Absolute Membrane Potential Recording with ASAP-Type Genetically Encoded Voltage Indicators Using Fluorescence Lifetime Imaging

The electrical membrane voltage (Vm) characterizes the functional state of biological cells, thus requiring precise, non-invasive Vm-sensing techniques. While voltage-dependent fluorescence intensity changes from genetically encoded voltage indicators (GEVIs) indicate Vm changes, variability in sensor expression confound determination of absolute Vm. Fluorescence lifetime imaging microscopy (FLIM) promises a solution to this problem, as fluorescence lifetime is expected to be unaffected by sensor expression and excitation intensity. By examining ASAP1, ASAP3, JEDI-1P, rEstus, and rEstus-NI (G138N:T141I) with one-photon excited FLIM measurements, we demonstrate that all sensors display a voltage-dependent lifetime. With the highest lifetime change in the Vm range of -100 to 50 mV of about 730 ps, ASAP3 and rEstus-NI are preferred for FLIM recordings. At a physiologically relevant Vm of -30 mV, the voltage sensitivity of rEstus-NI (6.6 ps/mV) is 3.6 and 1.4 times greater than that of ASAP1 and rEstus, respectively. As a proof of concept, we successfully used rEstus-NI to estimate absolute resting Vm in HEK293T, A375 melanoma, and MCF7 breast cancer cells and quantified spontaneous Vm fluctuations in A375 cells. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=89 SRC="FIGDIR/small/669310v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@1eaff8borg.highwire.dtl.DTLVardef@12f1a54org.highwire.dtl.DTLVardef@169ad83org.highwire.dtl.DTLVardef@fe9f44_HPS_FORMAT_FIGEXP M_FIG C_FIG