三维单分子超分辨显微镜能够获取活细胞内生物分子的纳米尺度结构和动态信息，已成为生命科学研究的重要工具。但对活体厚样品的成像，其在成像深度、时空分辨率、多分子同时示踪等方面存在诸多挑战。为此，本项目重点研究基于数字扫描光片照明的活细胞三维单分子超分辨成像方法：发展基于高速数字微镜的波前动态调制方法，产生数字扫描超薄晶格光片，对荧光分子进行轴向选择性激发，抑制非焦面荧光背景噪声，降低光毒性、提高成像效率和分辨率；发展基于双螺旋点扩散函数的多焦面显微技术，提高单分子定位的轴向探测深度和定位精度；基于双螺旋点扩散函数的轴向定位能力，发展基于多重测量矢量模型的稀疏贝叶斯学习三维压缩感知单分子高密度快速定位算法，提高单分子定位速度和分子识别密度，从而提高成像速度；在此基础上，结合多色标记构建多色三维单分子超分辨荧光动态成像平台，实现活细胞内高时空分辨三维多分子成像和示踪，对生命科学研究具有重要的意义。

Three-dimensional (3D) single-molecule super-resolution microscopy, which can now be used to image the nanoscale structures and dynamics of biomolecules in living cells with nanometer spatial precision, has become an essential tool in life science research. Though there existing advantages applying this technology in thick biological samples, such as a 10 micrometer thick whole cell, still need to solve some problems on improving the imaging depth, spatiotemporal resolution and the ability to track only a few molecules at one time. This project is to develop live-cell three-dimensional single molecule super-resolution imaging methods based on digital scanned light-sheet illumination. Specific aims are to 1) develop a new method by using digital scanned lattice light sheet illumination using wavefront modulation based on high-speed digital micromirror device(DMD) to confine photoactivation to the vicinity of the focal plane and reduce out-of-focus background fluorescence crosstalk, which improves imaging depth , imaging efficiency and localization accuracy;2)develop strategies combining multifocus microscopy and double-helix point spread function (DH-PSF) using PSF engineering to increase the depth of field for 3D nano-resolution dynamic fluorescence microscopy imaging; 2)develop a fast and high density DH-PSF 3D compressed sensing algorithm based on sparse Bayesian learning to further improve the localization accuracy and identified emitter density. Finally, the project will provide a multicolor 3D single-molecule super-resolution fluorescence dynamic imaging platform that can supports 3D imaging and tracking of fast moving multiple single molecules of the whole cell. This project can be used to study dynamic processes in living cells at the single molecule level, which is of great significance to life sciences.