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

The electrical membrane voltage ( V m ) characterizes the functional state of biological cells, thus requiring precise, noninvasive V m -sensing techniques. While voltage-dependent fluorescence intensity changes from genetically encoded voltage indicators (GEVIs) indicate V m changes, variability in sensor expression confounds the determination of absolute V m . 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. Based on the highest lifetime change in the V m range of −120 to 60 mV, rEstus-NI (798 ps) and ASAP3 (726 ps) are preferred for FLIM recordings. At a physiologically relevant V m 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 V m in HEK293T, A375 melanoma, and MCF7 breast cancer cells and quantified spontaneous V m fluctuations in A375 cells.