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 non-invasive manner. Voltage-sensitive fluorophores (VF dyes) that utilize a photoinduced electron transfer-based mechanism to detect membrane potential (Vm) are a powerful method for non-invasive 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 Vm probe. In this work, we develop the second generation of ReverseVFs through a combination of computation and synthesis, 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 carboVF enables two-color voltage imag-ing in cells and action potential detection with cellular resolution across multiple neurons.