Research On Super Resolution Microscopy Based On The Single Molecule Centroid Localization

Fluorescent microscopy iswidely applied in modern biomedical research. . However, the diffraction limited resolution (~200 nm) has poses a major hurdle on its application in studying sub-cellular structures which are usually below 100 nm. In recent years, the emerging super-resolution microscopic techniques have breaken the diffraction resolution limit. Among these techniques, the STORM, PALM and fPALM, which are based on photoswitchable flourophore and single molecule localization, have achieved nanometer resolution (20-50 nm) demonstrating great potential applications in biomedical research.The research presented in this dissertation aims to optimize the STORM technique. The first goal is to implement a STORM imaging microscopy using mercury lamp as the illumination source for achieving simplicity and reducing the total cost. The second goal is to develop new strategy for enhance the signal of photoswitchable fluorophore, e.g. Cy5. We have implemented the single fluorophore detection and photo induced switch of Cy5 fluorophore using an inverted fluorescence microscopy with mercury lamp. A new method was proposaled to improve the centrod localization method used in STORM. Most importantly, we have discovered that the total emitted photons of Cy5 can be increased 3-fold through a molecular partner in a close proximity. This novel phenomena could lead to synthesis a long lifetime flurophore in the future.