Research On Super-resolution Fluorescence Microscopy Method And System

Due to its unique advantages, including minimal perturbation, ultrahigh resolution, and versatility and simplicity of usage, super-resolution optical microscopy has attracted increasingly high attention in recent years. Based on the analysis of the concept of Stimulated Emission Depletion Microscopy (STED), this project built up a high speed STED system, which is the first one in China. The system has been demonstrated to achieve a spatial resolution of below100nm, which is far beyond the diffraction barrier, with an imaging speed of2frames per second. Moreover, some methods were also proposed in this project to further enhance the performance of the imaging system.In this thesis, I first gave a brief review on those popular super-resolution imaging techniques, especially the development and current research status of STED microscopy. Then, the main concept of STED, together with several related key techniques and the method to realize high speed imaging in STED, were introduced in detail. After that, I built up the high speed STED system and demonstrated its performance in practical through a series of experiments on various samples. In order to further enhance the performance of the system, several methods were proposed in this thesis, including optimizing the resolving ability of the system in three dimensions by beam shaping, axial displacement measurement and compensation based on asymmetrical illumination beam and double-helix beam to improve the stability of the system, and also realizing super-resolution imaging at low light level through Fluorescence Emission Difference Microscopy (FED) which could be readily integrated with the STED system. Finally, I concluded the work in this project and also pointed out the possible directions of the future research.