Research On Multifocal Structured Illumination Microscopy Based On Single-helix Point Spread Function

Multifocal Structured Illumination Microscopy(MSIM)provides twice the spatial resolution of a conventional wide-field microscope,also has strong optical sectioning ability and large penetration depth of confocal microscopy.So MSIM is suitable for three-dimensional(3D)imaging of thick biological samples.However,for3 D imaging,MSIM requires collecting multiple 2D scans taken at a series of axial positions by using a nano-stage,which greatly increases the acquisition time of the original data,and the labeled fluorescent probes are easily photobleached after multiple scanning of biological samples.In addition,MSIM has a large amount of three-dimensional raw data,which results in a large amount of post-processing workload.In order to solve the current challenges of MSIM in 3D imaging,in this thesis,we combines MSIM with single-helix point spread function(SH-PSF)for 3D sub-diffraction resolution imaging of biological samples.The main work of this thesis is listed as follows:(1)The advantages and disadvantages of MSIM are summarized,and SH-PSF-based MSIM(SH-MSIM)imaging method is developed.Furthermore,two super-resoluton reconstruction methods of optical sectioning images obtained by SH-MSIM using digital refocusing and pixel reassignment presented.(2)A SH-MSIM imaging system based on a spatial light modulator was designed and built using existing MSIM system in our laboratory.And the SH-PSF phase plate and control programs for the system were designed.The system parameters were calibrated and optimized by testing fluorescent beads.(3)The mitochondrial imaging experiments in Hela cells by using the SH-MSIM system were carried out.Two corresponding programs of super-resolution reconstruction using digital refocusing and pixel reassignment were written,respectively.Finally,the experiment results confirm the 3D imaging capability of our setup,provides theoretical and experimental basis for further research on dynamic processes in intact cells.The main innovations in this thesis are as follows:(1)Based on SH-PSF with nanometer-scale axial localization,SH-MSIM that has the ability to collect 3D information from axial planes adjacent to the nominal focal plane by a single 2D scan is developed,which can improve the 3D imaging speed of MSIM.(2)Two super-resolution reconstruction strategies for processing the collected raw data obtained by SH-MSIM by using digital refocusing and pixel reassignment are proposed.