亚细胞结构间的动态相互作用在各种生理过程中发挥重要作用并有可能为相关疾病的诊断和治疗提供理论基础和新的突破点。结构光照明超分辨显微成像技术（SIM）为长时程研究活细胞内亚细胞结构间的快速动态相互作用提供了一种非常有效的工具并已成为当前研究热点之一。但因现有SIM有机荧光探针存在易光漂白、背景高、能透膜探针数量有限等问题，极大限制了SIM技术在长时程亚细胞结构动态相互作用研究中的应用并成为关键瓶颈之一。本项目将在国际上首次利用非共价连接的细胞穿膜肽运送本身不透膜的SIM有机荧光探针进入活细胞内，拟得到一系列能在活细胞内特异标记各种亚细胞结构、能进行长时程、多色SIM成像的有机荧光探针，并应用于活细胞内亚细胞结构间的长时程动态相互作用研究。本项目将极大拓展活细胞SIM有机荧光探针的种类和数量，为活细胞超分辨有机荧光探针设计提供一种新策略，同时为进一步揭示和理解亚细胞结构间动态相互作用奠定基础。

Increasing evidence indicates that intracellular organelles are integrated into cellular networks and collaborate on various cellular tasks rather than acting as isolated entities. The dynamic physical interactions between intracellular organelles are considered to play pivotal roles in many important cellular processes and characterization of these long-term nanometer-scale dynamic physical interactions is essential for understanding functions of intracellular organelles. The recent development of structured illumination microscopy (SIM), which offers substantially improved spatiotemporal resolution, provides a powerful tool for characterizing the dynamics of subcellular structures in live cells. However, limitations of the existing fluorescent probes for intracellular organelles, such as photobleaching and a nonspecific background, hinder the characterization of dynamic physical interactions between intracellular organelles in live cells by SIM, leading to an incomplete understanding of functions of intracellular organelles. In this proposal, we will develop a new strategy for delivering cell-impermeable SIM organic fluorescent probes into live cells by using new types of cell-penetrating peptides (CPP) after a simple co-incubation procedure. Our new delivery strategy will not require a covalent bond between CPPs and organic fluorescent probes, which will significantly expanding the number of organic fluorescent probes available for live-cell SIM super-resolution multi-color imaging. We will utilize our new delivery strategy and organic fluorescent probes to label intracellular organelles and obtain long-term dual-color SIM super-resolution images in live cells. We anticipate that these fluorescent probes and the delivery strategy will be helpful tools for SIM super-resolution imaging and substantially benefit characterizations of dynamic physical interactions between intracellular organelles in living cells. Our results will possibly open up new avenues for designing organic fluorescent probes for live-cell long-term SIM super-resolution imaging and provide new tools for understanding the function and dynamic interplay of intracellular organelles in live cells.