| Obtaining high-resolution three-dimensional structural images of living biological samples and achieving long-term observation of them is crucial for the development of life science research.High spatiotemporal resolution 3D imaging requires microscopic imaging technology with 3D tomography capabilities,as well as optimal imaging time and quality.Wide-field fluorescence microscopy imaging technology based on dynamic speckle illumination utilizes dynamically changing speckle patterns to illuminate biological tissues or cell samples in the entire field.Through the extraction algorithm of tomographic images,three-dimensional structural fluorescence tomographic images of the tested samples with high temporal and spatial resolution are obtained.It has the characteristics of simple structure,low cost,and easy operation,and has broad application prospects in research fields such as biology,medicine,and life sciences.The research work in this thesis combines the wide field fluorescence microscopic imaging technology based on dynamic speckle with the line laser optical tweezers technology.Based on the principle of the wide field fluorescence microscopic imaging method based on optical tweezers light manipulation and dynamic speckle illumination,indepth research work has been carried out in simulation,system design,system construction,experimental measurement,and experimental results analysis.The main contents are as follows:1.Through simulation,the main factors affecting the imaging quality of dynamic speckle illumination fluorescence tomography images were analyzed,including the number of original fluorescence images recorded by CCD,the particle size of scatters,and the relationship between three different imaging algorithms and imaging quality.The simulation results show that the imaging quality of fluorescence tomography images first increases and then tends to saturation with the number of original fluorescence images,and then decreases with the particle size of scatters.Taking into account factors such as imaging quality and imaging time,when the number of original fluorescence images used to extract fluorescence tomography images is 60,and the particle size of the grit is about 1000,a high spatial resolution fluorescence tomography image with an image contrast of more than 85% can be obtained.Theoretical analysis and simulation research work provide theoretical basis and guidance for the design,implementation,and optimization of the system structure of wide field fluorescence microscopy based on dynamic speckle illumination.2.Based on theoretical research work,a dynamic speckle illumination wide field fluorescence microscopic imaging experimental system was established.Three experimental samples-resolution plates,fluorescent beads,and animal cell mitotic sections-were tested,and fluorescence tomography images of the samples were obtained.The main performance parameters of the system were calibrated.3.The organic combination of line laser tweezers manipulation technology and dynamic speckle illumination wide field fluorescence microscopy technology has enabled researchers to shift from passive observation to active manipulation.Active light manipulation of the rotation angle of the fluorescent bead sample using linear laser tweezers,and obtaining fluorescence tomography images of the sample at different angles through dynamic speckle illumination wide field fluorescence microscopy. |
