Proteins of the green fluorescent protein (GFP) family are well known owing to their unique biochemistry and extensive use as in vivo markers. We discovered that GFPs of diverse origins can act as light-induced electron donors in photochemical reactions with various electron acceptors, including biologically relevant ones. Moreover, via green-to-red GFP photoconversion, this process can be observed in living cells without additional treatment.

Photoinduced electron transfer in fluorescent proteins from the GFP-family can be regarded either as an asset facilitating new applications or as a nuisance lead- ing to the loss of optical output. Photooxidation commonly results in green-to-red photoconversion called oxidative redding. We discovered that yellow FPs do not undergo redding, however, the redding is restored upon halide binding. Calculations of the energetics of one-electron oxidation and possible ET pathways suggested that excited-state ET proceeds through a hopping mechanism via Tyr145. In YFPs, the π-stacking of the chromophore with Tyr203 reduces its electron donating ability, which can be restored by halide binding. Point mutations confirmed that Tyr145 is a key residue controlling ET. Substitution of Tyr145 by less efficient electron acceptors resulted in highly photostable mutants. This strategy — calculation and disruption of ET pathways — may represent a new approach towards enhancing photostability of FPs.

Photostability is one of the most important characteristic of a dye for fluorescence microscopy. Recently we demonstrated that vitamins present in imaging media dramatically accelerate photobleaching of Enhanced Green Fluorescent Protein (EGFP) and many other green fluorescent and photoactivatable proteins. Here we tested all vitamins of commonly used media (such as Dulbecco's Modified Eagle Medium, DMEM) one-by-one and found that only two vitamins, riboflavin and pyridoxal, decrease photostability of EGFP. Thus, DMEM without riboflavin and pyridoxal can be used as an imaging medium, which ensures high photostability of GFPs at the expense of minimal biochemical disturbance. Then, we tested some antioxidants and found that a plant flavonoid rutin greatly enhances photostability of EGFP during live cell microscopy. In complete DMEM, rutin increased EGFP photostability up to the level of vitamin-depleted DMEM. Moreover, being added to vitamin-depleted DMEM, rutin was able to further suppress EGFP photobleaching. Potentially, new medium formulations can be widely used for fluorescence microscopy of GFP-expressing cells and model multicellular organisms in a variety of imaging applications, where photostability represents a challenge.

Fluorescence lifetime imaging microscopy (FLIM) measures fluorescence decay rate at every pixel of an image. FLIM can separate probes of the same color but different fluorescence lifetimes (FL), thus it is a promising approach for multiparameter imaging. However, available GFP-like fluorescent proteins (FP) possess a narrow range of FLs (commonly, 2.3–3.5 ns) which limits their applicability for multiparameter FLIM. Here we report a new FP probe showing both subnanosecond fluorescence lifetime and exceptional fluorescence brightness (80% of EGFP). To design this probe we applied semi-rational amino acid substitutions selection. Critical positions (Thr65, Tyr145, Phe165) were altered based on previously reported effect on FL or excited state electron transfer. The resulting EGFP triple mutant, BrUSLEE (Bright Ultimately Short Lifetime Enhanced Emitter), allows for both reliable detection of the probe and recording FL signal clearly distinguishable from that of the spectrally similar commonly used GFPs. We demonstrated high performance of this probe in multiparameter FLIM experiment. We suggest that amino acid substitutions described here lead to a significant shift in radiative and non-radiative excited state processes equilibrium.