Enhancing the photostability of red fluorescent proteins through FRET with Si-rhodamine for dynamic super-resolution fluorescence imaging

Abstract

Red fluorescent proteins (RFPs) are extensively utilized in biological imaging. However, their susceptibility to photobleaching restricts their effectiveness in super-resolution imaging where high photostability is crucial. In this study, we substantially improved the photostability of RFPs by incorporating a hybrid Förster resonance energy transfer (FRET) pair, utilizing RFPs as the energy donor and a photostable fluorophore, tetramethyl-Si-rhodamine (TMSiR), as the acceptor. TMSiR was selectively introduced through fusion with the HaloTag protein linked to the RFPs. We constructed a series of mApple/mCherry–TMSiR pairs with varying FRET efficiencies. Our findings reveal that higher FRET efficiency in the mApple/mCherry–TMSiR complexes correlates with enhanced photostability of RFPs. FRET competes with the singlet-to-triplet state transition of RFPs, while the spatial barrier introduced by the HaloTag protein prevents interaction between sensitized reactive oxygen species near Si-rhodamine and red fluorescent protein, enhancing the photostability of red fluorescent protein. The nearly 6-fold enhancement in mCherry's photostability allows for extended durations of dynamic structured illumination microscopy (SIM) imaging in living cells, facilitating the capture of finer details in organelle interactions. Leveraging the photostable mCherry protein, we tracked various mitochondrial fission processes and their interactions with lysosomes and the endoplasmic reticulum (ER). Interestingly, we observed the involvement of ER in all mitochondrial fission events, whereas lysosomes participated in only 66% of them.