Zhou Xuelian, Miao Lu, Zhou Wei, Chen Yonghui, Ruan Yiyan, Wang Xiang, Wang Guangying, Bao Pengjun, Qiao Qinglong, Xu Zhaochao
Dalian Institute of Chemical Physics, Chinese Academy of Sciences 457 Zhongshan Road Dalian 116023 China
School of Chemistry, Dalian University of Technology 2 Linggong Road Dalian 116024 China.
Chem Sci. 2025 May 5. doi: 10.1039/d5sc02442k.
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.
红色荧光蛋白（RFPs）广泛应用于生物成像。然而，它们易受光漂白的特性限制了其在超分辨率成像中的有效性，而超分辨率成像中高光稳定性至关重要。在本研究中，我们通过引入一种混合Förster共振能量转移（FRET）对，将RFPs作为能量供体，光稳定荧光团四甲基 - Si - 罗丹明（TMSiR）作为受体，大幅提高了RFPs的光稳定性。通过与连接到RFPs的HaloTag蛋白融合，选择性地引入了TMSiR。我们构建了一系列具有不同FRET效率的mApple/mCherry - TMSiR对。我们的研究结果表明，mApple/mCherry - TMSiR复合物中较高的FRET效率与RFPs光稳定性的增强相关。FRET与RFPs的单重态到三重态跃迁竞争，而HaloTag蛋白引入的空间位阻阻止了硅罗丹明附近敏化的活性氧物种与红色荧光蛋白之间的相互作用，从而增强了红色荧光蛋白的光稳定性。mCherry光稳定性提高了近6倍，使得在活细胞中进行动态结构光照显微镜（SIM）成像的时间得以延长，有助于捕捉细胞器相互作用中更精细的细节。利用光稳定的mCherry蛋白，我们追踪了各种线粒体裂变过程及其与溶酶体和内质网（ER）的相互作用。有趣的是，我们观察到内质网参与了所有线粒体裂变事件，而溶酶体仅参与了其中的66%。
