Multiplexed FRET to Image Multiple Signaling Events in Live Cells
Multiplexed FRET to Image Multiple Signaling Events in Live Cells
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DOI：
10.1529/biophysj.108.139204
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发表时间：
2008-11-15
影响因子：
3.4
通讯作者：
French, Paul M. W.
中科院分区：
文献类型：
--
作者：
Grant, David M.;Zhang, Wei;French, Paul M. W.
我们报告什么，我们的知识是一种新的方法，同时成像的两个不同的福斯特共振能量转移（FRET）传感器在同一个细胞中，最小的光谱串扰。以前的方法的基础上的光谱比率成像的两个FRET传感器已被限制的第二个FRET对和光谱串扰时，测量在四个光谱窗口中产生的适当明亮的受体的可用性。与光谱比率成像相反，荧光寿命成像（FLIM）仅需要测量供体荧光，并且与受体的发射无关。通过将新型红移的TagRFP/mPlumFRET对的FLIM-FRET与ECFP/Venus对的光谱比率成像相结合，我们因此能够最大化我们选择的荧光团之间的光谱分离，同时克服远红受体mPlum的低量子产率。使用这种技术，我们可以读出一个TagRFP/mPlum分子间FRET传感器，用于报告表皮生长因子刺激后活细胞中的小Ras GTP酶激活，以及ECFP/Venus Cameleon FRET传感器，用于监测相同细胞内的钙瞬变。因此，ECFP/Venus的光谱比率成像和TagRFP/mPlum的高速FLIM-FRET的组合可以增加可用的光谱带宽，并提供同一细胞内多个FRET传感器的稳健成像。此外，由于FLIM不需要相等的化学计量的供体和受体，这种方法可以用于报告单分子FRET生物传感器和蛋白质-蛋白质相互作用与同一细胞。
We report what to our knowledge is a novel approach for simultaneous imaging of two different Forster resonance energy transfer (FRET) sensors in the same cell with minimal spectral cross talk. Previous methods based on spectral ratiometric imaging of the two FRET sensors have been limited by the availability of suitably bright acceptors for the second FRET pair and the spectral cross talk incurred when measuring in four spectral windows. In contrast to spectral ratiometric imaging, fluorescence lifetime imaging (FLIM) requires measurement of the donor fluorescence only and is independent of emission from the acceptor. By combining FLIM-FRET of the novel red-shifted TagRFP/mPlumFRET pair with spectral ratiometric imaging of an ECFP/Venus pair we were thus able to maximize the spectral separation between our chosen fluorophores while at the same time overcoming the low quantum yield of the far red acceptor mPlum. Using this technique, we could read out a TagRFP/mPlum intermolecular FRET sensor for reporting on small Ras GTP-ase activation in live cells after epidermal growth factor stimulation and an ECFP/Venus Cameleon FRET sensor for monitoring calcium transients within the same cells. The combination of spectral ratiometric imaging of ECFP/Venus and high-speed FLIM-FRET of TagRFP/mPlum can thus increase the spectral bandwidth available and provide robust imaging of multiple FRET sensors within the same cell. Furthermore, since FLIM does not require equal stoichiometries of donor and acceptor, this approach can be used to report on both unimolecular FRET biosensors and protein-protein interactions with the same cell.