Rapid global fitting of large fluorescence lifetime imaging microscopy datasets.
Rapid global fitting of large fluorescence lifetime imaging microscopy datasets.
复制标题
DOI：
10.1371/journal.pone.0070687
复制
发表时间：
2013
期刊：
影响因子：
3.7
通讯作者：
French PM
中科院分区：
文献类型：
--
作者：
Warren SC;Margineanu A;Alibhai D;Kelly DJ;Talbot C;Alexandrov Y;Munro I;Katan M;Dunsby C;French PM
荧光寿命成像（FLIM）被广泛应用于从荧光信号中获得定量信息，特别是使用Förster共振能量转移（FRET）测量来绘制蛋白质-蛋白质相互作用等。提取FRET效率或群体分数通常需要将数据拟合到复杂的荧光衰减模型，但是这样的实验通常是光子受限的，特别是对于活细胞或体内成像，并且当在逐像素的基础上分析数据时，这导致不可接受的误差。然而，寿命和群体分数可以使用全局分析来更鲁棒地提取，以在寿命分量跨图像（数据集）不变的假设下同时将图像或数据集中的所有像素的荧光衰减数据拟合到多指数模型。这种方法通常被认为是非常缓慢和/或计算昂贵，但我们在这里提出了一个计算效率高的全球分析算法的时间相关单光子计数（TCSPC）或时间门控FLIM数据的分析的基础上可变投影。它有效地利用了计算机处理器和存储器资源，在标准个人计算机上分析时间序列和多孔板数据集以及数百张FLIM图像所需的时间不到一分钟。这种寿命分析考虑了重复激发，包括由早期脉冲激发的荧光光子对拟合的贡献，并且能够适应时变背景和仪器响应函数。我们证明，这种全球性的方法使我们能够很容易地适应时间分辨的荧光数据复杂的模型，包括一个四指数模型的FRET系统，其中的FRET效率的两个物种的双指数供体的链接，和偏振分辨的寿命数据，其中的荧光强度和双指数各向异性衰减模型应用于活细胞的同源FRET数据的分析。实现该算法的软件包FLIMfit可通过开放显微镜环境在开源许可下获得。
Fluorescence lifetime imaging (FLIM) is widely applied to obtain quantitative information from fluorescence signals, particularly using Förster Resonant Energy Transfer (FRET) measurements to map, for example, protein-protein interactions. Extracting FRET efficiencies or population fractions typically entails fitting data to complex fluorescence decay models but such experiments are frequently photon constrained, particularly for live cell or in vivo imaging, and this leads to unacceptable errors when analysing data on a pixel-wise basis. Lifetimes and population fractions may, however, be more robustly extracted using global analysis to simultaneously fit the fluorescence decay data of all pixels in an image or dataset to a multi-exponential model under the assumption that the lifetime components are invariant across the image (dataset). This approach is often considered to be prohibitively slow and/or computationally expensive but we present here a computationally efficient global analysis algorithm for the analysis of time-correlated single photon counting (TCSPC) or time-gated FLIM data based on variable projection. It makes efficient use of both computer processor and memory resources, requiring less than a minute to analyse time series and multiwell plate datasets with hundreds of FLIM images on standard personal computers. This lifetime analysis takes account of repetitive excitation, including fluorescence photons excited by earlier pulses contributing to the fit, and is able to accommodate time-varying backgrounds and instrument response functions. We demonstrate that this global approach allows us to readily fit time-resolved fluorescence data to complex models including a four-exponential model of a FRET system, for which the FRET efficiencies of the two species of a bi-exponential donor are linked, and polarisation-resolved lifetime data, where a fluorescence intensity and bi-exponential anisotropy decay model is applied to the analysis of live cell homo-FRET data. A software package implementing this algorithm, FLIMfit, is available under an open source licence through the Open Microscopy Environment.
登录
查看更多内容
影响因子：
3.3
作者：
Demachy, I;Ridard, J;Lévy, B
通讯作者：
Lévy, B
影响因子：
3.5
作者：
Chang, Ching-Wei;Mycek, Mary-Ann
通讯作者：
Mycek, Mary-Ann
影响因子：
4.4
作者：
BERBERANSANTOS, MN;VALEUR, B
通讯作者：
VALEUR, B
影响因子：
--
作者：
CRUTZEN, M;AMELOOT, M;DESCHRYVER, FC
通讯作者：
DESCHRYVER, FC
影响因子：
6.5
作者：
Christensen, T. M.;Hurn, A. S.;Lindsay, K. A.
通讯作者：
Lindsay, K. A.