2019 Optogenetic Technologies and Applications Conference
Genetically Encoded Voltage Imaging in Induced Pluripotent Stem Cell Cardiomyocytes
Authors
Benjamin Kim - Presenter, Stanford University
Michael Lin, Stanford University
Haodi Wu, Stanford University
Joseph Wu, Stanford University
Characterization of induced pluripotent stem cell derived cardiomyocytes (iPSC-CMs) usually requires looking for beating frequency, membrane potential changes, shape, and movement. The gold standard for characterizing iPSC-CMs in cell culture is patch-clamp electrophysiology, but this method is low-throughput and destructive. An alternative method may be to use fluorescent voltage indicators. Preferentially, fluorescent voltage indicators would be ratiometric so that changes in membrane potential can be discriminated from motion-induced changes in fluorescence. Here we report a ratiometric variant of the voltage indicator ASAP3 in which the green fluorescent ASAP3 is fused to a new cyan-excitable fluorescent protein, mCyRFP2. ASAP3-mCyRFP2 enables ratiometric voltage imaging using single excitation dual emission microscopy. Additionally, we demonstrate we can faithfully report drug-induced fluorescence dependent voltage changes by adding isoproterenol. Finally, we show that upon movement of a beating cell, the change in fluorescence ratios can correct for motion artifacts.