宽场单分子荧光轴向纳米定位及三维超分辨成像研究

Fluorescence Imaging With One Nanometer Accuracy,known as "FIONA", can localize the lateral positions of singe molecules with precision unlimited by optical diffraction. It also promoted the invention of one mostly-used type of super-resolution fluorscence microscope. However, so far there is no precise and easy-to-use method to localize single molecules along axial direction, which limits the application of single molecule fluorescence and super-resolution microscopy in biology. Based on our previsous study of Standing Wave Axial Nanometery (SWAN) in confocal geometry, this project proposes a novel method to localize single molecules axially in a wide-field geometry. It will achieve localizaition of single molecules with nanometer accuracy and precision in all three directions. Further combined stochostic excitation of fluorophores, super-resolution imaging with same resolution in three directions will be realized. This project will provide a novel single molecule fluorescence imaging and super-resolution microscopy system as a powerful research tool for biology and fundermental medical study.

单分子荧光纳米定位技术（FIONA）能以纳米精度确定单分子在横向平面内的位置，突破了光学衍射极限的限制，并带动了超分辨荧光显微镜的发明和发展。但是轴向的单分子定位目前还没有简单易行的方法可以达到纳米的精度，这限制了单分子荧光和3维超分辨显微技术在生物医学领域的应用。本项目在共聚焦的驻波轴向纳米定位研究的基础上，提出一种在宽场成像模式下对单分子荧光进行轴向定位的新方法，实现对视场内所有单分子同时进行XYZ三个方向的纳米精确定位，为利用单分子荧光技术研究生物单分子动力学和单分子追踪增加了一个新的维度。进一步结合荧光分子随机激发，实现三个方向相同分辨率的超分辨荧光成像。本项目的实施将研制出新型单分子荧光和超分辨显微成像系统，为生物学和基础医学相关学科提供强有利的研究工具。

突破衍射极限的超分辨荧光显微成像由于其在纳米尺度观测亚细胞结构的能力，从而在分子细胞生物学领域有着广泛的应用前景。本项目一方面开发了在宽场成像模式下对单分子荧光进行三维定位的新方法，搭建了相关的显微成像系统，并进一细胞骨架为例进行超分辨显微成像的应用示范。项目在原驻波激发的概念基础上，进一步开展了激光干涉的结构光照明超分辨成像， 实现了三维的结构光光场，并开发了响应的图像重建算法，进行了分辨率标定和细胞内质网的精细结构成像。 本项目实现了我国高端光学显微镜的自主研发，将推动相关的产业发展和分子细胞生物基础研究。