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

• DOI: 10.1038/ncomms6262
• Publication Date: 2014-10-31T10:39:02Z
• Authors: Jiahui Wu; Ahmed S. Abdelfattah; Loïs S. Miraucourt; Elena Kutsarova; Araya Ruangkittis...; Hang Zhou; Klaus Ballanyi; Geoffrey Wicks; Mikhail Drobizhev; Aleksander Rebane; Edward S. Ruthazer; Robert E. Campbell
• Keywords: 0301 basic medicine; Microscopy, Confocal; Molecular Structure; Optical Imaging; Brain imaging; Protein Engineering; Rats; Rats, Sprague-Dawley; Luminescent Proteins; 03 medical and health sciences; Ca2+ imaging; Animals; Humans; Calcium; Indicators and Reagents; Protons; HeLa Cells; Red Fluorescent Protein

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.