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 and 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 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 2P illumination. This work is a critical foundation for application of VF-FLIM to record absolute membrane potential signals in thick tissue.