Seeing the long tail: A novel green fluorescent protein, SiriusGFP, for ultra long timelapse imaging

BACKGROUND: Fluorescent proteins (FPs) have widespread uses in cell biology. However, the practical applications of FPs are significantly limited due to their rapid photobleaching and misfolding when fused to target proteins. NEW METHOD: Using a combination of novel and known mutations to eGFP, we developed a well folded and very photostable variant, SiriusGFP. RESULTS: The fluorescence spectrum indicated that the excitation and emission peaks of SiriusGFP were red-shifted by 16 and 8 nm, respectively. Co-operative effects of two key mutations, S147R and S205 V, contribute to its photostability. SiriusGFP tagged to the mitochondrial outer membrane protein Omp25 showed sustained fluorescence during continuous 3D-scanning confocal imaging (4D confocal) compared to eGFP-tagged Omp25. Furthermore, with super-resolution structured illumination microscopy (SIM) we demonstrate marked improvements in image quality and resolution (130 nm in XY axis, and 310 nm in Z axis), as well as, decreased artifacts due to photobleaching. COMPARISON WITH EXISTING METHOD(S): Compared to eGFP. SiriusGFP shows a 2-fold increase in photostability in vitro, and folds well when fused to the N- and C- termini of cytoplasmic and membrane proteins. While its quantum yield is ˜3 fold lower than eGFP, its decreased brightness was more than compensated by its increasedphotostability in different experimental paradigms allowing practical experimentation without dynamic adjustment of light intensity or fluorescence sampling times. CONCLUSIONS: We have developed a variant of eGFP, SiriusGFP, that shows over a two fold increase in photostability with utility in methods requiring sustained or high intensity excitation as in 4D confocal or SIM imaging.