3-D And Parallelized Fluorescence Difference Microscopy And Instrument

Owing to optical diffraction limit,the resolution of the regular microscopy is around 230nm,which is unable to meet the needs of nano-scale observations and study.Breaking the diffraction limits means resolving the two points within the diffraction limit,rather than magnifying the nanoscale object.Although electron microscopy,scanning tunneling microscopy,atomic force microscopy and other related technologies can achieve nano-scale resolution,these techniques either use short wavelengths or are based on near-field detection methods,which do not break the diffraction limit in a real sense.Furthermore,these techniques require some conditions,such as vacuum environment,high-irradiation or conductive samples.Therefore,non-invasive imaging or depth imaging cannot be achieved by those techniques.In summary,breaking the diffraction limit is of great significance for the further development of nano-optics and life sciences.In recent years,with the continuous development of fluorescence super-resolution microscopy technology,researchers and engineers focus on not merely the breaking of the diffraction limit,but also the comprehensive performance improvement of microscopy technology and related instruments,which includes the imaging speed,multi-dimension super-resolution imaging,resistance to bleaching and so on.This dissertation is based on vector light field theory and point spread function engineering.Firstly,the regular fluorescence emission difference(FED)microscopy is expanded to three dimension imaging,in order to break the axial diffraction limit and improve the axial resolution.Secondly,a new subtraction method is proposed to enhance the image quality of FED microcopy,which can also improve the bleaching problem in saturation FED to some degree.Thirdly,a parallel FED method is proposed based on wide field excitation and detection.Finally,comparative investigation is conducted on several methods based on parallel detection.The main content and innovation points of this dissertation includes:In Chapter 1,the background of this dissertation is described,which includes the development history of microscopes,the development of super-resolution technology,and the condition of related commercial instruments,especially the domestic condition.In Chapter 2,the basic research of this dissertation is described,which includes confocal theory,commercial instrument prototype of four-color confocal microscopy and fluorescence emission difference microscope(FED).In Chapter 3,the 3D-FED microscopy theory and instrument prototype are demonstrated,which is the first innovation point of this dissertation.In Chapter 4,a method to improve FED imaging quality is proposed by using conjugated vortex phase mask,which is the second innovation point of this dissertation.In Chapter 5,a wide field FED microscopy is proposed based an optical lattice and using CCD detection,which is the third innovation point of this dissertation.In Chapter 6,a comparative investigation on several microscopy methods based on parallel detection is conducted,as well as their advantages and limitations.In Chapter 7,a summary is made,and the outlook of this project is also briefly discussed.