Abstract
A ratiometric genetically encoded voltage indicator (GEVI) would be desirable for tracking transmembrane voltage changes in cells that are undergoing motion. To create a high-performance ratiometric GEVI, we explored the possibility of adding a voltage-independent red fluorophore to ASAP3, a high-gain green fluorescent GEVI. We performed combinatorial multi-site mutagenesis on the cyan-excitable red fluorescent protein mCyRFP1 to enhance brightness and monomericity, creating mCyRFP2. Among red fluorescent proteins tested, mCyRFP2 proved to be the least perturbing when fused to ASAP3. We demonstrate that the red fluorescence of ASAP3-mCyRFP2 provides an effective reference channel to remove motion artifacts from voltage-induced changes in green fluorescence. Finally we use ASAP3-mCyRFP2 to visualize membrane voltage changes throughout the cell cycle of motile cells.
Competing Interest Statement
The authors have declared no competing interest.