新型活细胞SIM超分辨有机荧光探针及其在亚细胞结构长时程动态相互作用研究中的应用

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.

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。

利用超分辨显微成像技术对活细胞中的亚细胞结构进行快速、长时程超分辨成像对于进一步理解和揭示亚细胞结构的生理功能具有重要意义。要实现对活细胞亚细胞结构的快速、长时程超分辨成像不仅需要先进的成像系统，还需要具有优秀光学性能的荧光探针来标记各种亚细胞结构。有机小分子染料相对于荧光蛋白在荧光强度、抗漂白能力、可改造性方面都具有很大的优势，但目前大多数性能优秀的荧光染料都无法透过活细胞细胞膜，极大地限制了有机小分子荧光探针在活细胞中的应用范围。目前也有一些技术尝试解决高性能荧光染料无法透膜的问题，但目前还没有取得很好的效果。为了解决上述问题，我们提出了一种利用肽型载体通过非共价结合方式投送非透膜有机荧光探针进入活细胞的标记策略。通过一系列筛选，我们成功找到了一种可以通过简单共孵育高效投送非透膜有机荧光探针进入活细胞的肽型载体PV-1，并发现PV-1主要是通过內吞作用中的大胞饮途径将不透膜荧光探针投送到活细胞。然后利用23种具有不同化学结构的非透膜荧光探针和PV-1共孵育，实现了对多种亚细胞结构的特异性标记。此外，PV-1还可以一次性投送多达三种非透膜有机小分子荧光探针进入活细胞实现多色标记。再次，在多种超分辨成像系统上，我们发现基于PV-1投送的高性能有机荧光探针可以很好的用于活细胞超分辨成像，该方法极大地扩展了活细胞超分辨成像探针的可选范围。利用上述高性能探针结合2D或3D超分辨成像系统，我们捕捉到了内质网与溶酶体、微管与溶酶体、线粒体与内质网等多种亚细胞结构间的精细动态相互作用。.综上所述，我们开发了一种基于肽型载体非共价结合方式投送非透膜有机小分子荧光探针进入活细胞的策略。PV-1结合这些性能优异的荧光探针在多种2D与3D超分辨成像系统上取得了很好的成像结果，极大地扩展了活细胞超分辨成像探针的可选范围，并为肽型载体的设计与应用开辟了新的领域