The Application Of The Hollow Beams In The Microscopy Systems

In recent years,hollow beams with a center of zero light intensity are often used in microscope systems to achieve super-resolution imaging or improve imaging quality.For example,in a commonly used super-resolution technique of stimulated emission depletion microscopy,a ring-shaped hollow focal spot is used to deplete the fluorescence around the focal spot and a fluorescent spot smaller than the diffraction limit is obtained.Meanwhile,hollow beams have also been used to improve the imaging quality of Bessel light-sheet fluorescence microscopy.This thesis focuses on the application of hollow beams in microscopic imaging systems,where the theoretical simulation analysis of hollow focal spots are studied,the use of hollow Bessel beams via half-circle half-wavelength retardation phase plate to improve the imaging quality of Bessel light sheet fluorescence microscopes is researched,and especially,the hollow focal spot is experimentally studied.The main work of this paper is as follows:(1)Theoretical study of the hollow focal spots.Based on the vector theory of the focus imaged by the high numerical aperture objective,the light intensity distributions of the hollow focal spot created by inserting a vortex phase plate,a central half-wavelen the plate or a semi-circular half-wavelength mask in the beams with different polarizations are studied,where four polarized beams like x-polarized light,y-polarized light,left-handed circularly polarized light,or right-handed circularly polarized light are used.(2)An experimental study of hollow focal spots.In this paper,a scanning microscope system is studied,where the software and hardware systems are built and the point scanning imaging is achieved.Based on the system,the transverse and axial intensity distribution of the hollow focal spot is analyzed by using the vortex phase plate and a quarter wave plate.(3)The study of application of the hollow beam to obtain high-contrast imaging of Bessel light-sheet fluorescence microscope.Due to the advantages of high axial resolution and wide field of view imaging,Bessel beams work well as an important excitation beams in the Bessel light-sheet fluorescence microscopy.However,fluorescence excitation caused by the side lobe of the Bessel beam reduces the signal-to-noise ratio of the image.To address this problem,a hollow Bessel beam via half-circle half-wavelength retardation phase plate is created and is used to remove the side lobes by the method of differential imaging in the Bessel light-sheet fluorescence microscopy.With this method,the images achieved by a solid Bessel beam and a hollow beam with a similar distribution of the side lobes are obtained.By the subtraction of these two images,the side lobes are eliminated.Therefore,the imaging quality is enhanced with both the signal-to-noise ratio and the contrast of the image are improved.