荧光寿命成像技术FLIM.docVIP

WORD格式 可编辑 专业知识整理分享 生物光子学大作业 作业名称：荧光寿命成像技术FLIM 姓名：曾扬舰 学号 完成日期：2016.6.28 荧光寿命成像技术FLIM 摘要：荧光寿命显微成像技术（FLIM）技术是一种新颖的荧光成像技术，具有其他荧光成像技术无法代替的优异性能，是生物医学工程领域的研究热点。频域调制、门控探测和时间相关单光子计数是FLIM的几种主要实现方法。综述了这些计数的原理、研究现状和已取得的部分成果，比较了这三种方法的时间分辨率和成像速度等参数的优劣。宽场FLIM更适用于延时成像和实时成像。荧光偏振各向异性成像和内窥镜FLIM技术都是FLIM技术很有前景的应用方向。 Fluorescence Lifetime Imaging Microscopy(FLIM) Why lifetime Imaging? The fluorescence lifetime is the signature of a fluorescent material ;It is the exponential decay in emission after the excitation of a fluorescent material has been stopped. FLIM is a technique to map the spatial distribution of lifetimes within microscopic images and it allows measurements in living cells as well as in fixed. Because of the fact that some phenomena do affect fluorescence lifetimes, the lifetime is used to detect these phenomena leading to various applications such as; lon imaging ,oxygen imaging, probing microenvironment, and medical dlagnosis. Frequency domain method The homodyne frequency domain FLIM method requires a modulated light source and a modulated detector.In the LIFE system these are the LED and the intensified CCD camere. Both are modulated at exactly the same frequency,but with an adjustable diffrence in phase.These two parameters depend on the fluorescence lifetime of the sample and the modulation frequency and are measured to calculate the flurescence lifetime in each pixel of the image. 1、基本概念 荧光是分子吸收能量后使得其基态电子被激发到单线激发态后由第一单线激发态回到基态时所发生的辐射复合发出的光。 荧光寿命是指分子受到光脉冲激发后返回基态之前在激发平均停留的时间，处于激发态的荧光分子在从激发到基态的过程中发射荧光释放能量。荧光寿命是荧光物质固有性质，与浓度，光漂白和激发强度无关，依赖于围绕荧光物质的离子和局部环境，比如蛋白质的束缚等。影响荧光寿命的因素：荧光物质的结构；所处微环境的极性、温度、粘度，键合离子等条件有关。荧光寿命成像可以区分荧光物质浓度和淬灭效应的影响，这使得荧光寿命成像显微学成为一个有力的工具进行细胞内的定量环境和分子动力学分析。 荧光寿命成像技术有两种：时间域和频率域。 （1）时域FLIM：需要脉冲光源，所以一般在双光子的系统上比较常见，因为激光是脉冲的而且买双光子的老师一般也搭配一个FLIM。光路调节也是一个问题，提供的厂家一般是BeckerHickl和PicoQuant德国公司。这个方法比较流行，也有很