Simultaneous live cell imaging using dual FRET sensors with a single excitation light.
Simultaneous live cell imaging using dual FRET sensors with a single excitation light.
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DOI：
10.1371/journal.pone.0006036
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发表时间：
2009-06-24
期刊：
影响因子：
3.7
通讯作者：
Oka K
中科院分区：
文献类型：
--
作者：
Niino Y;Hotta K;Oka K
荧光蛋白之间的荧光共振能量转移（Fluorescence resonance energy transfer， FRET）是研究活细胞信号转导的有力工具，近年来，研究人员报道了单细胞内双荧光共振能量转移对的成像策略。然而，这需要在两个不同波长的激发光之间进行改变，以避免光谱重叠，从而导致具有滞后时间的顺序检测。因此，为了跟踪高运动细胞的快速信号动态或信号变化，应该需要单激发双fret方法。在这里，我们通过使用单激发光的四色成像和随后的线性解混来区分荧光蛋白报道了这一点。我们将蓝宝石/RFP与CFP/YFP结合，构建了新的FRET传感器，实现了单细胞中cAMP和cGMP的同时成像。我们证实，我们的双FRET测量的信号幅度是相当的传统的单FRET测量。最后，我们证明了在高运动性心肌细胞中监测细胞内Ca2+和cAMP。为了消除由细胞运动引起的伪影，该方法扩展了双FRET传感器对高运动性细胞样品的组合使用的适用性。
Fluorescence resonance energy transfer (FRET) between fluorescent proteins is a powerful tool for visualization of signal transduction in living cells, and recently, some strategies for imaging of dual FRET pairs in a single cell have been reported. However, these necessitate alteration of excitation light between two different wavelengths to avoid the spectral overlap, resulting in sequential detection with a lag time. Thus, to follow fast signal dynamics or signal changes in highly motile cells, a single-excitation dual-FRET method should be required. Here we reported this by using four-color imaging with a single excitation light and subsequent linear unmixing to distinguish fluorescent proteins. We constructed new FRET sensors with Sapphire/RFP to combine with CFP/YFP, and accomplished simultaneous imaging of cAMP and cGMP in single cells. We confirmed that signal amplitude of our dual FRET measurement is comparable to of conventional single FRET measurement. Finally, we demonstrated to monitor both intracellular Ca2+ and cAMP in highly motile cardiac myocytes. To cancel out artifacts caused by the movement of the cell, this method expands the applicability of the combined use of dual FRET sensors for cell samples with high motility.
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