摘要摘要双光子荧光显微成像技术是开展微观生命科学研究的重要手段和工具，使用该技术可以观察生物体内的精细结构、动态追踪生物体内组织、细胞、细胞核、蛋白、小分子等不同尺度的生命活动过程。其中，研究深层组织高时空分辨率荧光显微成像技术，是当前成像领域一个前沿问题。由于生物组织通常是非透明、非均匀结构，激发光和发射荧光在生物组织内传播时均会由于折射、散射、吸收等作用使得光波波前发生明显畸变，使得成像质量较差。借助自适应光学技术可对上述经由生物组织传播产生的波前畸变进行实时探测和精确校正，从而提升激发光照明和发射荧光成像的空间分辨率和深度。本文主要围绕基于自适应光学的双光子显微成像系统进行研究，主要研究内容包括如下：1,研究了双光子显微成像技术理论，对双光子显微成像的物理机制、双光子显微成像系统的组成、成像特点以及系统优化的基本方法进行详细分析，为后续光路的设计提供理论依据。另外，通过对成像特点的分析，利用自适应光学技术进行像差校正，从而提高双光子显微成像的分辨率以及探测深度。2,研究了自适应光学的原理，推导出了前十五个Zernike多项式以及在光学波前误差中的含义，并且用matlab对前十个Zernike多项式进行仿真。当利用Zernike多项式定义波前时，通过对PV值和RMS分析，得出在自适应光学中，通常用RMS来表征波前像差，RMS越小，自适应闭环效果越高。3,设计了一套基于自适应光学的双光子显微成像系统，在上面的理论基础上，首先对照明光路用光学仿真软件Zemax进行仿真。之后用点列图分析方法对光路进行分析，在整个扫描视场内，均能达到衍射极限。其次，在进行探测光路的设计时，要使得荧光最大限度的被探测设备收集。另外，分光源模块、自适应模块、二维扫描模块、缩扩束模块以及荧光收集模块分别介绍了系统中所用的关键器件以及性能参数。4,搭建了一套基于自适应光学的双光子显微成像系统，从获得红光准直光源、哈特曼标定、照明光路搭建、显微物镜调节以及收集光路搭建五个部分介绍光路搭建的过程。之后对用红光光源替代飞秒光源，用普通透镜和20X显微物镜来验证整个系统的自适应闭环效果。最后，对老鼠肾脏切片荧光标本进行显微成像。关键词：双光子显微成像技术，自适应光学，荧光成像，高分辨率ABSTRACTABSTRACTTwo-photon fluorescence microscopy imaging technology is an important meansand tool for conducting microscopic life science research.Using this technology,youcan observe the fine structure in vivo,and dynamically track biological activities atdifferent scales such as internal tissues,cells,nuclei,proteins,and small molecules.Among them,the study of high temporal and spatial resolution fluorescence microscopyimaging technology in deep tissues is a frontier issue in the current imaging field.Sinceliving biological tissues are usually non-transparent,non-uniform,and anisotropiccomplex three-dimensional structures,both excitation light and emitted fluorescent lightwill undergo significant distortion due to refraction,scattering,absorption,etc.in thebiological tissue.Poor quality.With the aid of adaptive optics,real-time detection andaccurate correction of wavefront distortions generated by biological tissue propagationas described above can be performed,thereby improving spatial resolution and depth ofexcitation light illumination and fluorescence imaging.This article mainly focuses onthe two-photon microscopy imaging system based on adaptive optics.The mainresearch contents include the following:1.The theory of two-photon microscopy imaging technology was studied.Thephysical mechanism of two-photon microscopy,the composition of two-photonmicroscopy imaging system,imaging characteristics,and the basic method of systemoptimization were analyzed in detail to provide a theoretical basis for the design ofsubsequent light paths.In addition,through the analysis of the imaging characteristics,the aberration correction using adaptive optics technology to improve the resolution anddepth of the two-photon microscopy imaging.2.The principle of adaptive optics is studied.The first fifteen Zernike polynomialsand their meanings in optical wavefront errors are derived.The first ten Zernikepolynomials are simulated by matlab.When Zernike polynomials are used to define thewavefront,the PV value and RMS analysis show that in adaptive optics,wavefrontaberrations are usually characterized by RMS.The smaller the RMS,the higher theadaptive closed-loop effect.3.A two-photon microscopy imaging system based on adaptive optics wasdesigned.Based on the above theory,the optical simulation software Zemax was firstsimulated for the illumination light path.Afterwards,the optical path was analyzed by