Görlitz Frederik, Kelly Douglas J, Warren Sean C, Alibhai Dominic, West Lucien, Kumar Sunil, Alexandrov Yuriy, Munro Ian, Garcia Edwin, McGinty James, Talbot Clifford, Serwa Remigiusz A, Thinon Emmanuelle, da Paola Vincenzo, Murray Edward J, Stuhmeier Frank, Neil Mark A A, Tate Edward W, Dunsby Christopher, French Paul M W
Photonics Group, Department of Physics, Imperial College London;
Photonics Group, Department of Physics, Imperial College London.
J Vis Exp. 2017 Jan 18(119):55119. doi: 10.3791/55119.
We present an open source high content analysis instrument utilizing automated fluorescence lifetime imaging (FLIM) for assaying protein interactions using Förster resonance energy transfer (FRET) based readouts of fixed or live cells in multiwell plates. This provides a means to screen for cell signaling processes read out using intramolecular FRET biosensors or intermolecular FRET of protein interactions such as oligomerization or heterodimerization, which can be used to identify binding partners. We describe here the functionality of this automated multiwell plate FLIM instrumentation and present exemplar data from our studies of HIV Gag protein oligomerization and a time course of a FRET biosensor in live cells. A detailed description of the practical implementation is then provided with reference to a list of hardware components and a description of the open source data acquisition software written in µManager. The application of FLIMfit, an open source MATLAB-based client for the OMERO platform, to analyze arrays of multiwell plate FLIM data is also presented. The protocols for imaging fixed and live cells are outlined and a demonstration of an automated multiwell plate FLIM experiment using cells expressing fluorescent protein-based FRET constructs is presented. This is complemented by a walk-through of the data analysis for this specific FLIM FRET data set.
我们展示了一种开源的高内涵分析仪器，它利用自动荧光寿命成像（FLIM），通过基于Förster共振能量转移（FRET）的读数来检测多孔板中固定或活细胞的蛋白质相互作用。这提供了一种筛选细胞信号传导过程的方法，该过程通过分子内FRET生物传感器或蛋白质相互作用（如寡聚化或异源二聚化）的分子间FRET读出，可用于识别结合伴侣。我们在此描述了这种自动化多孔板FLIM仪器的功能，并展示了我们对HIV Gag蛋白寡聚化的研究以及活细胞中FRET生物传感器的时间进程的示例数据。然后参考硬件组件列表和用µManager编写的开源数据采集软件的描述，提供了实际操作的详细说明。还介绍了FLIMfit（一种基于MATLAB的OMERO平台开源客户端）在分析多孔板FLIM数据阵列中的应用。概述了固定细胞和活细胞成像的协议，并展示了使用表达基于荧光蛋白的FRET构建体的细胞进行自动化多孔板FLIM实验。针对这个特定的FLIM FRET数据集的数据分析过程也进行了详细介绍。
