基于特殊结构暗场照明光的显微成像技术研究

The structured illumination microscopy (SIM) is one of wide-field imaging techniques, which has been applied in studying bio-medicines, chemistry and materials. Non-linear SIM is able to achieve 50 nm resolution. However, the high power density from the focusing laser to stimulate the non-linear effect of florescence is likely to damage the sample and cause the photo-bleach in non-linear SIM. Linear SIM is exempt from the above shortcomings, but the resolution is poorer than the former one. To improve the resolution of the linear SIM, specially structured illumination based on the dark-field microscopy is proposed. The difference between it and the traditional linear SIM lies in the following two aspects: 1. the illumination and image systems are linear and are limited by the Abbe’s definition; 2. the propagation of the light after being scattered by the object is non-linear. The non-linear propagation will make the higher frequency object information enter the imaging system, and finally improves the resolution. The dark-field illumination enhances the image contrast without fluorescence labeling. The main contents which are included in this proposal are: 1.the non-linear propagation theory that describes the relationship between the structured illumination and the sample will be studied; 2. the method to retrieve the lost low frequency information, and the technique to synthesize the low and high frequency information will be discussed; 3. the aberration correction techniques of the recording and illuminating systems will be investigated under specially structured illumination. It is possible to pave a new way for the superresolution technology.

结构光照明显微术作为一种宽场超分辨成像方法，被应用于生物医学、化学、材料科学等领域。基于荧光技术的非线性结构光照明可以获得50 nm的空间分辨率，但用于激发非线性的光源易造成样品损伤、漂白。线性结构光显微术虽不存在上述缺点，但是分辨率远不及前者。为提高线性结构照明光显微术分辨率，本项目提出研究特殊结构光暗场照明显微术。该技术与传统的线性、非线性结构光照明显微成像术区别在于其照明和成像系统部分保持线性，而使携带物体信息的散射光非线性传输，令超高频成分参与成像，获得高分辨率。暗场照明方式可以保证在非荧光标记下，获得高对比度。本项目主要研究内容包括：1.研究特殊结构光与样品之间产生非线性传输的原理机制和方法；2.研究暗场照明下，低频信息的重拾方法，以及高、低频相位信息的融合方法；3.在新型照明方式下，研究照明和成像系统像差分析及校正方法。本项目拟开展的工作将为光学显微超分辨技术开辟新途径。

结构光照明显微术作为一种宽场超分辨成像方法，被应用于生物医学、化学、材料科学等领域。为提高线性结构照明光显微术分辨率，同时，优化成像质量。本项目提出研究特殊结构光暗场照明显微术。. 本项目主要在以下四方面取得研究成果：（1）特殊结构光的生成和测量以及传输特性研究。通过全息编码研究了在三维空间产生多维度新型矢量光场和阵列的方法；提出了一种利用椭圆Airy相位掩模测量涡旋光束拓扑荷数的方法；研究特殊光场在空气以及浑浊介质中的传输特性；（2）研究了在特殊光场照明下，基于偏振复用和主成分分析的实时明场超分辨成像技术；设计了明、暗场同时成像系统，并基于数理统计理论，建立了明、暗场数字切换成像的网络框架模型，利用任意一幅低分辨率明场图像可以高效转换输出其对应的高分辨率暗场图像；（3）利用深度学习的网络框架对系统像差、成像噪声也进行了校正和祛除，并能完成自动聚焦；（4）在成像系统中，通过调制照明光，同时设计滤波器，可获得对比度参数可控、方向具有选择性的类暗场成像效果。本项目取得的研究成果将为光学显微超分辨技术开辟新思路和新方法，在项目的支持下课题组共发表25篇学术论文，5篇会议论文，获得1项发明专利授权。