本文選題：熒光壽命 + 時(shí)間相關(guān)單光子計(jì)數(shù)��； 參考：《深圳大學(xué)》2017年碩士論文

【摘要】：熒光顯微成像技術(shù)可通過探測熒光信號的強(qiáng)度、偏振、光譜、壽命等信息來獲取被標(biāo)記物的結(jié)構(gòu)和功能信息。由于熒光分子的熒光壽命對激發(fā)態(tài)分子與周圍環(huán)境的相互作用和能量轉(zhuǎn)移非常敏感,且通常不受探針濃度、激發(fā)光強(qiáng)度、光漂白等因素的影響,因此對樣品中熒光分子的熒光壽命進(jìn)行探測和成像的熒光壽命顯微成像(Fluorescence Lifetime Imaging Microscopy,FLIM)技術(shù)可用于定量測量熒光探針?biāo)幬h(huán)境中的許多生物物理和生物化學(xué)參量,如各種離子濃度、pH值、氧含量、溶液疏水性及猝滅劑分布等。故FLIM技術(shù)在生物學(xué)和微生物學(xué)研究中有著廣泛的應(yīng)用。時(shí)間相關(guān)單光子計(jì)數(shù)(Time-Correlated Single Photon Counting,TCSPC)技術(shù)是目前FLIM中最常用的熒光壽命探測方法。TCSPC-FLIM技術(shù)采用脈沖激光激發(fā)樣品,探測并記錄樣品每次被激發(fā)后產(chǎn)生的熒光光子到達(dá)探測器的時(shí)間,通過多次重復(fù)累積后得到每個(gè)像素?zé)晒夤庾訑?shù)隨到達(dá)時(shí)間的分布,從而利用熒光強(qiáng)度衰減曲線擬合計(jì)算樣品熒光壽命及其分布。這種方法具有信噪比高、時(shí)間分辨率高、靈敏度高且動態(tài)范圍廣等特點(diǎn),因此成為應(yīng)用最廣泛的FLIM技術(shù)。然而,傳統(tǒng)的TCSPC-FLIM通常采用振鏡作為掃描模塊,由于振鏡在運(yùn)動過程中存在機(jī)械慣性,且掃描方式不夠靈活,一定程度上制約了成像速度,使TCSPC-FLIM尚不能很好地實(shí)現(xiàn)對活細(xì)胞內(nèi)的一些快速變化過程進(jìn)行實(shí)時(shí)記錄,更無法實(shí)現(xiàn)對細(xì)胞內(nèi)運(yùn)動粒子的追蹤與動態(tài)壽命測量。實(shí)際上,當(dāng)研究的對象是運(yùn)動粒子時(shí),激光掃描的范圍應(yīng)當(dāng)盡量小且掃描位置需要跟隨粒子的運(yùn)動而變化,才能實(shí)現(xiàn)對該粒子的追蹤和動態(tài)壽命探測,這就需要一種更加靈活且穩(wěn)定的掃描方法。為此,本論文采用基于聲光偏轉(zhuǎn)器(Acousto-Optic Deflector,AOD)代替振鏡作為掃描模塊的AOD-FLIM技術(shù),并在此基礎(chǔ)上研究運(yùn)動粒子熒光壽命追蹤技術(shù)。為了實(shí)現(xiàn)粒子追蹤熒光壽命探測,本論文首先根據(jù)AOD-FLIM技術(shù)原理搭建了AOD-FLIM系統(tǒng),然后引入了寬場探測模塊、粒子追蹤算法和反饋控制機(jī)制,并通過LabVIEW圖形化編程語言將分別編寫的寬場探測控制程序、質(zhì)心定位算法、反饋控制程序和AOD尋址掃描控制程序等進(jìn)行整合,結(jié)合TCSPC探測和圖像數(shù)據(jù)分析,構(gòu)建了一套粒子追蹤熒光壽命動態(tài)探測系統(tǒng)。利用該系統(tǒng)可以同時(shí)獲得粒子的運(yùn)動軌跡和動態(tài)壽命信息,為研究細(xì)胞內(nèi)運(yùn)動的生物大分子與其周圍環(huán)境或結(jié)構(gòu)的相互作用等細(xì)胞生物學(xué)問題提供了一種新的研究手段。本論文的主要工作如下:1.根據(jù)AOD-FLIM技術(shù)原理搭建了一套可對任意感興趣區(qū)域進(jìn)行快速尋址掃描的雙光子AOD-FLIM系統(tǒng),通過對比分析單路同步和三路同步實(shí)驗(yàn)結(jié)果,為運(yùn)動粒子熒光壽命追蹤優(yōu)化了同步方式,并通過熒光珠標(biāo)定實(shí)驗(yàn),驗(yàn)證了系統(tǒng)的可靠性以及采集運(yùn)動粒子壽命以反映運(yùn)動軌跡微環(huán)境變化的可行性。2.通過在AOD-FLIM系統(tǒng)中引入寬場探測模塊、粒子追蹤算法和反饋控制機(jī)制,提出并實(shí)現(xiàn)了一種運(yùn)動單粒子熒光壽命快速獲取技術(shù),利用LabVIEW編程整合了寬場探測、粒子定位、反饋控制、尋址掃描、同步觸發(fā)等程序模塊,從而實(shí)現(xiàn)了對運(yùn)動粒子的追蹤掃描,并通過與TCSPC熒光壽命探測和數(shù)據(jù)處理模塊的結(jié)合同時(shí)獲取了其運(yùn)動軌跡和動態(tài)壽命信息。3.以在純甘油中做布朗運(yùn)動的熒光珠為例進(jìn)行了運(yùn)動粒子追蹤熒光壽命探測的驗(yàn)證實(shí)驗(yàn),重構(gòu)出同時(shí)反映粒子運(yùn)動軌跡和動態(tài)壽命變化的壽命軌跡圖,驗(yàn)證系統(tǒng)具有追蹤記錄布朗運(yùn)動粒子壽命的能力。本論文的創(chuàng)新點(diǎn)如下:1.提出并實(shí)現(xiàn)了一種結(jié)合AOD尋址掃描、TCSPC壽命探測、寬場探測、單粒子追蹤和反饋控制機(jī)制對運(yùn)動粒子進(jìn)行追蹤掃描并獲取其動態(tài)熒光壽命信息的方法;2.提出基于運(yùn)動粒子追蹤和實(shí)時(shí)熒光壽命探測的微環(huán)境測量方法,有望為特定的細(xì)胞生物學(xué)問題提供一種新的研究手段。
[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.
【學(xué)位授予單位】：深圳大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：O439

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 屈軍樂,牛憨笨,郭寶平;熒光壽命成象顯微技術(shù)及其應(yīng)用[J];光子學(xué)報(bào);1997年09期

，

本文編號：2098115