为了拓展荧光辐射差分(Fluorescence Emission Difference,FED)显微术的应用, 使得该方法可以同时对生物样品的不同组织结构进行超分辨成像, 本文对双色FED显微系统展开了研究。FED的基本原理是将实心光斑扫描得到的共焦显微图像减去空心光斑扫描得到的负共焦图像, 以此获得超分辨显微图像。在对单色FED显微系统进行研究后, 本文提出了一种可行的双色FED显微成像系统方案。实验结果表明, 在488 nm和640 nm激发光下, 该系统在荧光颗粒上分别实现了135 nm和160 nm的空间分辨率, 另外也能对生物样品的不同组织进行多色同时超分辨显微成像, 满足了实际应用的要求。

To perform super-resolution imaging of different tissue structures of biological samples using fluorescence radiation differential microscopy simultaneously, a dual-color FED microscopy system is studied in this paper. The basic principle of the FED is to remove the confocal microscopy image obtained by scanning the solid spot from the confocal microscopy image obtained by scanning the hollow spot to obtain a super-resolution microscopy image. Based on the study of the monochromatic FED microscopy system, a feasible dual-color FED microscopy imaging system is proposed and imaging experiments are performed on fluorescent particles in this paper. The experimental results indicate that under excitation light of 488nm and 640nm, the system realizes spatial resolution of 135 nm and 160 nm of the fluorescent particles respectively. In addition, this system can also perform multi-color super-resolution microscopy imaging simultaneously on different tissues of biological samples, which meets the requirements of practical applications.