Study On The Digital Line Scanning Fluorescence Microscopic Imaging System And Its Application

Based on laser scanning confocal microscope,line scanning fluorescence microscopy uses line scanning configuration instead of the process of point raster scanning to further improve the image acquisition speed.It has a simple system structure,fast imaging speed and low phototoxicity.So,it is more suitable for fast highresolution imaging of living thick samples,which is of great significance for research in life sciences and biomedicine.However,there are still many challenges such as system flexibility,imaging speed,spatial resolution and optical sectioning ability to overcome.Therefore,the research and development of new line scanning microscopy technology and related image reconstruction algorithm to further improve its performance have important scientific significance and application value.In order to solve the above problems,this thesis focuses on the imaging method,system optimization,experiment and three-dimensional(3D)image reconstruction methods of digital micromirror device(DMD)-based digital line scanning fluorescence microscopy(DLSFM)based on the existing multifocal structured illumination microscope(MSIM)in our laboratory.The main work of this thesis is as follows:(1)The advantages and disadvantages of the current line scanning fluorescence microscopy are analyzed.And the basic imaging theory of DMD-based DLSFM and the main factors affecting its imaging resolution are studied.Further,several DLSFM image restoration algorithms are compared and analyzed in detail: 3D deconvolution algorithm,Virtual-slit-based image reconstruction method and the Max Min approach.Then,a new strategy-Mean-variance-based image reconstruction method is proposed.(2)Based on the current MSIM,the excitation light module,detection light module and system control module of DLSFM were optimized and designed,and the system parameters were calibrated to achieve the multi-line parallel illumination digital scanning fluorescence microscopy imaging.(3)Using DLSFM,a 3D microscopy imaging experiment of mouse slice samples was carried out,and the corresponding image reconstruction program is written to achieve the 3D high-resolution imaging.The experimental results prove the capability of our system,which provides the theoretical and technical basis for further research on whole living cells and other thick samples.