A long Stokes shift red fluorescent Ca 2+ indicator protein for two-photon and ratiometric imaging

Abstract

The introduction of calcium ion (Ca 2+) indicators based on red fluorescent proteins (RFPs) has created new opportunities for multicolour visualization of intracellular Ca 2+ 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 Ca 2+ 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 Ca 2+ 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 Ca 2+ and glutamate imaging in organotypic cultures.