基于虚拟点扩散函数的无伪像超分辨差分显微成像

Far-field super-resolution optical microscopy is the major tool for the observation on the subcellular structure of living cells. However, there are barriers to be overcome for widespread applications of existing techniques, for example, the requirement of the intricate optical system, time-consuming data processing, specialized sample preparation, expensive instrument, etc. Subtraction microscopy, based on point spread function engineering, is a simpler super-resolution technique with higher applicability, but excessive subtraction for higher resolution will lead to deformation of the image, which restricts the usage of this technique in observation on the subcellular structure of living cells. In this project, we propose a deformation-free subtraction microscopy base on virtual point spread function. First, we will study the novel method for generating virtual hollow point spread function with abundant high-frequency components. At the same time, the influence of detector point spread function modulation will be studied, and pixel reassignment method and virtual pinhole are used to modulate the effective point spread function. Finally, by means of solving the optimization model, the high similarity between the linear combination of solid point spread function and virtual hollow point spread function is achieved. This project will realize a deformation-free super-resolution microscopy with a lateral resolution of 100nm, which is a novel research tool for living cell research.

远场光学超分辨显微技术是研究活细胞精细亚细胞结构的主要研究工具，但大多存在光学系统复杂、成像处理时间长、通用性较低、成本较高等问题。成像差分显微术是一种基于点扩散函数工程的超分辨成像技术，它具备通用性强及设备简单等优点，但是在进一步提高分辨率的过程中会出现伪像，限制了其在活细胞精细亚细胞结构检测中的应用。本项目，提出一种基于虚拟点扩散函数的无伪像成像差分显微技术。首先，研究新的含有丰富高频分量的虚拟环形照明点扩散函数生成方法。同时，研究探测点扩散函数调制对差分成像系统点扩散函数的影响，采用像素重分配方法及虚拟针孔对系统有效点扩散函数进行调制。最终，通过解优化模型的方式，实现实心点扩散函数的线性组合外围与虚拟环形点扩散函数的高相似度，获得无伪像前提下约100nm的横向分辨率，进而为生物医学研究提供新的工具。

基于点扩散函数工程优化显微成像是远场光学超分辨显微技术的重要研究热点。本项目基于矢量积分模型，研究了多种调制方法对点扩散函数的影响，基于径向偏振光照明及其相位调制，通过获得的环形点扩散函数设计了差分成像方法，在0.3Au的针孔下采用图像差分成像的方法，其横向成像分辨率比传统的共聚焦提高将近1.90倍，轴向分辨率比传统共聚焦提高了1.26倍。针对成像中相位调制后光子利用率低的问题，通过迭代傅里叶变换算法对相位进行优化，优化后的点扩散函数的主瓣能量更强，侧瓣能量大大减弱，更适合于光子有限的应用。在此基础上，基于菲涅尔区方法设计并优化相位，生成了可增大轴向定位范围的点扩散函数，实现了轴向~10µm的景深，大幅度提高了三维成像的时间分辨率。.通过项目的研究，提供了一种通用且成本较低的超分辨成像技术，有利于超分辨技术的普及及相关基础研究的发展。