Beyond Membrane Potential: Exploiting Signal Complexity in Genetically Encoded Voltage Indicators.

Genetically encoded voltage indicators (GEVIs) have long promised optical access to membrane potential, yet their adoption has lagged significantly behind genetically encoded calcium indicators. A central but underappreciated reason is that the metrics used to evaluate and compare GEVIs-fractional fluorescence change (ΔF/F), kinetics, and signal-to-noise ratio-rest on an assumption that is frequently violated: that GEVI fluorescence reflects a single underlying process. In this perspective, we argue that GEVI signals are composite optical measurements, arising from the superposition of voltage-dependent fluorescence, intracellular and nonresponsive signal, background, and contributions from neighboring cells. Under these conditions, ΔF/F is not a measure of sensor sensitivity but a contrast metric whose value depends on baseline fluorescence composition, optical sampling, and imaging configuration. This reinterpretation has two key consequences. First, it explains a substantial source of variability in GEVI performance that is currently attributed to noise or experimental inconsistency. Second, and more importantly, it reveals that the complexity of GEVI signals is not a limitation to be minimized but a resource to be exploited. By resolving composite signal components, GEVIs can report multiplexed physiological variables, expose hidden conformational states of voltage-sensing domains, probe membrane organization, and reveal intracellular and intercellular electrical coupling. We propose that realizing the full potential of GEVIs requires treating ΔF/F not as a gold standard for sensor performance, but as one interpretable component of a richer optical measurement whose structure encodes multiple layers of cellular physiology.