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

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. We use fluorescence lifetime imaging of a small molecule voltage sensitive dye (VF2.1.Cl) to estimate mV values of absolute membrane potential. 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. We demonstrate that VF-FLIM is a robust and portable metric for V _mem across imaging platforms and under both one-photon and 2P illumination. This work is a critical foundation for application of VF-FLIM to record absolute membrane potential signals in thick tissue.