Double-helical Point Spread Function Microscopic Imaging Method Based On Light Sheet Illumination And Its Application Research

Double-helix point spread functions(DH-PSF) microscopy provides high resolution three-dimensional(3D) imaging over a long depth-of-field. It has been widely used in the field of super resolution fluorescence microscopy, 3D single particle tracking(SPT) etc, and become an important tools in many areas of life science. However, to study thick biological samples, this technique still faces many challenges,such as improving the signal to noise ratio of the image, improving the spatial and temporal resolution and the imaging depth etc. In order to overcome these limitations, we combine the light-sheet illumination with DH-PSF microscopy to propose a new microscopic imaging method for nanoscale 3D imaging and tracking of single particles in thick samples. At the same time, it has the advantages of high image contrast, high temporal and spatial resolution, low background noise and deep imaging depth. The main research work of this thesis is as follows.(1) Analyzed the advantages and disadvantages of 3D SPT, and then propose the DH-PSF microscopy method based on light-sheet illumination for suppressing the background fluorescence noise and improving the signal to noise ratio. Achieved the 3D nanometer resolution imaging and tracking of single particles.(2) Illustrated the basic theory and technology problems of the DH-PSF microscopy.Used MATLAB to perform the numerical simulation verification and design the high efficiency DH-PSF phase plate. Presented a new method to realize phase plate by using the spatial light modulator.(3) Based on the combination of Powell prism and lighting objective lens, proposed a new method to generate a light-sheet with a large field of view and uniform intensity distribution. Accordingly, designed and set up a light sheet based DH-PSF microscopy. Then optimized and calibrated the system by a series of experiments, demonstrated the design is in consistent with the theoretical results.(4) Accomplished the 3D SPT experiments of single fluorescent beads in glycerol solution of different concentration using the system. These experimental results proved thereliability of the system. And provided a theoretical and experimental basis for the further study of the dynamic process of the molecules in the thick samples such as intact cells.