Fig. 1. Electrochromic imaging of MDA-MB-231 cells with voltage dye di-4-AN(F)EP(F)PTEA.

a Schematic of the widefield epifluorescence imaging system with two-color excitation. b MDA-MB-231 cell stained with di-4-AN(F)EP(F)PTEA with simultaneous voltage clamp c The blue and red lines show di-4-AN(F)EP(F)PTEA’s excitation and emission spectra²⁹, respectively. Fluorescence is excited by blue and green LEDs on opposite sides of the excitation maximum (black vertical dashed lines). Decreasing or increasing V_m causes the excitation spectra to shift right or left, respectively. Decreasing V_m, therefore, causes reduced emission in response to blue channel excitation, and increased fluorescence with green channel excitation and vice versa for increasing V_m. d Waveforms commanding the MDA-MB-231 whole-cell voltage clamp for calibration of the ratio of blue- to green-excited fluorescence with respect to the baseline (ΔR/R₀). e Recorded ΔR/R₀ signal in response to the injected V_m waveforms. f Average ΔR/R₀ change with membrane voltage change and a linear fit to the data indicating the imaging sensitivity (% change in ratio per 100 mV membrane voltage change). g Measured sensitivities for different patched cells with a gaussian kernel estimate (blue envelope). h Example time course of a spontaneously active MDA-MB-231 cell displaying typically observed transient V_m hyperpolarisations (indicated by red ticks). Gray, unfiltered time course, black, gaussian filtered time course, sigma = 3 sampling points (0.6 s).