement of ground-state recovery times in red fluorescent proteins

The field of bioimaging and biosensors has been revolutionized by the discovery of fluorescent proteins (FPs) and their use in live cells. FPs are characterized with rich photodynamics due to the presence of nonfluorescent or dark states which are responsible for fluorescence intermittency or "blinking", which has been exploited in several localization-based super-resolution techniques that surpass the diffraction-limited resolution of conventional microscopy. Molecules that convert to these dark states recover to the ground states either spontaneously or upon absorption of another photon, depending on the particular FP and the structural transition that is involved. In this work, we demonstrate time- and frequency-domain methods for the measurement of the ground-state recovery (GSR) times of FPs both in live cells and in solutions. In the time-domain method, we excited the sample with millisecond pulses at varying dark times to obtain percent-recovery. In the frequency-domain method, dark-state hysteresis was employed to obtain the positive phase shift or "phase advance". We extracted the GSR time constants from our measurements using calculations and simulations based on a three-state model system. The GSR time constants of the red FPs studied in these experiments fall in the range from μs to msec time-scales. We find that the time- and frequency-domain techniques are complementary to each other. While accurate GSR times can be extracted from the time-domain technique, frequency-domain measurements are primarily sensitive to the rates of dark-state conversion (DSC) processes. A correlation between GSR times, DSC, and photobleaching rates for the red FPs mCherry, TagRFP-T, and Kriek were observed. These time- and frequency-domain methods can be used in high-throughput screening and sorting of FPs clones based on GSR time constant and photostability and will therefore be valuable for the development of new photoswitchable or photoactivatable FPs.