Controlling Electron Flow in Carbofluorescein Voltage Indicators

Interrogating biochemistry and biophysics with fluorescent reporters that respond to environmental cues is a powerful way to study dynamic processes in living systems in a noninvasive manner. Voltage-sensitive fluorophores (VF dyes) that utilize a photoinduced electron transfer-based mechanism to detect membrane potential (V_m) are a powerful method for noninvasive monitoring of bioelectrical signaling. We recently showed that VF dyes can “run in reverse” (ReverseVF) by introducing an electron withdrawing group to flip the direction of electron flow in the system. This first generation of ReverseVFs possessed both a low voltage sensitivity and signal-to-noise ratio (SNR), prompting further exploration of the system to develop a more sensitive V_m probe. In this work, we develop the second generation of ReverseVFs, addressing several hypotheses about the physical organic processes that drive the voltage sensitivity of VF probes. Here, we highlight the novel 4-NO₂ carbofluorescein VF: it displays a turn-on response to membrane hyperpolarization, with a nearly 4-fold increase in voltage sensitivity and 10-fold increase in SNR compared to previous generations. The high brightness and sensitivity of 4-NO₂ carbofluorescein VF enables both two-color voltage imaging in cells and action potential detection with cellular resolution across multiple neurons.