A long Stokes shift red fluorescent Ca2+ indicator protein for two-photon and ratiometric imaging.
A long Stokes shift red fluorescent Ca2+ indicator protein for two-photon and ratiometric imaging.
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DOI：
10.1038/ncomms6262
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发表时间：
2014-10-31
影响因子：
16.6
通讯作者：
Campbell RE
中科院分区：
文献类型：
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作者：
Wu J;Abdelfattah AS;Miraucourt LS;Kutsarova E;Ruangkittisakul A;Zhou H;Ballanyi K;Wicks G;Drobizhev M;Rebane A;Ruthazer ES;Campbell RE
基于红色荧光蛋白（RFPs）的钙离子（Ca 2+）指示剂的引入为细胞内Ca 2+动态的多色可视化创造了新的机会。然而，这些指示剂的一个缺点是，它们在组织对光最透明的近红外窗口（650- 1，000 nm）之外具有最佳的双光子激发。为了解决这一缺点，我们开发了一种基于长斯托克斯位移RFP的Ca 2+指示剂REX-GECO 1，其最佳双光子激发波长<1,000 nm。REX-GECO 1在480 nm或910 nm处分别通过单光子或双光子过程激发时在585 nm处发荧光。我们证明，REX-GECO 1可以被用来作为一个比率或强度Ca 2+指示器在器官型海马切片文化（单光子和双光子）和视觉系统的白化病蝌蚪（双光子）。此外，我们证明了单一激发波长双色Ca 2+和谷氨酸成像在器官型培养。
The introduction of calcium ion (Ca2+) indicators based on red fluorescent proteins (RFPs) has created new opportunities for multicolour visualization of intracellular Ca2+ dynamics. However, one drawback of these indicators is that they have optimal two-photon excitation outside the near-infrared window (650–1,000 nm) where tissue is most transparent to light. To address this shortcoming, we developed a long Stokes shift RFP-based Ca2+ indicator, REX-GECO1, with optimal two-photon excitation at <1,000 nm. REX-GECO1 fluoresces at 585 nm when excited at 480 nm or 910 nm by a one- or two-photon process, respectively. We demonstrate that REX-GECO1 can be used as either a ratiometric or intensiometric Ca2+ indicator in organotypic hippocampal slice cultures (one- and two-photon) and the visual system of albino tadpoles (two-photon). Furthermore, we demonstrate single excitation wavelength two-colour Ca2+ and glutamate imaging in organotypic cultures.