DNA纳米结构在荧光共振能量转移显微镜突破光学衍射极限成像的应用研究

For the reason that the study of life science considerably relies on the observation and exploration of microcosmic world of cells, there is an urgent need for the microscopes with imaging resolution in the range of a few tens of nanometers. To meet the requirements, the work in this study is focused on developing a new method to improve the resolution of far-field microscopes, called DNA nanostructures-based saturated fluorescence resonance energy transfer microscopy (SFM). This technique relies on the intrinsic size of several nanometers of the DNA nanostructures. We design and prepare ultrahigh-FRET-efficiency DNA probes through conjugating a molecular-pair of the donor and acceptor to the DNA nanostructure. By applying these probes and taking advantage of nonlinear effect of these probes, the spot of the fluorescence of the donor is reduced through modifying the intensity of the excitation laser and the imaging with diffraction barrier broken is achieved in SFM. This system is hoped to provide a resolution enhancement of 2-3 compared with that of the confocal microscope. Moreover, further researches of dual-color fluorescence nanoscopy by SFM as well as SFM imaging on living cells will be carried out in this work. Our study is hopeful to advance the growth of the science of superresolution imaging in our country and also is helpful to provide a new sight for the use of DNA nanostructures.

生命科学研究在很大程度上依赖于对细胞这一微观世界的观察与探索，因此现代生物学的发展对发展超高分辨率的显微系统提出了迫切的需求。本项目正是面向这一学科发展的重要需求，希望开发出具有自主知识产权的新型超分辨光学显微系统，即基于DNA纳米结构的饱和荧光共振能量转移显微镜。本项目拟借助于DNA结构纳米尺度的优势，将FRET荧光分子对与DNA链结合，设计和制备多种高效率FRET过程的DNA荧光探针。随后利用这些探针对激发光产生的非线性响应，控制激发光强度来压缩供体分子的荧光发射点，将现有的激光扫描共聚焦显微镜(LSCM)平台的空间分辨率提高2-3倍，将其发展成为一套克服光学衍射极限成像的饱和荧光共振能量显微镜（SFM）系统，并在此基础上，开展其双色和活细胞成像研究。本项目的实施有望推动我国超分辨显微技术研究的发展，也能够为DNA纳米结构开拓新的应用领域。

本项目关注于发展突破光学衍射极限的远场超分辨成像技术，实现几十纳米的空间分辨率以用于生命科学的超高分辨率成像，以促进生命科学的深入发展。主要研究内容为：（1）超分辨成像技术能够突破光学成像中的分辨率极限的全部奥秘就在于巧妙地借助各种非线性效应。本项目设计和使用DNA纳米荧光探针，利用荧光探针对激发光产生的非线性响应，发展多种超分辨技术。（a）发展了饱和荧光共振能量转移显微镜的相关理论研究。实验上，基于DNA纳米结构，设计合成了新型的高转移效率的荧光共振能量转移探针，开展其应用在受激发射损耗显微镜系统中的超分辨成像研究，进一步提高受激发射损耗显微镜的空间分辨率约1.4倍。（b）提出并实验验证了基于光致荧光漂白的非线性效应的超分辨显微镜，在实验室普遍使用的激光共聚焦扫描显微镜上实现了超分辨成像。设计和构建双色标记的DNA纳米结构探针，实现荧光光致漂白显微镜的两维超分辨成像。实验表明，该技术实现了空间分辨率约80 nm，将普通激光共聚焦扫描显微镜的分辨率提高2-3倍。（2）建立连续光和脉冲光的受激发射损耗显微镜超分辨成像系统，实现了双色超分辨成像功能。利用多种纳米尺寸的样品对系统的分辨率进行了测量，表明空间分辨率约60-70 nm，将原有的普通激光共聚焦成像分辨率提高了3-4倍。（3）将超分辨显微镜用于细胞生物学研究，实现了端粒、DNA四面体等多种纳米结构在亚细胞水平的高精度观测。