| With the advance of life science, it is necessary to development novel imaging methods which can observe the single living cell in real-time and in situ at single molecule level to understand the interaction between materials and the life process. In recent years, as the development of laser techniques, molecular probe preparation techniques, fluorescence labeling techniques, and weak signal detection techniques, fluorescence microscopy has become indispensable tool for cell biology research. However, because of the diffraction limitation, the resolution of traditional light microscopy is restricted to ~200 nm laterally and ~500 nm axially, which can not meet the requirement for biological study. Thanks for the new fluorescent probes and novel imaging theories, various super-resolution imaging techniques have been developed, which break the diffraction barrier and greatly improve the spatial resolution to nanometer level. However, spatial resolution and temporal resolution are mutually restricted each other. Single molecule localization method is to obtain the ultrahigh spatial resolution at the expense of temporal resolution, which is imaged using fixed cell. And single molecule tracking method is to obtain the dynamic process of molecules in whole cell require not only high spatial but also high temporal resolution. Through the combination of the two methods allowed biologists to study of the interaction and physiological processes in cell, protein, and biological macromolecules became reality.Despite these breakthroughs in super resolution imaging, the living cell imaging method still faces many challenges, such as how to improve the depth of field of system for acquirement of the complete dynamic feature information inside living cell. The present method can be implemented with dynamic imaging within intact cells are mostly based on improved multi focal plane microscopy(MFM). E.g., the MFM based on the distorted grating and the MFM based on the multi-detectors. The low coefficient of utilization for light energy, high complexity of systems, or high cost restricts the application widely of these methods. In this paper, a new idea is put forward to image the whole cell at super resolution. In order to realize nano-resolution imaging in extended depth of field, the DH-PSF are combined with the distorted grating and applied in the nano-imaging system. The multiple molecules in the whole living cell were tracked successfully at high- precision by this 3D system. The main work is as follows:1. Designed and established the three-dimensional localization imaging system based on double helix point spread function.Double helix point spread function(DH-PSF)is a continuous rotating double helix pattern by transforming the point pread function, to obtain the corresponding 3D position in three-dimension by the location and rotated angle, thus the method is extremely useful way to realize three dimensional nano-resolution imaging. The thesis conducts the theory simulation of the DH-PSF, and improves the original DH-PSF light energy utilization through the optimization algorithm. The simulation results show that the peak intensity of DH-PSF is 30 times higher than the original. Finally, the 3D localization system based on DH-PSF is established by high efficient DH-PSF mask prepared by lithography method. The result of system calibration, our system can realize axial depth of up to 4 μm and position precision of 10 nm in lateral and 20 nm in axial direction, which verifies the feasibility of the system to be used in super resolution imaging.2. Designed and established the imaging system with extended depth-of-field combined the distorted grating(DG) function and DH-PSF function.Separately using DH-PSF system cannot satisfy the 3D localization imaging in whole cell; especially in thick cell samples such as a cell monolayer that is ~10 μm thick. Therefore it needs to combine with other extended depth of field. The thesis puts forward the way of extension of the distorted grating that is actually an off-axis Fresnel zone plate, which makes the mask capable of diffracting light in different directions as an ordinary optical grating. At the same time, the mask can focus beams with different strengths by introducing different phase contribution into different diffraction orders. In practice, the grating can be positioned anywhere within the detection light path. Simulation analysis of the distorted grating imaging pattern, verified the feasibility of imaging of thick samples of different depths. Considering the light effective utilization rate of the original amplitude distorted grating is low, we improve the binary phase formation of distorted grating, The special design of the phase steps it can form three equal diffraction efficiency in orders, improving the light utilization rateIn order to obtain the complete dynamic imaging of biological molecules in whole cell, we design and setup a super resolution imaging system based on combination of the extended DOF of the distorted grating(DG) function and the 3D nano-localization of DH-PSF, so the approach is named DG and DH-PSF combination microscopy(DDCM). By lithography techniques, we obtain composite phase plate and establish three-dimensional imaging system. The experimental results show that the system has the high localization precision. Using this system, the brown motion of fluorescent beads can be tracked and imaged in glycerol solution. And tracking capabilities of system is verified by calculating the diffusion coefficient of fluorescent beads. Forther more, we have sucessful acquired dynamic tracking results of multi-molecules in glycerin solution, which establish the foundation of realize the molecular dynamic process in intact cells..3. Developed the dynamic tracking experimental research in the living cells.Some fundamental researches on molecular dynamic tracking in cell are carried out based on the DH-PSF and DDCM system. In this thesis we choose RAW and HEP-2 cell as object, to obtain different dynamic status of the molecules across the membrane and intracellular movement by reason of the different endocytic mechanism of the cells. Finally, we have successfully tracked the dynamics process of multi-molecule in cell, and got their different motion pattern.The main innovation in this thesis is that, a method of nano-resolution imaging with extended depth-of-field has been proposed. And a key component, dual-function phase mask was designed and fabricated by combining the DG and DH-PSF, which could extend the depth of field in our imaging depth to 12 μm. Based on this ground, we establish the fluorescent dynamic imaging system with nano-resolution to track the multiple molecules inside living cells. This method can be used to obtain all the dynamic information in the intact cell with nano-resolution, without scanning and sectioning, which provides very useful tools for biologists. |
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