Wu, JiahuiAbdelfattah, Ahmed S.Miraucourt, Loïs S.Kutsarova, ElenaRuangkittisakul, ArayaZhou, HangBallanyi, KlausWicks, GeoffreyDrobizhev, MikhailRebane, AleksanderRuthazer, Edward S.Campbell, Robert E.2014-12-102014-12-102014-10Wu, Jiahui, Ahmed S. Abdelfattah, Loïs S. Miraucourt, Elena Kutsarova, Araya Ruangkittisakul, Hang Zhou, Klaus Ballanyi et al. "A long Stokes shift red fluorescent Ca2+ indicator protein for two-photon and ratiometric imaging." Nature Communications 5 (2014). Nature Communications 5, Article number: 5262 http://dx.doi.org/10.1038/ncomms62622041-1723http://dx.doi.org/10.1038/ncomms6262https://scholarworks.montana.edu/handle/1/8749CC BY 4.0 license (http://creativecommons.org/licenses/by/4.0/).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.NeursciencesBiologyArticle