Research On Single Molecules Axial Location In Three-dimensional Stochastic Optical Reconstruction Microscopy

Super-resolution microscopy greatly improves the fundamental research of biology science and provides an important tool to reveal the essence of life. Stochastic Optical Reconstruction Microscopy(STORM), based on single molecule localization technology, is a key method for super-resolution imaging. The resolution of STORM can be approximately 20~50nm in lateral and 70 nm in axial direction. Despite these breakthroughs, there is no precise and easy-to-use method to localize single molecules along axial direction, which limits the application of single molecule fluorescence and super-resolution microscopy in biology.In this paper the relationship between the ellipticity of point spread function and the focal length of cylindrical lens has been investigated. We completed the simulation of imaging and built a STORM-SIM microscope. Then the parameters of cylindrical lenses was optimized. As the demonstration, we reconstructed 3D-STORM and SIM image of actin filaments.The first section mainly introduces the 3D-STORM imaging algorithm and simulation process. In this section, we discussed the difference imaging algorithm, include Centroid algorithm and Guassian fitting. With MATLAB, we finished the 3D-STORM imaging process using Guassian fitting.The second part mainly focuses on setting up the three-dimensional super-resolution microscopy, based on optical astigmatism method with cylindrical lenses and optimized cylindrical lens.The imaging system was built up based on Olympus’ s inverted fluorescence, including light source module, laser selection moduel, total internal reflection subsystem, and fluorescence imaging subsystem. With three different focal length lenses, the point spread function in custom built stochastic optical reconstruction microscopy instruments has been measured. A method to evaluate the cylindrical lens was developed based on linear region and localization error. The results show that 90 nm axial resolution can be achieved with suitable focal length.The third part is about the SIM imaging mode of STORM-SIM system.Firstly introduced the SIM imaging process and algorithm in detail. The grating is generated by digital micro-mirror device(DMD). With fourier transformation, ?1-order laser beams are generated. Then, the two beams of laser interference occur in the focal plane and generate cosine fringes to illuminate samples. At last, the SIM-images of actin are demonstrated.In last part, the paper made a summary. The shortcomings of our experiment were analyzed. Then possible solutions were proposed. Finally, some prospects for super-resolution microscopy were introduced.