Abstract
Background:
Membrane potential (Vmem) exerts physiological influence across a wide range of time and space scales. To study Vmem in these diverse contexts, it is essential to accurately record absolute values of Vmem, 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 <0.5 ps/mV in the slope and <50 ps in the y-intercept). We also demonstrate that VF2.1.Cl lifetime reports absolute Vmem under two-photon (2P) illumination with better than 20 mV of Vmem resolution, a nearly 10-fold improvement over other lifetime-based methods.
Conclusions:
We demonstrate that VF-FLIM is a robust and portable metric for Vmem 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.
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Supplementary Material
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