运动粒子荧光寿命追踪技术研究

发布时间：2018-07-05 00:25

本文选题：荧光寿命 + 时间相关单光子计数　；参考：《深圳大学》2017年硕士论文

【摘要】：荧光显微成像技术可通过探测荧光信号的强度、偏振、光谱、寿命等信息来获取被标记物的结构和功能信息。由于荧光分子的荧光寿命对激发态分子与周围环境的相互作用和能量转移非常敏感,且通常不受探针浓度、激发光强度、光漂白等因素的影响,因此对样品中荧光分子的荧光寿命进行探测和成像的荧光寿命显微成像(Fluorescence Lifetime Imaging Microscopy,FLIM)技术可用于定量测量荧光探针所处微环境中的许多生物物理和生物化学参量,如各种离子浓度、pH值、氧含量、溶液疏水性及猝灭剂分布等。故FLIM技术在生物学和微生物学研究中有着广泛的应用。时间相关单光子计数(Time-Correlated Single Photon Counting,TCSPC)技术是目前FLIM中最常用的荧光寿命探测方法。TCSPC-FLIM技术采用脉冲激光激发样品,探测并记录样品每次被激发后产生的荧光光子到达探测器的时间,通过多次重复累积后得到每个像素荧光光子数随到达时间的分布,从而利用荧光强度衰减曲线拟合计算样品荧光寿命及其分布。这种方法具有信噪比高、时间分辨率高、灵敏度高且动态范围广等特点,因此成为应用最广泛的FLIM技术。然而,传统的TCSPC-FLIM通常采用振镜作为扫描模块,由于振镜在运动过程中存在机械惯性,且扫描方式不够灵活,一定程度上制约了成像速度,使TCSPC-FLIM尚不能很好地实现对活细胞内的一些快速变化过程进行实时记录,更无法实现对细胞内运动粒子的追踪与动态寿命测量。实际上,当研究的对象是运动粒子时,激光扫描的范围应当尽量小且扫描位置需要跟随粒子的运动而变化,才能实现对该粒子的追踪和动态寿命探测,这就需要一种更加灵活且稳定的扫描方法。为此,本论文采用基于声光偏转器(Acousto-Optic Deflector,AOD)代替振镜作为扫描模块的AOD-FLIM技术,并在此基础上研究运动粒子荧光寿命追踪技术。为了实现粒子追踪荧光寿命探测,本论文首先根据AOD-FLIM技术原理搭建了AOD-FLIM系统,然后引入了宽场探测模块、粒子追踪算法和反馈控制机制,并通过LabVIEW图形化编程语言将分别编写的宽场探测控制程序、质心定位算法、反馈控制程序和AOD寻址扫描控制程序等进行整合,结合TCSPC探测和图像数据分析,构建了一套粒子追踪荧光寿命动态探测系统。利用该系统可以同时获得粒子的运动轨迹和动态寿命信息,为研究细胞内运动的生物大分子与其周围环境或结构的相互作用等细胞生物学问题提供了一种新的研究手段。本论文的主要工作如下:1.根据AOD-FLIM技术原理搭建了一套可对任意感兴趣区域进行快速寻址扫描的双光子AOD-FLIM系统,通过对比分析单路同步和三路同步实验结果,为运动粒子荧光寿命追踪优化了同步方式,并通过荧光珠标定实验,验证了系统的可靠性以及采集运动粒子寿命以反映运动轨迹微环境变化的可行性。2.通过在AOD-FLIM系统中引入宽场探测模块、粒子追踪算法和反馈控制机制,提出并实现了一种运动单粒子荧光寿命快速获取技术,利用LabVIEW编程整合了宽场探测、粒子定位、反馈控制、寻址扫描、同步触发等程序模块,从而实现了对运动粒子的追踪扫描,并通过与TCSPC荧光寿命探测和数据处理模块的结合同时获取了其运动轨迹和动态寿命信息。3.以在纯甘油中做布朗运动的荧光珠为例进行了运动粒子追踪荧光寿命探测的验证实验,重构出同时反映粒子运动轨迹和动态寿命变化的寿命轨迹图,验证系统具有追踪记录布朗运动粒子寿命的能力。本论文的创新点如下:1.提出并实现了一种结合AOD寻址扫描、TCSPC寿命探测、宽场探测、单粒子追踪和反馈控制机制对运动粒子进行追踪扫描并获取其动态荧光寿命信息的方法;2.提出基于运动粒子追踪和实时荧光寿命探测的微环境测量方法,有望为特定的细胞生物学问题提供一种新的研究手段。
[Abstract]:Fluorescence microscopy can obtain the structural and functional information of the labeled materials by detecting the intensity, polarization, spectrum, and life of the fluorescence signals. The fluorescence lifetime of the fluorescent molecules is very sensitive to the interaction and energy transfer between the excited molecules and the surrounding environment, and is usually free from the concentration of the probe, the excitation of light intensity, and the photobleaching. The Fluorescence Lifetime Imaging Microscopy (FLIM) technology can be used to quantitatively measure many biophysical and biochemical parameters in the microenvironment of the fluorescent probe, such as the concentration of various ions, pH values, and oxygen content, as the fluorescence lifetime of the fluorescent molecules in the sample is detected and the imaging of the fluorescence lifetime microscopy (FLIM) technology. FLIM technology has been widely used in biological and microbiological research. Time related single photon counting (Time-Correlated Single Photon Counting, TCSPC) is the most commonly used method of fluorescence lifetime detection in FLIM.TCSPC-FLIM technology, which uses pulse laser to stimulate samples, detect and remember. The fluorescence photons produced after each excitation of the sample arrive at the time of the detector, and the distribution of the number of photons of each pixel with the arrival time is obtained by repeated cumulation, and the fluorescence lifetime and distribution of the sample are calculated by the fluorescence intensity attenuation curve. This method has high signal to noise ratio, high time resolution and high sensitivity. Moreover, it is the most widely used FLIM technology. However, the traditional TCSPC-FLIM usually uses the vibrating mirror as a scanning module. Because of the mechanical inertia of the vibrating mirror during the motion process, and the scanning mode is not flexible enough, the imaging speed is restricted to a certain extent, so that the TCSPC-FLIM can not be well realized in the living cell. In fact, when the object of the study is moving particles, the range