Study On Super-resolution Fluorescence Microscopy Imaging Method And System Based On Multifocal Structured Illumination

As a new super-resolution(SR)fluorescence microscopy technology,multifocal structured illumination microscopy(MSIM)not only achieves twofold spatial resolution improvement over wide-field imaging,but also has strong capability of biological tissue penetration and optical section.Compared with traditional wide-field structured illumination microscopy(WF-SIM),MSIM is more suitable for threedimensional(3D)SR imaging of thick samples.In addition,it can be combined with most point spread function(PSF)engineering methods or multiphoton technology to improve system performance.Therefore,MSIM has great application potential in life science research.However,the limited spatial resolution and the relatively slow speed for volumetric imaging so far still restrict its application in nano-resolution dynamic imaging of living cells.In order to solve the above-mentioned problems existing in MSIM,this work firstly improved the spatial resolution of MSIM by improving the SR image reconstruction algorithm.Then,the detection path is reconstructed by combining multifocal plane microscopy and double helical PSF engineering to improve the temporal resolution of 3D-MSIM imaging and expand its capacity of spectral imaging.The main work of this thesis is listed as follows:1)The MSIM SR image reconstruction method based on sparse Bayesian learning was studied.The MSIM experimental system was built and the data processing program was coded.The factors affecting the imaging quality of MSIM were analyzed through biological sample experiments.On this basis,the sparse Bayesian learning(MSBL)based on multi-measurement vector model was applied to the SR image reconstruction of MSIM.The simulation experiments and the biological sample imaging experiments demonstrated that this method can achieve a lateral resolution of 90 nm,which is 1.5 times of the conventional MSIM.2)Combining the multi-value phase encoding technique with the traditional design of distorted grating,a design method of distorted multi-value pure-phase grating(DMVPPG)was proposed.The grating designed by this method can distribute the incident light intensity signals emitted by objects in different axial positions evenly to a required diffraction level.The simulation imaging experiment results showed that the optical system composed of DMVPPG and lenses can realize multi-focal plane imaging of the sample and maintain the consistency of imaging intensity.3)A spectrally resolved multi-focal MSIM imaging methodwas proposed,and the related imaging principles,spectrum and image reconstruction methods were studied.In the detection path of the MSIM system,a multi-foccal plane imaging system based on DMVPPG was introduced.The MSIM image reconstruction program was modified accordingly,which realized simultaneous SR imaging of multi-focal planes and improved the volumetric image speed of MSIM.In addition,the spectral detection and processing of MSIM were realized by utilizing the linear dispersion of grating and the additional dispersion calibration setup and data processing program.4)MSIM based on double helix phase engineering(MSIMH)was proposed,and the related imaging principles and image reconstruction methods were studied.On this basis,a double-helix PSF(DH-PSF)phase mask was designed,and the MSIMH SR imaging system was built.A program for system control and image reconstruction was compiled to restore the 3D sample structure and depth information in the imaging area.The experiment results showed that MSIMH can obtain SR structure information of samples in the depth range of 2 ?m through a single two-dimensional(2D)scan.Compared with conventional MSIM,the scanning times required for 3D imaging by MSIMH were reduced to 1/10.The main innovation in thesis is as follows:1)MSBL compressed sensing algorithm is proposed to reconstruct SR image from experimental data acquired by the MSIM system,which simplifies the steps of MSIM image reconstruction and further improves the spatial resolution.2)A multi-focal plane MSIM imaging method with spectral resolution is proposed.The detection optical path of MSIM is modified by using self-designed multi-value pure-phase distorted grating.The multi-focal plane SR imaging and spectral detection of MSIM are realized.3)MSIM based on DH-PSF is developed.The optical transfer function of the MSIM system is modulated by the DH-PSF phase mask to realize axial localization.The 3D information of samples can be obtained by 2D fast digital scanning of DMD,and the rapid 3D SR imaging of MSIM can be realized.