Fig. 1: Components, setup, and functionality of the OVC.

A Molecular OVC components: Two counteracting optogenetic actuators for de- and hyperpolarization (GtACR2, Chrimson; blue star: mCerulean), and a voltage indicator. B Hardware setup and communication: Membrane voltage is monitored via a fast and sensitive (sCMOS or EMCCD) camera; gray values are processed by live control software (Supplementary Code 1). Based on the difference to the set holding value, a feedback of light from a monochromator is sent to the optogenetic actuators. C OVC feedback mechanism: After selection of clamping parameters and calibration for bleaching correction, ΔF/F₀ is calculated and compared with the set value, to adapt the wavelength accordingly, in closed-loop. D OVC (QuasAr2 and BiPOLES) in BWMs. E Confocal fluorescence z-projection. BiPOLES (mCerulean) and QuasAr2 in BWM membranes, scheme (dashed lines represent muscle plasma membranes). Scale bar, 50 µm. Representative image (from n = 22). F OVC four-step protocol (0, −5, 5, and again 0% ΔF/F₀) in BWMs; insets and below: close-up. Wavelength shown in the respective color, holding values and tolerance range (gray boxes) are indicated for each step. Orange shade in lower panel: transition period to reach tolerance range. ADU: analog-to-digital units. G Upper panel: Overlay of mean (±S.E.M.) wavelength and (lower panel) fluorescence traces (n = 24; holding values: 0, −5, 5, 0% ΔF/F₀). H Times required for the indicated 5 and 10% ΔF/F₀ transitions (n = 24). I Membrane resistance of BWM cells, before (n = 13), and during 637 nm laser illumination (orange, n = 13), and during laser + calibration light (green, n = 12). One-way ANOVA with Bonferroni correction (laser/dark: p = 0.65; laser+cal./dark: p = 0.39; laser+cal./laser: p = 1). In H, I, box plots (median, 25th–75th quartiles); open dot: mean; whiskers: 1.5× IQR. n—numbers refer to independent biological replicates (animals) (G–I). Source data are provided as a Source data file.