Fluorescence 3D Microscopic Imaging Based On Self-Interference Holographic Super-Resolution Localization

The research on the accurate distribution of organelles,viruses and proteins in cells in three-dimensional(3D)space is helpful to understand the life process and the pathogenesis of diseases better.The single-molecule microscopic localization technique combined with the optical switching properties of fluorescent particles can achieve super-resolution microscopic imaging beyond the diffraction limit.However,there are many problems in the super-resolution microscopic imaging technology based on single molecule localization,such as limited axial positioning accuracy and imaging depth,and the imaging system is sensitive to aberration.Fresnel incoherent correlation holography(FINCH)uses the characteristics of spatial self-interference after splitting the light from the same point.Through appropriate wave splitting techniques,the light wave emitted from the same point on the object interferes in the detector plane after beam splitting,so as to obtain the corresponding point source hologram(PSH)of the object.The 3D spatial position information of the corresponding object point is encoded in PSH.This characteristic of self-interference PSH makes it possible to obtain the 3D position information of the original object point through the reconstruction of hologram(such as diffraction propagation algorithm)or other appropriate algorithms to realize the localization of the original object point.Combined with the idea of SMLM,FINCH technology can be used as the localization method.The lateral localization and axial localization of particles in object space are independent.It is expected to realize isotropic 3D localization,so as to realize super-resolution imaging.This thesis mainly studies the method of realizing 3D super-resolution localization,the accuracy and influence factors of 3D localization,and the method of realizing superresolution microscopic imaging according to 3D localization of fluorescent particles based on FINCH.The main contents and results of this thesis are as follows:1、The basic principle of PSH to code the 3D position of object points was elucidated.And the method based on the combination of constructing a fluorescent selfinterference PSHs Library and cross-correlation algorithm to achieve high-precision 3D localization of fluorescent particles was developed.The relationship between particle3 D localization accuracy and imaging resolution was clarified.2、The effects of signal-to-noise ratio(SNR)and aberration of the PSH on the 3D localization accuracy of the object were studied.A fluorescence holographic microscopic imaging experimental system(FINCH)was built.The fluorescence selfinterference digital holographic microscopic imaging system(FINCH)was built,and the fluorescent microspheres were used as samples for experimental research.The experimental results verified the correctness of the theoretical analysis and the performance of the particle localization algorithm,and clarified the localization accuracy and the achievable imaging resolution.The research showed that the localization method based on self-interference hologram cross-correlation can achieve high-precision 3D localization of fluorescent particles in the presence of aberration and noise.3、The method to optimize the 3D localization accuracy of fluorescence selfinterference was developed.The 3D microscopic imaging of samples was realized based on the super-resolution localization of fluorescent particles.The localization accuracy and density of particles were improved,by optimizing the cross-correlation reconstruction algorithm and matching the appropriate noise reduction and filtering algorithm.The simultaneous localization of multiple sparsely distributed fluorescent particles in the same field of view was realized.The super-resolution imaging experiment of fluorescent microspheres based on fluorescence self-interference holography was completed.