面向活细胞结构和功能的三维单分子成像研究

The ability to visualize cellular structures and their spatial relationship at single-molecule level (nanometer scale) is important for life science research. The diffraction barrier, however, limits the imaging resolution of conventional light microscopy to 200 to 300 nm in the lateral dimensions, leaving many intracellular organelles and molecular structures unresolvable. Recently, the super-resolution microscopy techniques have been used to break this diffraction limit and have achieved resolution of tens of nanometers, which is helpful in better understanding cellular physico-chemical mechanism. However, fast, three-dimensional (3-D) super-resolution imaging at whole-cell level remains a challenge. In addition, the current super-resolution imaging approaches only used to image the structure information of live cells and the corresponding functional information is difficult to be acquired by these approaches. To overcome these limitations, in this study, we propose a new imaging approach and establish a new super-resolution microscopy system for imaging the 3-D structure and function of live cells at single-molecule level, which are achieved by taking advantage of the optical tomographic reconstruction method and the forster (or fluorescence) resonance energy transfer (FRET) technology. To achieve the goal, firstly, based on the strategy of optical tomography, an imaging mathematics model is established, which is used to image the 3-D structure information of live cells at single-molecule level. Secondly, a photo-switchable FRET pair is established and then is applied to the three-cube FRET method, which is used to quantitatively image the function information of live cells at single-molecule level. Finally, by combining the optical reconstruction model with FRET method, a super-resolution fluorescence microscopy imaging system is established. When applied to the live cell experiments, the new imaging system not only can image the 3-D structure information, but also can image the 3-D function information at single-molecule level. This will greatly enhance our understanding of molecular interactions and processes in live cells.

单分子水平（纳米尺度）的活细胞成像对于生命科学的发展具有重要推动作用。传统的荧光显微技术不能用于单分子成像。超分辨荧光显微技术在进行单分子成像时还存在问题，很难在全细胞水平，进行快速、三维成像；此外，现有超分辨技术仅能反映细胞器或分子的空间结构信息，不能描述功能信息。因此，本项目基于光学断层重建与荧光共振能量转移（FRET）策略，研究建立适用于活细胞结构和功能成像的新型三维单分子成像系统及方法。主要研究内容包括：（1）将光学三维重建思想与超分辨成像结合，建立重建数学模型，实现单分子水平的快速、三维结构成像。（2）构建光可开关的FRET供体-受体对，并结合FRET定量算法，实现单分子水平的功能成像。（3）整合三维重建模型与FRET技术，建立新型超分辨成像系统，实现单分子水平的三维结构和功能活细胞成像。项目的完成有助于在纳米尺度上研究活细胞内关键分子的分布及功能，具有重要的理论和实际应用意义。

超高分辨荧光显微成像对于生命科学的发展具有重要推动作用。然而，现有超分辨成像技术难以在全细胞水平，进行快速、三维成像；此外，现有超分辨技术仅能反映亚细胞器或大分子的空间结构信息，难以描述功能信息。针对上述问题，本项目主要工作是基于光学断层重建策略，并将其与FRET技术相结合，实现超快地三维结构/功能超分辨成像。通过四年的研究，我们完成了如下工作。1）建立了单视图超分辨三维重建算法，并结合光传播模型，实现了基于光学重建策略的超快三维超分辨成像；同时，利用结构、功能、时间、组等先验信息，进一步改善了单视图三维重建成像的性能。2）构建了双边光可开关的FRET供体-受体对（rsTagRFP-Dronpa），结合3-cube方法，实现了FRET定量计算；在此基础上，结合电生理，揭示L型钙通道门控新机制。此外，进一步构建了新型探针GCaMP-X，提高了成像性能。3）将上述断层重建方法与定量FRET技术相结合，实现了超快地三维结构/功能成像；进一步，构建了适用于双标同时成像的超高分辨荧光显微成像系统。基于该系统，开展了生物学实验。此外，在项目研究过程中，我们扩宽了研究领域，将该项目研究的核心技术单视图重建方法应用于X射线发光成像领域，在此基础上，项目申请人获批国家自然科学基金面上项目资助；此外，我们亦将项目研究中建立的超分辨定位方法应用于超声领域，实现了超高分辨的超声成像并已取得了阶段性的成果。.本项目已在《eLife》、《Optics Letters》、《IEEE Transactions on Biomedical Engineering》、《Biomedical Optics Express》、《Journal of Biomedical Optics》、《科学通报》等国内外主流学术期刊和会议上共发表文章（含录用）16篇，其中，SCI检索11篇，影响因子累计38；EI检索4篇；此外，参编英文著作1部，ELSEVIER出版社出版；申请国内发明专利2项，其中授权1项。