The Study On The Fluorescence Emission Difference Microscopy With Pupil Filters

This thesis focuses on the theory of fluorescence emission difference(FED)Microscopy.Based on confocal microscope,FED microscopy realizes super-resolution imaging by taking advantages of the difference between two differently modulated confocal images achieved by scanning with solid focal spot and hollow focal spot.The easily accessible annular pupil is proposed to enhance the resolution of the FED imaging.Meanwhile,diaphragm with higher numerical aperture is permitted for the beam of solid focal spot.Under the dual effects of these two aspects,the spatial resolution of the FED microscopy is increased.A super-resolution of 0.18? with ? being the excitation wavelength is achieved,which improves the imaging resolution is improved by 14.3%.Because the azimuthal polarization beam can produce a smaller hollow focal spot,azimuthal polarization beam and a three-zone amplitude-type pupil filter are used,where a leaky filter further reduces the size of the hole of the azimuthal polarized beam.By designing appropriate filter parameters,the resolution is further increased to 0.1700 ?,which is 8.6% higher than that of the normal FED with the azimuthal polarization without filter.The research shows that applying appropriate pupil filters can improve the imaging resolution of the FED microscopy and provide guidance for the practical experiments to a certain extent.FED microscopy is a super-resolution imaging technology built on confocal laser scanning imaging microscope.In this thesis,the confocal laser scanning microscope system is experimentally studied.In confocal microscope,a conjugate system between the point illuminating pinhole and detecting through the pinhole is used,which greatly eliminates the signals from the out-of-focus plane and sectional imaging of objects can be achieved.Confocal microscopy is widely used in the biomedical field because of its high-resolution and three-dimensional imaging of living cells.We investigate the basic principle and development status of confocal microscopy.A confocal laser scanning imaging system is built which includes the modules of excitation system,detection system and scanning system.By using the home-built software control system,two-dimensional imaging and three-dimensional imaging are realized with various samples.The data shows that the lateral imaging resolution is about 448 nm.