高精度荧光寿命成像方法及应用

发布时间：2018-06-15 16:25

本文选题：时间相关单光子技术(TCSPC) + 荧光寿命成像　；参考：《华中科技大学》2013年博士论文

【摘要】：荧光寿命成像技术因其具有对微环境敏感、空间分辨率高、不依赖于荧光物浓度和激发光强度等特点,使它成为生物微环境测量的一种重要工具。然而,基于TCSPC的FLIM技术存在成像信噪比低、可测量最短寿命受响应函数宽度限制等问题,导致其测量精度较低。本文通过增强激发荧光效率、降低系统噪声和优化分析方法三个方面发展出高精度荧光寿命成像方法,并将该方法用于荧光鼠脑样本制备方案筛选、FRET效率准确性测量和超短荧光寿命测量的研究中。本文内容如下： (1)发展了一种简单易行荧光寿命成像系统的性能评估及优化方法。定量评估了TCSPC-FLIM的空间分辨率、成像信噪比、测量准确性等,发现成像信噪比对系统的成像精度影响最大。提出了利用降低系统噪声和增强激发荧光效率的方法两种方法来提高成像信噪比。结果表明对探测器制冷使图像信噪比提高了4倍,单光子激发比双光子激发显著增强了荧光激发效率。 (2)提出将高精度荧光寿命成像方法用于荧光鼠脑样本制备方案筛选应用中,该方法相比于荧光强度成像方法使测量更精确、更快捷。定量比较了三种常用固定剂和包埋剂对荧光特性的影响,发现利用4%多聚甲醛固定和GMA(乙二醇甲基丙烯酸酯)包埋的鼠脑脑片背景荧光最弱。该方法大大缩短了实验样本量,使实验周期更短。 (3)针对高精度荧光寿命成像技术在荧光共振能量转移研究中的应用,对我们的TCSPC-FLIM系统进行了优化,使其适用于高精度研究CFP-YFP FRET效率变化。研究表明,我们的TCSPC-FLIM系统可以测量3%的FRET效率变化,初步证明了高精度TCSPC-FLIM方法在微小FRET效率变化测量中的有效性。 (4)研究了系统极限时间分辨率与荧光信号强度之间的关系,建立了一种超高精度寿命分析方法。比较了一阶力矩法(M1)与传统的最小二乘拟合法的荧光寿命定量分析能力,发现M1方法的测量精度仅受信号强度影响,而与时间通道无关。进一步发现,M1方法在下面两种情况下明显优于拟合法：寿命为70ps-3ns；信号强度小于103个光子。最后,对超短荧光寿命样品(CdS纳米线)进行定量分析,证明了M1方法在研究短寿命样本时比拟合法更优。该方法为精确测量短寿命荧光样品或是微小荧光寿命变化方面的研究提供新的途径。
[Abstract]:Because of its sensitivity to microenvironment, high spatial resolution, no dependence on fluorescence concentration and intensity of luminescence, fluorescence lifetime imaging technology makes it an important tool for the measurement of biological microenvironment. However, the FLIM technology based on TCSPC has the problems of low imaging signal to noise ratio and the limitation of the shortest lifetime of the response function, such as the width of response function. In this paper, the high precision fluorescence lifetime imaging method is developed in this paper by enhancing the excitation fluorescence efficiency, reducing the noise of the system and optimizing the analysis method in three aspects. This method is used in the screening of fluorescent rat brain sample preparation scheme, FRET efficiency accuracy measurement and ultra short fluorescence lifetime measurement. Below:
(1) the performance evaluation and optimization method of a simple and easy fluorescence lifetime imaging system is developed. The spatial resolution, image signal to noise ratio and accuracy of TCSPC-FLIM are evaluated quantitatively, and the imaging accuracy of the imaging signal noise ratio system is found to be the most influential. Two methods to reduce the noise of the system and enhance the efficiency of the excitation fluorescence are proposed. The method is used to improve the signal to noise ratio of the imaging. The results show that the signal to noise ratio of the image is increased by 4 times for the detector refrigeration, and the fluorescence excitation efficiency is significantly enhanced by the single photon excitation than the two-photon excitation.
(2) a high precision fluorescence lifetime imaging method is proposed for the screening of fluorescent rat brain samples. Compared with the fluorescence intensity imaging method, this method makes the measurement more accurate and faster. The quantitative comparison of the effects of three common fixative and embedding agents on the fluorescence characteristics, and the use of 4% polyoxymethylene and GMA (ethylene glycol methyl C) The background fluorescence of rat brain slices embedded in enolates is the weakest. This method greatly reduces the number of experimental samples and makes the experimental cycle shorter.
(3) in view of the application of high precision fluorescence lifetime imaging technology in the study of fluorescence resonance energy transfer, our TCSPC-FLIM system is optimized so that it is suitable for high precision study of CFP-YFP FRET efficiency change. The study shows that our TCSPC-FLIM system can measure the FRET efficiency of 3%, and the high precision TCSPC-FLIM method has been proved preliminarily. The effectiveness of the measurement of small FRET efficiency changes.
(4) the relationship between the limit time resolution of the system and the intensity of the fluorescence signal is studied. A super high precision life analysis method is established. The quantitative analysis ability of the first order moment method (M1) and the traditional least square method is compared. It is found that the measurement precision of the M1 method is only influenced by the intensity of the signal, but is independent of the time channel. One step is to find that the M1 method is obviously superior to the fitting method in the following two cases: the lifetime is 70ps-3ns and the signal intensity is less than 103 photons. Finally, the quantitative analysis of the ultrashort fluorescent life sample (CdS nanowires) proves that the M1 method is more effective than the short life sample. This method is a precise measurement of short life fluorescence samples or It provides a new way for the study of micro fluorescence lifetime changes.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R310

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