Research On Structured Tilt Illumination Microscopy Based On Mirror Transform Spectrum Expansion

At present,structured illumination microscopy(SIM)is one of the mainstream super resolution optical imaging techniques.SIM plays an important role in research on biological sample super resolution imaging owing to its advantages such as lower excitation light intensity,weak photo toxicity,no requirement for special fluorescent dyes.The application field of SIM has expanded from fluorescent labeled samples to label-free samples.However,there are still the following problems in SIM:(1)SIM needs to rotate the structured illumination field direction several times for sample spectrum omnidirectional extension.Multiple rotations make the operation complex and accumulate rotation angle errors.As a result,multiple rotations of structured illumination field direction affect the imaging speed and quality severely.(2)The situation that SIM stripe frequency is lower than cutoff frequency due to the limitation of device parameters exists in actual operation.Under this condition,the vertical illumination of the sample in SIM results in limited high frequency information collected by imaging system.As a result,vertical illumination affects the super resolution imaging ability of label-free samples and the resolution can not be increased to 2 times.This thesis of“Research on structured tilt illumination microscopy based on mirror transform spectrum expansion”carries out the theoretical analysis,method research and experimental verification for the above problems.The thesis research improves the imaging process of SIM through the synthetic spectrum algorithm based on mirror transform and tilt illumination.The purpose of the research is to increase the imaging speed,the resolution ability of label-free samples and the noise robustness.This thesis focuses on the application requirements of SIM and provides theoretical basis and technical reservation for SIM to achieve fast super resolution imaging of biological samples.The research of this thesis can promote the rapid development of optical microscopy and label-free microscopy instrument.The main contents of this article are as follows:(1)The thesis proposes a super resolution image reconstruction method of SIM based on mirror transform spectrum expansion(MTSIM)to solve the problem that multiple rotations of structured illumination field direction affect the imaging speed and quality.According to the symmetry of real part and imaginary part in one dimensional Fourier transform,the mirror transform based on symmetry is proposed.On the basis of single direction structured illumination and sample high frequency spectrum demodulation calculation,whole domain spectrum data is constructed by synthetic spectrum algorithm based on mirror transform.The sample high frequency spectrum corresponding to that direction is expanded and the synthetic high frequency spectrum in complementary angle direction of structured illumination is obtained.In this way,MTSIM achieves the omnidirectional extension of the sample spectrum and super resolution image reconstruction.This method only needs to rotate the structured illumination field of 45~oand perform single direction structured illumination for super resolution imaging.MTSIM overcomes the disadvantage that traditional SIM needs to rotate the structured illumination field direction several times.This method reduces the amount of SIM image acquisition and accumulation of rotation angle errors to increase the imaging speed and quality.Numerical simulation and experimental results show that MTSIM can achieve the same resolution as traditional SIM when imaging fluorescent labeled samples and label-free samples.MTSIM increases the imaging speed to 3 times and has stronger robustness to shot noise compared with traditional SIM.When the structured illumination field rotation angle errors are the same,MTSIM has higher imaging quality than traditional SIM for label-free samples and fluorescent labeled samples.(2)The thesis establishes the theoretical model of coherent imaging to label-free samples by structured tilt illumination and proposes structured multi-angle tilt illumination microscopy(ti SIM)to solve the problem that vertical illumination of structured illumination field affects the super resolution imaging ability to label-free samples and the resolution can not be increased to 2 times with the SIM stripe frequency lower than cutoff frequency.When the SIM stripe frequency is lower than cutoff frequency,ti SIM expands the coherent transfer function bandwidth through structured tilt illumination and moves the high frequency information undetectable during vertical illumination to the measurable range of the imaging system.Aiming at the problem that it is difficult to measure the tilt angle of structured illumination field in the experiment,a computing technology for calculating the tilt angle of incident light in lens optical imaging system is proposed.The computing technology uses the digital image correlation method and optical flow method to calculate the center displacement of the image and then determine the tilt angle.On this basis,the calculation accuracy of digital image correlation method and optical flow method is compared.ti SIM achieves the omnidirectional extension of sample spectrum through single direction structured multi-angle tilt illumination,spectrum splicing and the synthetic spectrum algorithm based on mirror transform.ti SIM breaks through the hardware limitation of generating high frequency SIM stripe and increases the imaging resolution to label-free samples with the SIM stripe frequency lower than cutoff frequency.Numerical simulation and experimental result show that optical flow method has higher accuracy in calculating the image displacement and the tilt angle than digital image correlation method.ti SIM(SIM field rotation angle 45~oand tilt angles as±1~o,±2~o)achieves the imaging resolution of 2.19μm to label-free samples and increases the resolution to 1.12 times and 2 times compared with traditional SIM and wide field microscopy respectively when the SIM stripe frequency is lower than cutoff frequency.