Research On Wavefrount Correction Of Two Photo Microscopy Based On Adaptive Optics

Two-photon microscopy techniques have special features that make them less sensitive to scattering and thus are well suitable for high-resolution 3D imaging of biological tissues.However,as the imaging depth increases,the refractive index mismatch will cause spherical aberration(SA)which seriously degrades the fluorescence intensity and system resolution.The SA limits the application of two-photon microscopy in thick tissue.In this thesis,we focus on the characteristics of the SA and propose two methods to correct the aberration in different situations.The experimental results verify the effectiveness of the methods.First of all,we introduce the background and basic concept of two-photon microscopy(TPM),wavefront aberration and adaptive optics.Because the wavefront aberrations significantly reduce the fluorescence intensity and three-dimensional resolution of two-photon microscopy,we propose the idea of using adaptive optics to correct the aberration.From the basic theory,we analyze the two-photon imaging characteristics and get the point spread function of two-photon microscopy with spherical aberration based on the Wolf vector diffraction formula.Due to those theories,an adaptive two-photon microscopy imaging system for spherical aberration correction is designed,after that the corresponding hardware and software system are built which will be described in this dissertation.Then we found the refractive index mismatch between objective medium and optical clearing agent can cause spherical aberration which degrades the fluorescence intensity and axial resolution.To solve this problem,we analyze the effect of SA at the focus,and built a SA compensation model based on objective characteristics(numerical aperture(NA),immersion media),the imaging depth and the sample refractive index.Then,we correcte the SA by incorporating a spatial light modulator(SLM)into TPM.The TPM images of fluorescent beads phantom and optical clearing brain tissues show considerable improvement of fluorescence intensity and axial resolution.The proposed correction process is simple and fast since it does not require repeated imaging.More importantly,it is suitable for different objectives and optical clearing agents.Further,we discuss the phenomena of multilayer refractive index mismatch.By analyzing the characteristics of aberration in this case,we propose a new AO correction method which combines the model-based and segment-based algorithm.We compared our method with other three popular methods for spherical aberration correction using simulation.The results show that our method can correct the circular symmetric aberration more accurately which has 8 times higher efficiency than others.Further experimental results show that the ring segment method can improve the fluorescence intensity of beads at the depth of 250 ?m by two times.The imaging of the brain tissue of the Thy1-GFP transgenic mice treated with the tissue light transparent agent shows that the method can effectively correct the spherical aberration,improve the fluorescence signal and reduce photodamage and photobleaching in the two-photon microscopy.