| The optical microscopy is an indispensable tool for exploration of the microscopic world.Due to the optical diffraction limit,the lateral resolution of the traditional optical microscopy cannot exceed the half of light wavelength.The emergence of superresolution fluorescence microscopy has increased the resolution of optical microscopy to sub-micron and even nano-scale.It makes possible to observe the fine structure of cells and the process of life activities,and promotes further development of many fields of the life science.Compared with other super-resolution fluorescence microscopies,the Structured Illumination Microscopy(SIM)has some advantages,such as high resolution,superior three-dimensional imaging capability,high system compatibility,and low phototoxicity.It is more suitable for 3D super-resolution imaging and dynamic imaging of live cells,and has gradually become an important imaging tool in biomedicine and other fields.Recently,SIM has been developed around the direction of spatial resolution improvement,imaging speed,and 3D imaging capability,especially achieving fast 3D super-resolution imaging of live cells.With the continuous development of super-resolution fluorescence microscopy,besides focusing on technical performance parameters,more attention is paid to make super-resolution microscopies better and more stably applied to actual imaging experiments.Aiming at the drawbacks of slow imaging speed and image artifacts faced by the current 3D SIM,a new method of asymmetric three-beam interference structured illumination and a 3D reconstruction algorithm based on Richard-Lucy were proposed.In this paper,the theoretical research,key technology research,system design and detection method research of SIM based on asymmetric three-beam interference(ATISIM)were carried out,and a prototype of ATI-SIM was built.Using the ATI-SIM,practical application experimental research on hot biological issues such as cell membrane and filament morphological changes in endocytosis was carried out.Specific content includes:1)The related concepts of super-resolution fluorescence microscopy and the principles of mainstream far-field super-resolution fluorescence microscopy were introduced,the current research of SIM technology and its limitations in the field of fast 3D super-resolution imaging were summarized.The purpose and significance of this research were explained.2)The basic principle of ATI-SIM was expounded.By analyzing the illumination light field characteristics of ATI-SIM,the structured light generation method based on spatial light modulator was optimized,and a new type of polarization modulation method with less energy loss and less time delay was proposed.3)A 3D reconstruction algorithm based on Richard-Lucy was proposed.The algorithm has low sensitivity to the original parameters of structured light,and can effectively suppress out-of-focus information,super-resolution image reconstruction artifacts were reduced.4)The design of apochromatic microscope objective was completed.The design of the optical,mechanical and electronic control units of the ATI-SIM was carried out.The prototype of the ATI-SIM was developed,and the system test was carried out.The lateral and axial resolutions of the ATI-SIM reached 103nm and 279.5nm,respectively,and the 3D imaging speed increased by 30%compared to conventional SIM systems.5)Using the self-developed ATI-SIM system,the biological imaging experiment research was carried out.A series of imaging experiments on microfilament morphological changes in macropinocytosis were completed,and the biological experiments showed that ATI-SIM has good application value and broad prospects in the field of biomedical imaging. |
