多模态结构光照明超分辨显微成像

Among various super-resolution imaging techniques, structured illumination microscopy (SIM) needs much less raw images and much lower illumination intensity than other techniques, so it is deemed as an ideal tool for super-resolution live cell imaging. Recently, total internal reflection SIM (TIRF-SIM), 3 dimensional SIM (3D-SIM), nonlinear SIM (NL-SIM) and optical section SIM (OS-SIM) have been developed, each of which improved a single aspect of the imaging performance via a specific method. However, the great diversity of life science research demands variety of imaging techniques, so a single of the aforementioned SIM imaging modalities cannot meet the diverse requirements. In this project, we first devise a new SIM imaging modality --- Grazing Incident SIM (GI-SIM), which can fulfill the imaging depth gap between TIRF-SIM and 3D-SIM. Therefore, it can image the intracellular organelles, such as endoplastic reticulum, microtubule, lysosome, etc., which could not be done in TIRF-SIM mode, at high spatiotemporal resolution and over long imaging time course. Second, we figure out a scheme to integrate these SIM imaging modalities into a single microscope, and simultaneously maintain the optimum imaging performance for each modality, also allow one to flexibly select the desired SIM imaging modality. Finally, we plan to utilize this multimodal SIM super-resolution imaging system to study the highly dynamic structures and functions of endoplastic reticulum.

在众多超分辨显微镜技术中，结构光照明超分辨显微镜所需原始图像数少且照明光强低，使其成为实现高时空分辨活细胞成像的理想技术。近年来，结构光技术先后发展出全反射结构光、三维结构光、非线性结构光，以及层析结构光照明等多种衍生技术方法，这些技术分别改进了成像性能的不同方面，且实现细节要求各异。然而，生命科学研究对成像技术的需求非常多样，上述单一模态技术适用面有限，无法满足多样化的需求。本项目首先提出了一种新的结构光成像模态---掠入射结构光成像，其有效填补了全反射结构光和三维结构光之间的成像深度差距，可以对内质网、微管、溶酶体等原先在全反射结构光模式下无法成像的细胞器进行高速超分辨长时程成像。其次，申请人提出了一套优化集成多种结构光成像模态的解决方案，可在同一显微镜系统中实现各模态的最优成像性能，且可在各个模态间灵活切换。最后，申请人将运用这一多模态结构光超分辨成像系统研究内质网的动态结构与功能。

本项目旨在开发新型结构光照明超分辨率显微成像技术，并将多种结构光照明超分辨成像模态整合成一套系统为生命科学研究提供良好通用性的超分辨率活细胞成像平台。在项目执行过程中，我们首先开发了全新的掠入射结构光照明超分辨模态，实现了对活细胞以97纳米分辨率，266幅每秒的成像速度连续成像几千幅超分辨图像，并有效观测了内质网与微管之间的细胞器动态互作。接着，我们将TIRF-SIM、GI-SIM、NL-SIM和3D-SIM等多种结构光照明超分辨成像模态有效集成，从而实现一套超分辨成像装置融合且可以无缝切换多种超分辨成像模态，并且成功开发出多模态结构光照明超分辨显微镜的工程样机。最后，将结构光照明超分辨率显微技术与传统牵引力显微镜相结合，开发出新型生物力显微镜，并利用此显微镜研究了细胞贴壁和细胞免疫反应等生物学过程。本项目开发出一套具有高通用性的超分辨率活细胞成像平台，为生命科学研究提供新的显微成像解决方案。