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A long Stokes shift red fluorescent Ca2+ indicator protein for two-photon and ratiometric imaging

2014; Nature Portfolio; Volume: 5; Issue: 1 Linguagem: Inglês

10.1038/ncomms6262

2041-1723

Jiahui Wu, Ahmed S. Abdelfattah, Loïs S. Miraucourt, Elena Kutsarova, Araya Ruangkittisakul, Hang Zhou, Klaus Ballanyi, Geoffrey Wicks, Mikhail Drobizhev, Aleksander Rebane, Edward S. Ruthazer, Robert E. Campbell,

Retinal Development and Disorders

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. Current calcium-sensitive probes based on red fluorescent proteins are unsuitable for two-photon excitation at the near-infrared wavelengths commonly used for green fluorescent probes. Wu et al. use a structure-guided approach to engineer a red fluorescent probe with optimal two-photon excitation at these wavelengths.