Research On Two-photon Micro-imaging System Based On Adaptive Optics

Two-photon fluorescence microscopy is an important tool for the development of microscopic biological research.The technique can be used to observe the fine structure in organisms and to dynamically track the life activities of tissues,cells,nuclei,proteins and small molecules in living organisms.The fluorescence microscopy with high spatial resolution in deep tissues is a frontier issue in the current imaging field.Since biological tissues have usually non-transparent and non-uniform structures,when the excitation light and fluorescence propagate in the biological tissue,the refraction,scattering,absorption can cause obvious wavefronts distortion,which makes the imaging quality poor.Using adaptive optics technology,wavefront distortions caused by light propagation in biological tissue can be detected in real-time and corrected accurately,hence the spatial resolution and depth of image have been improved.This article mainly focuses on the two-photon microscopy system based on adaptive optics.The main research contents are as follows:1.The theory of two-photon microscopy is analyzed.The physical mechanism of two-photon microscopy,the composition of two-photon microscopy system,characteristics of images captured by system and the basic method of system optimization are analyzed in detail and and all of this are the basic theory for designing optical paths.With the analysis of the imaging characteristics,the resolution of the two-photon microscopy are improved by using adaptive optics technology to correct phase difference.2.The principle of adaptive optics is studied.The first fifteen Zernike polynomials are derived and their meanings in optical wavefront aberration are introduced.The first ten Zernike polynomials are simulated by Matlab.Though analyzing PV value and RMS,it is obtained that in adaptive optics,wavefront aberrations are usually characterized by RMS,and the smaller the RMS stands for the better the adaptive closed-loop effect.3.A two-photon microscopy system based on adaptive optics is designed.Based on the above theory,Zemax software is used to simulate for the illumination optical path.Afterwards,the optical path is analyzed by the dot-mapping analysis method,and within the entire scanning field of view,the system approaches the diffraction limit.Second,in the design of the detection light path,the fluorescence should be maximized by the detection equipment.4.A two-photon microscopy system based on adaptive optics is set up.The system is constructed in five procedures: obtaining a collimated light source,Hartmann calibration,setting up the illumination light path,adjusting the objective,and setting up the collecting light path.After replacing the femtosecond light source with a red light source,the adaptive closed-loop effect of the entire system is tested.Finally,micrographs are taken of the mouse kidney section based on the system and verify the effectiveness of the system.