Research On The Theory And Experiment Of Structured Illumination Microscopy For Super-resolution And Optical Sectioning

In recent years,with the development of fluorescence labeled techniques,a series of far-field super-resolution are proposed,making light microscope break the diffraction limit.Among these super-resolution microscopies,SIM which has the merits of high spatial-temporal resolution,low photo-toxicity and three dimensional capability is very suitable for dynamic observation with super-resolution.Therefore,since the method appears at the beginning,it has received the widespread attention at home and abroad.This thesis studied theoretically and experimentally the SIM in the context of optical super-resolution and optical sectioning.The majority of the work is outlined as below:1.The theory underlying the relationship between SR-SIM and OS-SIM is found.In order to demonstrate and verify the basic principles,the formulas of the structured illumination microscopy are derived in detail according to the imaging process.A unified SIM theory is established by examining the relation between SR-SIM and OS-SIM in the terms of the illuminated patterns distribution and phase-shifting.The simulations showed clearly the ability of SIM in super-resolution and optical sectioning,helped to analyze the parameters of structured illumination affecting the imaging results.2.DMD-based LED illumination SIM is designed and built.At present,laser beams interference realized by spatial light modulator(SLM)is the general way to produce illuminated patterns.However,laser will lead to speckle noise to deteriorate the imaging.And the SLM gives rise to some problems of complex controlling in polarization of beams and low refreshing rate.In order to avoid these problems,we proposed a DMD-based LED illumination SIM(D-SIM)scheme.The speckle noise and complex polarization controlling are eliminated by using of high brightness LED light source.Comparing to SLM,DMD provides higher light efficiency and faster modulating rate.After calibration,the resolution of D-SIM is about 90 nm which beyond the diffraction limit by 2 times,the imaging speed is at least over ten times than that of laser scanning confocal microscopy(LSCM).Moreover,a custom software is also developed to realize the driving of all the component devices,the raw data acquisition,post data processing and image rendering.3.Several improved reconstruction algorithms of SIM are proposed.The reconstruction algorithms are the core technology of SIM.It requires reconstruction algorithms to obtain super-resolution and optical sectioning images.In this thesis,three new algorithms are proposed: Image Recombination Transform(IRT),Sequence Hilbert Transform(SHT)and Sectioned Super-Resolution(SSR)algorithm.IRT algorithm is robust in precise measurement of initial phase which is essential in SR-SIM reconstruction process even in the case of low-level contrast of illuminated patterns.SHT algorithm is proved in efficiency of generating sectioning slices than other available algorithms,reducing nearly 1/3 raw data in amount.The principle of SSR algorithm is based on the proposed united SIM theory,it combines the capability of super-resolution and optical sectioning.4.To tiny animal samples,a large field-of-view,rapid,high resolution microscopy is realized by the proposed D-SIM system.Currently,the available 3D microscopy is challenged in imaging speed and resolution to tiny animal samples.A modified D-SIM with an adaptive stitching algorithm of field-of-view is applied in the imaging of these large size of specimens.Through measurement,the field-of-view is reached to 0.79mm?0.54 mm,resolution is 500 nm and consume time is declined from several hours to a few minutes.We expect D-SIM to become as a routine tool in zoology studies.