| Two-photon fluorescence scanning microscopy with the advantages of high tissue penetration,long imaging depth,low optical bleaching and light-damage has become one of the most important tools in life science research.In neuroscience field,we can get useful information by labeling and observing the calcium ion to observe the regular activity of neurons.However,calcium signal in neurons changes too fast to be captured,the calcium imaging system needs to scan one time within one millisecond to obtain the information released by the calcium signal.The scanning rate of acoustical optic deflector(AOD)can be much faster than millisecond scale,with its random scanning mode,so it can meet the scanning rate requirement of above research.The laser beam with ultrashort pulse through AOD triggers dispersion and ultimately damages the imaging quality.Currently,only partial dispersion can be compensated due to its uneven spread,which limits the field of view(FOV)of neural network.In this paper,an innovative dispersion compensation method is proposed to scan wider range of neural network.To achieve full dispersion compensation,the dispersion of 2D AOD is firstly compensated by the prism,and then the relay optical path is used to compensate the residual space dispersion.Objective lens with wide FOV needs to be used when scanning wide field.The objective lens of the imaging system covers not only the scanning wavelength,but also the fluorescence wavelength,thus the objective lens needs a large FOV and wide operating wavelength range.In this paper,an objective lens is designed,which can meet the above requirements.The research contents are as follows:(1)The principle of two-photon scanning microscopy imaging system is summarized.Firstly,the principle of two-photon fluorescence,high-speed scanning technologies and methods are introduced.Then,the main structure of the two-photon scanning microscopy system,the operating principal and primary parameters of AOD are studied.Finally,the dispersion of AOD is calculated.The conclusion on the regularity of dispersion spread is obtained.The angular dispersion on the edge of FOV is 0.21 mrad/nm which resulting in imaging spot distortion of 7.9 ?m.(2)The dispersion compensation method of 2D AOD is proposed and verified by simulation.Firstly,the dispersion of the prism is introduced and the dispersion of 2D AOD is compensated by prism.The residual space dispersion is calculated and analyzed,and the conclusion of the residual spatial dispersion on the center of the field of symmetry distribution is obtained.On the edge of large scanning view,the remaining angular dispersion is 0.0627 mrad/nm which resulting in imaging spot distortion of 2.3 ?m.Secondly,the residual optical dispersion is compensated by the relay optical path composed of the diffractive lens.The dispersion compensation result is analyzed theoretically.Finally,the light path of dispersion compensation is simulated by Zemax.The compensation result of the residual dispersion is verified by Zemax,the full-field imaging spot diameter after dispersion compensation is less than 1.13 ?m and the distortion of the spot is suppressed.(3)A microscopic objective lens for two-photon scanning microscopy imaging system is designed.The working wavelength of the objective lens is 400~1100 nm,the FOV radius is 0.3 mm,the spot diameter is less than 1.1 ?m,which is smaller than the spot diameter 1.13 ?m of the imaging system.The designed objective lens meets the needs of the two photon fluorescence imaging system. |
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