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  1. The advancement of super-resolution imaging (SRI) relies on fluorescent proteins with novel photochromic properties. Using light, the reversibly switchable fluorescent proteins (RSFPs) can be converted between bright and dark states for many photocycles and their emergence has inspired the invention of advanced SRI techniques. The general photoswitching mechanism involves the chromophore cis-trans isomerization and proton transfer for negative and positive RSFPs and hydration–dehydration for decoupled RSFPs. However, a detailed understanding of these processes on ultrafast timescales (femtosecond to millisecond) is lacking, which fundamentally hinders the further development of RSFPs. In this review, we summarize the current progress of utilizing variousmore »ultrafast electronic and vibrational spectroscopies, and time-resolved crystallography in investigating the on/off photoswitching pathways of RSFPs. We show that significant insights have been gained for some well-studied proteins, but the real-time “action” details regarding the bidirectional cis-trans isomerization, proton transfer, and intermediate states remain unclear for most systems, and many other relevant proteins have not been studied yet. We expect this review to lay the foundation and inspire more ultrafast studies on existing and future engineered RSFPs. The gained mechanistic insights will accelerate the rational development of RSFPs with enhanced two-way switching rate and efficiency, better photostability, higher brightness, and redder emission colors.« less
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  9. We discovered a novel fluorophore by incorporating a dimethylamino group (–NMe2) into the conformationally locked green fluorescent protein (GFP) scaffold. It exhibited a marked solvent-polarity-dependent fluorogenic behavior and can potentially find broad applications as an environment-polarity sensor in vitro and in vivo. The ultrafast femtosecond transient absorption (fs-TA) spectroscopy in combination with quantum calculations revealed the presence of a twisted intramolecular charge transfer (TICT) state, which is formed by rotation of the –NMe2 group in the electronic excited state. In contrast to the bright fluorescent state (FS), the TICT state is dark and effectively quenches fluorescence upon formation. We employedmore »a newly developed multivariable analysis approach to the FS lifetime in various solvents and showed that the FS → TICT reaction barrier is mainly modulated by H-bonding capability instead of viscosity of the solvent, accounting for the observed polarity dependence. These deep mechanistic insights are further corroborated by the dramatic loss of fluorogenicity for two similar GFP-derived chromophores in which the rotation of the –NMe2 group is inhibited by structural locking.« less