Research On Differential Super-resolution Micro-Scopy And System Based On Parallel Detection

Compared with wide field fluorescence microscopy,confocal fluorescence microscopy has the advantages of low background noise,high contrast,high signal-to-noise ratio,and relatively high lateral and axial resolutions,which is one of the most common microscopic technologies in biological research.The parallel detection pixel reassignment method can further improve the SNR and resolution of confocal microscopy.Additionally,fluorescence emission difference(FED)super-resolution microscopy was proposed in 2013 and has been widely studied,which developed from confocal fluorescence microscopy.These methods inherit almost all the advantages of confocal fluorescence microscopy,which can be widely used in biomedical research.However,there are still some problems and shortcomings in these technologies,such as pixel reassignment with uncorrected detectors' position,low axial resolution and slow imaging speed.In addition,as an important supplement to confocal microscopy,the emerging FED super-resolution microscopy with universal fluorescence labeling can well bypass the limitations of foreign patents,which is very suitable for the research and popularizing of advanced optical microscopy equipment.In this dissertation,parallel detection pixel reassignment microscopy and FED super-resolution microscopy are deeply studied according to normalized cross-correlation method,point spread function engineering and grating diffraction theory to further improve the system resolution and imaging speed,and make them more versilte in life science research.In addition,this dissertation proposes the FED super-resolution microscopy equipment with modular design,which promotes the process of multi-mode super-resolution fluorescence microscopies instrument equipment.The major contents and innovations of this dissertation are listed below:1.The influence of the pixel position in the parallel detector array on the super-resolution microscopy based on pixel reassignment is studied in depth.Theoretical analysis and simulation of the related content are carried out.The normalized orthogonal correlation algorithm is used to obtain accurate information of detector positions to realize the correct pixel position reassignment,and the fluorescence microscope system of parallel detection based on pixel reassignment is built.The experimental results show that this method can accurately obtain the information of the detector positions to eliminate the influence of pixel dislocation in the process of the manufacturing and installation of the parallel detector array,and achieve the optimum imaging result of pixel reassignment.2.The method of the parallel detection based on pixel reassignment and the three-dimension difference are introduced into the differential super-resolution microscopy system.By using spatial light modulator(SLM)to modulate the phase of the p-polarized and s-polarized excitation light,respectively.The hollow spots in both the transverse and axial directions can be obtained simultaneously.Combined with the parallel detection based on pixel reassignment,the three-dimension resolution of the differential super-resolution microscope system can be further improved.Through the theoretical analysis and simulation of the related content,a differential super-resolution microscopy system based on SLM modulation as well as parallel detection is built.The experimental results show that,compared with the confocal microscopy and parallel detection pixel reassignment microscopy,the proposed method achieves 1.85 times and 1.33 times improvement in lateral resolution as well as 1.48 times and 1.24 times improvement in axial resolution,respectively.3.The imaging speed limitation of differential super-resolution microscopy is deeply studied.By loading the blazed grating on SLM and adjusting the period of the grating to control the blazed angle,the controllable dislocation of the solid focal spot and the hollow focal spot on the focal plane can be realized,and then the dual focal spot can scan at the same time.A differential super-resolution microscopy system based on parallel scanning is designed and built.The experimental results show that the imaging speed of the differential super-resolution microscopy can be doubled by this method.4.The miniaturization and modularization of various differential super-resolution microscopy methods are deeply studied and analyzed.The modular design and three-dimensional layout optimization of the microscopy instrument are carried out here,and the self-complied software of the system imaging is designed.Three sets of the advanced optical microscopy instruments based on differential microscopy are constructed.