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.

Photoconvertible fluorescent proteins (PCFPs) are widely used as markers for the visualization of intracellular processes and for sub-diffraction single-molecule localization microscopy. Although wild type of a new photoconvertible fluorescent protein SAASoti tends to aggregate, we succeeded, via rational mutagenesis, to obtain variants that formed either tetramers or monomers. We compare two approaches: one is based on the structural similarity between SAASoti and Kaede, which helped us to identify a single point mutation (V127T) at the protein’s hydrophobic interface that leads to monomerization. The other is based on a chemical modification of amino groups of SAASoti with succinic anhydride, which converts the protein aggregates into monomers. Mass-spectrometric analysis helped us to identify that the modification of a single ε-amino group of lysine K145 in the strongly charged interface AB was sufficient to convert the protein into its tetrameric form. Furthermore, site-directed mutagenesis was used to generate mutants that proved to be either monomeric or tetrameric, both capable of rapid green-to-red photoconversion. This allows SAASoti to be used as a photoconvertible fluorescent marker for in vivo cell studies.

SAASoti is a unique fluorescent protein (FP) that combines properties of green-to-red photoconversion and reversible photoswitching (in its green state), without any amino acid substitutions in the wild type gene. In the present work, we investigated its ability to photoswitch between fluorescent red (‘on’) and dark (‘off’) states. Surprisingly, generated by 400 nm exposure, the red form of SAASoti (R1) does not exhibit any reversible photoswitching behavior under 550 nm illumination, while a combination of prior 470 nm and subsequent 400 nm irradiation led to the appearance of another—R2—form that can be partially photoswitched (550 nm) to the dark state, with a very fast recovery time. The phenomenon might be explained by chemical modification in the chromophore microenvironment during prior 470 nm exposure, and the resulting R2 SAASoti differs chemically from the R1 form. The suggestion is supported by the mass spectrometry analysis of the tryptic peptides before and after 470 nm light exposure, that revealed Met164 oxidation, as proceeds in another dual phototransformable FP, IrisFP.