1、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
2、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
3、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
4、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ABSTRACTTwo-photon fluorescence microscopy imaging technology is an important means and tool for conducting microscopic life science research. Using this technology, you can observe the fine structure in vivo, and dynamically track biological activities at di
5、fferent scales such as internal tissues, cells, nuclei, proteins, and small molecules. Among them, the study of high temporal and spatial resolution fluorescence microscopy imaging technology in deep tissues is a frontier issue in the current imaging field. Since living biological tissues are usuall
6、y non-transparent, non-uniform, and anisotropic complex three-dimensional structures, both excitation light and emitted fluorescent light will undergo significant distortion due to refraction, scattering, absorption, etc. in the biological tissue. Poor quality. With the aid of adaptive optics, real-
7、time detection and accurate correction of wavefront distortions generated by biological tissue propagation as described above can be performed, thereby improving spatial resolution and depth of excitation light illumination and fluorescence imaging. This article mainly focuses on the two-photon micr
8、oscopy imaging system based on adaptive optics. The main research contents include the following:1. The theory of two-photon microscopy imaging technology was studied. The physical mechanism of two-photon microscopy, the composition of two-photon microscopy imaging system, imaging characteristics, a
9、nd the basic method of system optimization were analyzed in detail to provide a theoretical basis for the design of subsequent light paths. In addition, through the analysis of the imaging characteristics, the aberration correction using adaptive optics technology to improve the resolution and depth
10、 of the two-photon microscopy imaging.2. The principle of adaptive optics is studied. The first fifteen Zernike polynomials and their meanings in optical wavefront errors are derived. The first ten Zernike polynomials are simulated by matlab. When Zernike polynomials are used to define the wavefront
11、, the PV value and RMS analysis show that in adaptive optics, wavefront aberrations are usually characterized by RMS. The smaller the RMS, the higher the adaptive closed-loop effect.3. A two-photon microscopy imaging system based on adaptive optics was designed. Based on the above theory, the optica
12、l simulation software Zemax was first simulated for the illumination light path. Afterwards, the optical path was analyzed by the dot-mapping analysis method, and the diffraction limit was reached within the entire scanning field of view. Second, in the design of the detection light path, thefluores
13、cence should be maximized by the detection equipment. In addition, the sub-light source module, the adaptive module, the two-dimensional scanning module, the condensed beam expansion module, and the fluorescence collection module respectively introduce the key components and performance parameters u
14、sed in the system.4. A two-photon microscopy imaging system based on adaptive optics was constructed. The light path was constructed in five parts: obtaining a red collimated light source, Hartmann calibration, setting up the illumination light path, adjusting the microscope objective, and collectin
15、g the light path. After replacing the femtosecond light source with a red light source, the adaptive closed-loop effect of the entire system was verified with an ordinary lens and a 20X microscope objective lens. Finally, micrographs were taken of the mouse kidney section fluorescence specimens.Keyw
16、ords: Two-photon microscopy，Adaptive Optics，Fluorescence imaging，High resolution目录目录第一章 绪论11.1 研究背景11.1.1 荧光显微成像技术11.1.2 共聚焦显微成像技术21.1.3 双光子激发荧光显微成像技术31.1.4 双光子荧光显微成像研究现状及局限41.2 自适应光学的发展与应用51.3 自适应光学在双光子显微成像中研究现状71.4 课题研究意义及论文主要安排8第二章 理论基础92.1 双光子显微成像的技术基础92.1.1双光子显微成像的物理机制92.1.2 双光子显微成像系统的基本组成132.1.3 成像特点142.1.4 系统优化的基本方法162.2 自适应光学原理182.2.1 利用泽尼克多项式来表征波阵面182.2.2 自适应光学结构212.3 本章小节23第三章