Research On Super-resolution Optical Fluctuation Microscopic Imaging Technology

Super resolution optical fluctuation imaging is based on statistical analysis of fluorescence fluctuations,accessing the super-resolution images.Fluorescence fluctuation was statistically analyzed by cumulative algorithm,and super-resolution images with different degrees were obtained according to different orders.SOFI has a lot of advantages such as faster imaging speed,higher signal-to-noise ratio,lower phototoxicity,lower system complexity,long time observation of living biological samples,and high-speed imaging of multiple fluorescent molecules.This research can improve the real-time performance of SOFI,expand its application range,and provide a technical basis for future research and development of instruments.Based on the research of SOFI technology,this paper studies the SOFI technology from the three aspects of SOFI imaging principle,image reconstruction algorithm and experimental device.The main research contents are as follows:(1)SOFI imaging theory and SOFI imaging conditions are deep researched.The principle of SOFI image reconstruction algorithm,the optical properties of samples and the effect of effective pixel size on image are analyzed.(2)Two image reconstruction algorithms,n order SOFI algorithm and Fourier SOFI algorithm,are proposed to improve the spatial resolution of SOFI images.The simulation data are verified and the advantages and disadvantages of the two algorithms are analyzed.(3)In order to improve the real-time performance of SOFI,an SOFI algorithm based on spatial disk filter is proposed.The multi-frame images are processed by the SOFI algorithm according to the temporal autocorrelation of the fluorescent particles in the multi-frame images,and the super-resolution images with high signal-to-noise ratio can be obtained quickly.The data analysis shows that SOFI algorithm based on spatial disk filter can obtain super-resolution image with high signal-to-noise ratio in low SNR image sequence.(4)Based on the inverted fluorescence microscope,the imaging optical path of the SOFI system was set up.A mercury lamp was used as the excitation light.Using commercial fluorescence quantum dot QDot525 as the sample,the sCMOS camera was used to record the broad field Microscopic image.The experimental results are analyzed to find the best image frame number,exposure time,effective pixel size.Combined with the proposed algorithm and the influence factors of SOFI image,the aim of obtaining the super-resolution image in the shortest time is achieved.