Super-Resolution Fluorescence Lifetime Imaging Method And System

A fluorescence microscope system has functions of fluorescence intensity imaging and fluorescence lifetime imaging, is a very useful tool to reflect micro-structure and micro-environment of observed sample. Thus, it can provide a direct, visible approach for researches such as biology, materialsscience, etc. This thesis proposes super-resolution microscope system which breaks through the optical diffraction-barrier by Stimulated Emission Depletion (STED). The system integrates Time-Correlated Single Photon Counting (TCSPC) technique to acquire the fluorescence lifetime.At first, the main methods of super-resolution microscopy and each method’s characteristics are introduced. The integrations of each super-resolution microscopy and fluorescence lifetime measurement are simply discussed.The theory and basic principle of STED (Stimulated Emission Depletion) microscopy are demonstrated. A doughnut-shaped depletion spot is the key to achieve super-resolution. Therefore, the simulation of hollow depletion spot generated by phase-modulated is presented. The techniques of fluorescence lifetime measurement are introduced. And one of them is selected to be integrated into STED microscopy to perform super-resolution fluorescence lifetime imaging.To prove the feasibility of fluorescence lifetime imaging, we first set-up confocal fluorescence lifetime imaging system and present fluorescence lifetime image at the resolution of200nm. Based on the confocal system, we introduced depletion beam into the beam path to squeeze the size of fluorescence. As expected, fluorescence lifetimes are imaged at the resolution of75nm with depletion power of200mW.Based on the super-resolution fluorescence lifetime imaging system, we simulate and set-up a sub-system to prove that the extending the functionality of the system.At the end of the thesis, the following efforts to achieve better resolution are indicated. The potential applications of the system are also discussed.