基于结构光照明和探测光场调控的大景深超分辨显微技术研究

Currently, real-time tracking and dynamic observation of living cells face two challenges: the confined depth-of-field (DOF) and the limited spatial resolution. 1) The former leads to difficulties in real-time imaging because samples often drift away from the focal plane. The successively axial scanning scheme is universally utilized to extend the DOF, which however seriously impedes the imaging speed. 2) The diffraction-limited resolution also prevents us from the hyperfine structure of the sample. To address these two issues, this project attempts to construct a hybrid microscope with an extended DOF and high temporal-spatial resolution by combining the super-resolution of structured illumination microscopy (SIM) and the extended DOF of the double-helix point spread function (PSF) engineering method. In the illumination path, we propose a novel method to generate structured light field by using a zero-order vortex half-wave retarder to modulate the polarization state of two interfered beams, avoiding the complex polarization controlling in general scheme; In the detection path, by simply loading the corresponding phase of the double helix PSF on the spatial light modulator, the sample’s 3D information can be encoded into single snapshot. By uniting the presented decoding algorithm and the super-resolution image reconstruction algorithm, we can obtain an extended DOF super-resolution image without axial scanning. This proposed approach based on the modulation of optical fields will provide a new tool for super-resolution real-time tracking and dynamic imaging of living cells with large DOF.

目前活体生物样品的实时跟踪与动态显微成像存在两个挑战：1）成像景深不够大，样品脱离焦面时导致跟踪和成像困难，尽管采用轴向扫描的办法可以扩展景深，但成像速度一般较慢；2）成像空间分辨率受衍射极限限制导致无法看清样品的超精细结构。针对这两个问题，本项目利用光场调控原理，首次提出将结构光照明超分辨显微技术（SIM）与基于双螺旋点扩散函数（PSF）调制扩展景深的方法相结合，实现大景深、高时空分辨的显微成像。在照明光路上，提出一种采用零级涡旋半波片调控双光束干涉偏振态的结构光场产生方案，避免复杂的光束偏振态控制；在探测光路中，使用空间光调制器加载双螺旋PSF调制相位，实现单次曝光中图像深度信息的编码；最后通过我们提出的快速深度解调算法和超分辨图像重构算法，获得大景深的超分辨图像。本项目提出的基于光场调控的显微成像技术将为活体生物样品的超分辨大景深实时跟踪与动态成像提供一种新的手段。

针对光学显微系统分辨率受限和景深不足这两个科学问题，项目开展了基于结构光照明和探测光场调控的大景深超分辨显微技术研究。该技术利用光场结构与样品结构的空间频率耦合实现超分辨成像（SIM）；同时，在探测光路上对点扩展函数（PSF）进行双螺旋结构调制，利用双螺旋PSF空间结构与轴向深度的对应关系，解析成像景深信息。经过三年研究，项目提出了在空间域直接重构SIM超分辨图像的理论，并实现了荧光纳米粒子布朗运动的实时超分辨动态监测；揭示了超分辨SIM与光切片SIM理论联系，统一了这两种理论，并提出了超分辨SIM和光切片SIM融合的显微方法；在项目技术基础上，进一步提出了结构光照明和探测光场混合PSF调制的高精度三维显微方法；发现了双螺旋PSF定量感应系统光学畸变的规律，通过分析双螺旋PSF的空间结构变化实现了显微畸变的矫正；借助卷积神经网络的深度学习技术，大幅精简了光切片SIM的原始数据采集量。项目完成了显微系统的搭建和优化，在波长561nm和100×/NA1.49物镜下，空间分辨率达到107nm（衍射极限值约217nm）、景深范围2μm（普通值约为0.38μm），成像速度30帧/秒（@1024×1024pixels）；共发表学术论文10篇，累计培养博士研究生3名，申请国家发明专利2项（已授权1项）。