We report on GCaMP-Rs, a new family of genetically encoded ratiometric calcium indicators that extend the virtues of the GCaMP proteins to ratiometric measurements. We have engineered a tandem construct of calcium-dependent GCaMP and calcium-independent mCherry fluorescent proteins. The tandem design assures that the two proteins localize in the same cellular compartment(s) and facilitates pixelwise ratiometric measurements; however, Förster resonance energy transfer (FRET) between the fluorophores reduces brightness of the sensor by up to half (depending on the GCaMP variant). To eliminate FRET, we introduced a rigid α-helix, the ER/K helix, between GCaMP and mCherry. Avoiding FRET significantly increases the brightness (notably, even at low calcium concentrations), the signal-to-noise ratio, and the dynamic range.
Bibliographical noteFunding Information:
This research was supported by NIH grant R01 GM85791 to R.H. Chow, R01 EY022931 (to J. Weiland and R.H. Chow), EY12155 EY027193 (to J. Chen), 1DP2 CA186752-01 (to S. Sivaramakrishnan) R01 AR47364, and AR60306 (to C.-M. Chuong).