of the laser scanning should be as small as possible and the scanning position should be changed to follow the motion of the particle, in order to achieve the tracking and dynamic life of the particle. Detection, this requires a more flexible and stable scanning method. For this purpose, this paper uses the Acousto-Optic Deflector (AOD) instead of the vibrating mirror as the scanning module's AOD-FLIM technology. On this basis, we study the motion particle fluorescence lifetime tracking technology. In order to realize the particle tracking fluorescence lifetime detection, this paper first is the first paper. First, the AOD-FLIM system is built according to the AOD-FLIM technology principle, and the wide field detection module, particle tracking algorithm and feedback control mechanism are introduced, and the wide field detection control program, the centroid positioning algorithm, the feedback control program and the AOD addressable scanning control program are integrated through the LabVIEW graphical programming language, and the TCS is integrated with the TCS. PC detection and image data analysis are used to construct a set of particle tracking fluorescence lifetime dynamic detection system. Using this system, the motion trajectory and dynamic life information of particles can be obtained at the same time. A new study is provided for the study of cell biological problems such as the interaction between the biological macromolecules in cell movement and their surrounding environment or structure. The main work of this paper is as follows: 1. based on the principle of AOD-FLIM technology, a set of two photon AOD-FLIM systems for fast addressing scanning in any region of interest is built. By comparing and analyzing the results of single and three road synchronization experiments, the synchronization method is optimized for the tracing of the fluorescence lifetime of the moving particles, and the real time calibration is made by the fluorescent beads. The reliability of the system and the feasibility of collecting the life of the moving particle to reflect the microenvironment change of the motion trajectory are verified..2. is introduced and realized by introducing the wide field detection module, the particle tracking algorithm and the feedback control mechanism in the AOD-FLIM system, and the integration of LabVIEW programming is implemented. The program modules such as wide field detection, particle location, feedback control, addressing scanning, synchronous trigger and so on, the tracking and scanning of the moving particles are realized, and by combining with the TCSPC fluorescence lifetime detection and data processing module, the example of the fluorescence beads of Brown movement in pure glycerin as an example is obtained by combining with the fluorescence lifetime detection and data processing module. The verification experiment of the motion particle tracking fluorescence lifetime detection is carried out. The life trajectory map which reflects the trajectory of the particle and the dynamic life change is reconstructed, and the system has the ability to track and record the life of Brown motion particles. The innovation points of this paper are as follows: 1. a kind of combination of AOD addressing scanning and TCSPC lifetime detection is proposed and realized. Wide field detection, single particle tracking and feedback control mechanism to track and scan motion particles and obtain their dynamic fluorescence lifetime information. 2. a micro environment measurement method based on moving particle tracking and real-time fluorescence lifetime detection is proposed. It is expected to provide a new research method for specific cell biology questions.
【学位授予单位】：深圳大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：O439

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