Membrane potential (V_mem) is a fundamental biophysical signal present in all cells. V_mem signals range in time from milliseconds to days, and they span lengths from microns to centimeters. V_mem affects many cellular processes, ranging from neurotransmitter release to cell cycle control to tissue patterning. However, existing tools are not suitable for V_mem quantification in many of these areas. In this review, we outline the diverse biology of V_mem, drafting a wish list of features for a V_mem sensing platform. We then use these guidelines to discuss electrode-based and optical platforms for interrogating V_mem. On the one hand, electrode-based strategies exhibit excellent quantification but are most effective in short-term, cellular recordings. On the other hand, optical strategies provide easier access to diverse samples but generally only detect relative changes in V_mem. By combining the respective strengths of these technologies, recent advances in optical quantification of absolute V_mem enable new inquiries into V_mem biology.

Membrane potential is a fundamental biophysical parameter common to all of cellular life. Traditional methods to measure membrane potential rely on electrodes, which are invasive and low-throughput. Optical methods to measure membrane potential are attractive because they have the potential to be less invasive and higher throughput than classic electrode based techniques. However, most optical measurements rely on changes in fluorescence intensity to detect changes in membrane potential. In this chapter, we discuss the use of fluorescence lifetime imaging microscopy (FLIM) and voltage-sensitive fluorophores (VoltageFluors, or VF dyes) to estimate the millivolt value of membrane potentials in living cells. We discuss theory, application, protocols, and shortcomings of this approach.