Sprenger Julia U, Perera Ruwan K, Götz Konrad R, Nikolaev Viacheslav O
Emmy Noether Group of the DFG, Department of Cardiology and Pneumology, European Heart Research Insitute Göttingen, Georg August University Medical Center, Göttingen, Germany.
J Vis Exp. 2012 Aug 20(66):e4081. doi: 10.3791/4081.
Förster resonance energy transfer (FRET) microscopy continues to gain increasing interest as a technique for real-time monitoring of biochemical and signaling events in live cells and tissues. Compared to classical biochemical methods, this novel technology is characterized by high temporal and spatial resolution. FRET experiments use various genetically-encoded biosensors which can be expressed and imaged over time in situ or in vivo. Typical biosensors can either report protein-protein interactions by measuring FRET between a fluorophore-tagged pair of proteins or conformational changes in a single protein which harbors donor and acceptor fluorophores interconnected with a binding moiety for a molecule of interest. Bimolecular biosensors for protein-protein interactions include, for example, constructs designed to monitor G-protein activation in cells, while the unimolecular sensors measuring conformational changes are widely used to image second messengers such as calcium, cAMP, inositol phosphates and cGMP. Here we describe how to build a customized epifluorescence FRET imaging system from single commercially available components and how to control the whole setup using the Micro-Manager freeware. This simple but powerful instrument is designed for routine or more sophisticated FRET measurements in live cells. Acquired images are processed using self-written plug-ins to visualize changes in FRET ratio in real-time during any experiments before being stored in a graphics format compatible with the build-in ImageJ freeware used for subsequent data analysis. This low-cost system is characterized by high flexibility and can be successfully used to monitor various biochemical events and signaling molecules by a plethora of available FRET biosensors in live cells and tissues. As an example, we demonstrate how to use this imaging system to perform real-time monitoring of cAMP in live 293A cells upon stimulation with a β-adrenergic receptor agonist and blocker.
作为一种实时监测活细胞和组织中生化及信号转导事件的技术，Förster共振能量转移（FRET）显微镜越来越受到关注。与传统生化方法相比，这项新技术具有高时间和空间分辨率的特点。FRET实验使用各种基因编码的生物传感器，这些传感器可以在原位或体内随时间表达并成像。典型的生物传感器可以通过测量荧光团标记的一对蛋白质之间的FRET来报告蛋白质-蛋白质相互作用，或者报告单个蛋白质的构象变化，该蛋白质含有与感兴趣分子的结合部分相连的供体和受体荧光团。用于蛋白质-蛋白质相互作用的双分子生物传感器包括，例如，设计用于监测细胞中G蛋白激活的构建体，而测量构象变化的单分子传感器广泛用于成像第二信使，如钙、环磷酸腺苷（cAMP）、肌醇磷酸和环磷酸鸟苷（cGMP）。在这里，我们描述了如何从单个市售组件构建定制的落射荧光FRET成像系统，以及如何使用Micro-Manager免费软件控制整个装置。这个简单但功能强大的仪器专为活细胞中的常规或更复杂的FRET测量而设计。采集的图像使用自编插件进行处理，以便在任何实验期间实时可视化FRET比率的变化，然后以与用于后续数据分析的内置ImageJ免费软件兼容的图形格式存储。这个低成本系统具有高度灵活性，并且可以通过活细胞和组织中大量可用的FRET生物传感器成功用于监测各种生化事件和信号分子。例如，我们展示了如何使用这个成像系统在β-肾上腺素能受体激动剂和阻滞剂刺激后对活的293A细胞中的cAMP进行实时监测。
