Open Source High Content Analysis Utilizing Automated Fluorescence Lifetime Imaging Microscopy.
Open Source High Content Analysis Utilizing Automated Fluorescence Lifetime Imaging Microscopy.
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DOI：
10.3791/55119
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发表时间：
2017-01-18
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影响因子：
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通讯作者：
French PM
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文献类型：
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作者：
Görlitz F;Kelly DJ;Warren SC;Alibhai D;West L;Kumar S;Alexandrov Y;Munro I;Garcia E;McGinty J;Talbot C;Serwa RA;Thinon E;da Paola V;Murray EJ;Stuhmeier F;Neil MA;Tate EW;Dunsby C;French PM
我们提出了一种开源的高含量分析仪器，利用自动荧光寿命成像（FLIM），使用基于Förster共振能量转移（FRET）的多孔板中固定或活细胞的读数来测定蛋白质相互作用。这提供了一种筛选使用分子内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读出的细胞信号传导过程的方法，其可用于鉴定结合伴侣。我们在这里描述了这种自动化多孔板FLIM仪器的功能，并提出了来自我们的研究的HIV Gag蛋白寡聚化和FRET生物传感器在活细胞中的时间过程的示例数据。然后，参考硬件组件列表和使用µManager编写的开源数据采集软件的描述，详细描述了实际实施。FLIMfit，一个开源的基于MATLAB的客户端的OMERO平台，分析阵列的多孔板FLIM数据的应用程序也被提出。成像固定和活细胞的协议概述和演示的自动化多孔板FLIM实验使用细胞表达荧光蛋白为基础的FRET结构。这是补充了一个步行通过这个特定的FLIM FRET数据集的数据分析。
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.