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

Abstract

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.

A Bright and Fast Red Fluorescent Protein Voltage Indicator That Reports Neuronal Activity in Organotypic Brain Slices

“…The structure reveals that the Lys80 (R-GECO1 numbering) side chain from the surface of the cpmApple-derived ␤-barrel engages in an electrostatic interaction with the phenolate oxygen of the chromophore. This structural evidence, combined with additional mutagenesis results (Wu et al, 2014), points to this interaction as the mechanistic basis for the response of R-GECO1. Because the lysine is part of the ␤-barrel (in contrast to GCaMP, in which the key interaction is with an arginine from CaM; Wang et al, 2008), we reasoned that the cpmApple domain is likely a self-contained unit that would retain its fluorescence modulation mechanism even if the Ca 2ϩ sensitive domain were replaced with an alternative sensing domain.…”

“…Red-shifted fluorophores that require longer wavelength excitation light provide the added advantages of lower phototoxicity, deeper tissue penetration, and lower autofluorescent background (Shu et al, 2009). Moreover, they are spectrally distinct from green fluorescent indicators and blue-light-excitable channelrhodopsin variants (Alford et al, 2013).…”

Novel uses of fluorescent proteins

“…Later, other red fluorescent Ca 2+ reporters appeared, namely RCaMP [39], R-CaMP1.07 [40], ratiometric REX-GECO1 [41], and low-affinity LAR-GECO for Ca 2+ imaging in mitochondria and ER [42].…”

Red fluorescent proteins (RFPs) and RFP-based biosensors for neuronal imaging applications

“…R-GECO1 was further optimized and engineered into spectrally diversified and low-affinity variants, including an improved R-GECO1.2, a blue-shifted O-GECO, a red-shifted CAR-GECO, 102 a highlightable GR-GECO, 103 an LSS REX-GECO 104 and low-affinity red-GECO variants. 105 RCaMP, a similar single RFP-based Ca 2þ biosensor, was engineered from the cpmRuby template.…”