Research On Self-Correcting,Long-term And High-Resolution Imaging Method For Confocal Microscope

The confocal laser scanning microscopy uses the pinhole to filter out the stray light on the non focal plane to obtain the fluorescence signal on the focal plane of the sample,which can realize the high-resolution imaging of the micro structure inside the cell or tissue.The laser confocal high-resolution imaging system can be used to analyze and track the pathological structure of the biological tissue in real time,so as to promote the biomedical application in the major diseases such as tumor diseases,cardiovascular and cerebrovascular diseases to explore the pathogenesis.However,confocal microscopy has the problems of blur and short effective time,which seriously limits the application of confocal imaging.In order to solve the above problems,this paper constructs a fast imaging system of confocal scanning image,construct an adaptive correction method,proposes a confocal highresolution imaging method based on image restoration technology,and designs a nonuniform laser scanning mode with adjustable illumination for long-range confocal imaging,and builds a corresponding confocal imaging verification platform.The main contents of this paper are as follows:(1)The high-speed confocal imaging system based on resonant galvanometer galvanometer plane scanning is studied.Based on the analysis of the principle of resonant galvanometer galvanometer scanning,a method of distortion correction and adaptive dislocation compensation for nonlinear scanning is proposed,which realizes the fast segmentation of continuous inter frame images.Compared with commercial confocal fluorescence imaging system,the effectiveness of the high-speed confocal imaging system is verified.(2)The high resolution imaging method of confocal based on image restoration is studied.Based on the prior distribution of gradient data of confocal image,a fully variational regular model with gradient field is constructed,which can effectively protect the details of reconstructed image and suppress noise.In order to solve the nonlinear problem of confocal image restoration,a plug and play inertial forward backward confocal restoration numerical framework is proposed,which achieves robust and efficient numerical convergence.The system test shows that the proposed model can effectively improve the imaging resolution by 35.3%.(3)The confocal long-term imaging method with adjustable scanning laser quantity is studied.A non-uniform laser scanning mode with adjustable illumination is proposed to realize the de bleaching of fluorescence image under the condition of point laser scanning.A weak fluorescence confocal image restoration model based on the space-time constraint is constructed based on the feature that the image structure between consecutive frames is continuous in space-time and the noise is discontinuous.Experimental results show that the proposed method can significantly improve the effective time history of confocal imaging.(4)The long-term high-resolution confocal imaging platform is constructed.The overall architecture of the platform and the specific structure of each core module are introduced.Based on the platform,the continuous slice sampling imaging,fluorescence image restoration and imaging effect under long-time observation are tested,which verifies the accuracy of the proposed method and model.In this paper,an adaptive confocal laser scanning microscopy fast imaging system is developed,which can complete the accurate image reconstruction of the collected fluorescence signal,realize the long-time,high-resolution imaging of the confocal fluorescence signal,and expand the application range of the high-speed confocal laser scanning microscopy imaging system.