Journal
ACS CHEMICAL BIOLOGY
Volume 12, Issue 4, Pages 1066-1074Publisher
AMER CHEMICAL SOC
DOI: 10.1021/acschembio.6b00883
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Funding
- NIH grant [R01 GM85791, R01 EY022931, EY12155, EY027193, 1DP2 CA186752-01, R01 AR47364, AR60306]
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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, Forster 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 alpha-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.
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