Optical Estimation of Absolute Membrane Potential Using One- and Two-Photon Fluorescence Lifetime Imaging Microscopy

Background Membrane potential (V mem ) exerts physiological influence across a wide range of time and space scales. To study V mem in these diverse contexts, it is essential to accurately record absolute values of V mem , rather than solely relative measurements. Materials & Methods We use fluorescence lifetime imaging of a small molecule voltage sensitive dye (VF2.1.Cl) to estimate mV values of absolute membrane potential. Results We test the consistency of VF2.1.Cl lifetime measurements performed on different single photon counting instruments and find that they are in striking agreement (differences of <0.5 ps/mV in the slope and <50 ps in the y-intercept). We also demonstrate that VF2.1.Cl lifetime reports absolute V mem under two-photon (2P) illumination with better than 20 mV of V mem resolution, a nearly 10-fold improvement over other lifetime-based methods. Conclusions We demonstrate that VF-FLIM is a robust and portable metric for V mem across imaging platforms and under both one-photon and two-photon illumination. This work is a critical foundation for application of VF-FLIM to record absolute membrane potential signals in thick tissue.