Abstract
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.
Original language | English (US) |
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Pages (from-to) | 1066-1074 |
Number of pages | 9 |
Journal | ACS Chemical Biology |
Volume | 12 |
Issue number | 4 |
DOIs | |
State | Published - Apr 21 2017 |
Bibliographical note
Publisher Copyright:© 2017 American Chemical Society.