Research On Light Sheet Microscopy And Instrument

During the development of fluorescence microscopy,the main focus of the research has always been changing.Initially,the resolution is what everyone concerns.Then,the imaging depth takes its place.In recent years,scientists care more and more about how to improve the microscope system through the perspective of biological application.Light sheet microscopy stands out because of its property of fast imaging and low damage on the sample and is gradually accepted as the main approach of observing long-term biological process.However,there exists a major problem in light sheet microscopy that high axial resolution and large field of view cannot be fulfilled at the same time.Based on the above background,this thesis has introduced several research on light sheet microscopy,as follows.(1)A light sheet microscopy system with large field of view and high axial resolution achieved by the combination of Bessel beam excitation and rolling shutter mode has been built.The work includes hardware programming and biological samples imaging.Eventually,the system has achieved field of view of 800 μm and axial resolution beyond 3 μm.(2)A lattice light sheet microscope with extremely high spatial resolution and small field of view has been built.The setup is redesigned to make the system more stable and easier for optical alignment through double-deck light path,inspection cameras and socket design.Besides,hardware control has been done.Finally,the design target has been achieved and the stability of the whole system has been largely improved.(3)Research on the improvement of axial resolution in different kinds of light sheet microscope has been made.For conventional light sheet microscope,on one hand,saturation effect and mode modulation are proposed to use to improve the axial resolution,which is verified by the results.On the other hand,lattice light sheet difference microscopy is proposed,which uses the idea in fluorescence emission difference microscopy to combine the results under conventional optical lattice and our newly proposed one.It is also shown that the axial resolution of lattice light sheet microscopy has been improved from 370 nm to 300 nm through lattice light sheet difference microscopy.The applicability of lattice light sheet difference microscopy has also been proved from aberration,polarization and subtraction factor.