用于活细胞超分辨成像的新型透膜靶向光激活荧光化学探针研究

The recent development of localization-based super-resolution microscopy has allowed the diffraction-limited resolution to be surpassed and biological structures to be imaged with resolution as high as about 20 nm. These super-resolution imaging approaches are providing new insights into cell biology, micro-biology,and neurobiology. However, to date, no photo-activatable fluorescent probes have been reported to be able to label intracellular endogenous proteins in living cells. In this proposal, we will develop a series of novel organic photo-activatable fluorescent probes, which not only are cell-permeable but also can specifically bind with intracellular endogenous proteins directly, for live-cell super-resolution imaging. These probes will have three components: (a) a targeting group that can selectively bind with the protein of interest, (b) a photo-activatable fluorophore for visualizing the bound proteins, and (c) an arginine-rich cell-penetrating peptide (CPP) to engender the cellular permeability of the probe. We will explore the behaviors and photochemical properties of these probes in living cells and utilize them to label targeted proteins to obtain super-resolution images of ultrastructures in living cells and tissues. To the best of our knowledge, the fluorescent probes presented in this proposal will be the first organic photo-activatable fluorescent probes that are capable of binding with intracellular endogenous proteins directly in living cells for single-molecule super-resolution imaging. We anticipate that these fluorescent probes will be helpful tools for super-resolution imaging and substantially benefit ultrastructural characterizations in living cells. Our resutls will possibly open up new avenues for designing organic fluorescent probes for live-cell super-resolution imaging.

定位超分辨显微成像技术能在细胞内进行纳米尺度的光学检测，其应用势必会极大推动生命科学的发展，并已成为当前研究热点之一。定位超分辨显微成像技术需要特殊的光激活荧光探针，而现有的光激活荧光探针都难以在活细胞内直接标记内源蛋白，极大限制了活细胞内的内源蛋白超分辨成像研究。本项目将在国际上首次将细胞穿膜肽引入光激活荧光探针的设计中，拟研究一系列新型透膜靶向光激活荧光化学探针，实现在活细胞内直接特异性标记内源蛋白，并应用于活细胞及组织中亚细胞结构的多色超分辨成像。本项目中的透膜靶向光激活探针将是国际上首类能实现对活细胞内的内源蛋白直接进行标记的光激活荧光探针，在活细胞超分辨成像领域有着广阔的应用前景。同时，本项目利用细胞穿膜肽的运载能力，携带不能透膜的荧光染料及特异性识别基团进入活细胞，突破现有光激活荧光化学探针中荧光染料及识别基团本身需要透膜的限制，将为活细胞超分辨成像化学探针设计提供一种新策略。

单分子定位超分辨显微成像技术（PALM/STORM）能在细胞内进行纳米尺度的光学检测，其应用势必会极大推动生命科学的发展，并已成为当前研究热点之一。单分子定位超分辨成像技术的应用需要特殊的能够用光进行“开（发光）”和“关（不发光）”状态转换的光激活荧光探针。并且光激活荧光探针的光学性质是决定超分辨成像的时间及空间分辨率的关键因素之一。因此，单分子定位超分辨成像技术的发展和应用极大地依赖于光激活荧光探针的发展。现有的光激活荧光探针都难以在活细胞内直接标记内源蛋白，极大限制了活细胞内的内源蛋白的超分辨成像研究。本项目在国际上首次将细胞穿膜肽引入光激活有机荧光探针的构建中，利用共价连接的细胞穿膜肽运送能力，针对活细胞中微丝和溶酶体，设计、合成了一系列包含有细胞穿膜肽、可用于活细胞超分辨成像的透膜光激活有机荧光探针，建立了简便、高效的合成路线，为活细胞超分辨成像有机荧光探针的构建提供一种具有普适性的新策略，将极大拓展可用于活细胞超分辨成像的光激活有机荧光探针的种类和数量。利用以上探针，在活细胞内分别实现了对微丝和溶酶体的PALM/STORM超分辨成像，并在活细胞内首次记录了微丝重新排列的动态过程，获得了目前活细胞单分子定位成像中微丝最高的时空分辨率。