Fig. 2: Bi-directional optical clamping and calibration of membrane voltage and currents in BWMs.
From: All-optical closed-loop voltage clamp for precise control of muscles and neurons in live animals
A APs in simultaneous patch-clamp/fluorescence recordings during OVC calibration (637 nm laser + calibration wavelength). Inset: close-up. B Simultaneous patch-clamp/fluorescence recording, OVC four-step protocol (0, −3, 3, 0% ΔF/F0). Upper: Fluorescence recording, wavelength adaptation and tolerance ranges indicated. Lower: Corresponding patch-clamp voltage recording. C Overlay of mean (± S.E.M.) wavelength, fluorescence (upper) and voltage traces (lower; n = 12) as in B. D Statistics of data in C; absolute voltages of transitions during simultaneous OVC measurements (n = 12, 12, 12, 12, 7 for cal., steps 1, 2, 3, 4; p = 1, 1, 1, 4.1E-6, 1.6E-7, 4.5E-16 for cal/step 1, cal/step 4, step 1/step 4, step 1/step 2, step 1/step 3, step 2/step 3). E Voltage modulation for respective steps of OVC protocol in D (n = 12, 12, 12, 7 for cal→step 1, step 1→step 2, step 1→step 3, step1→step 4; and p = 7.51E-11, 1.04E-21, 1.13E-10, 1 for cal→step1/step 1→step 2, step 1→step2/step1→step3, step1→step3/step1→step 4, cal→step1/step 1→step 4). F Distinct ΔF/F0 steps, presented by the OVC (lower: blue shades, tolerance ranges; black trace, actual ΔF/F0 mean ± S.E.M.), and the required wavelengths (upper: mean ± S.E.M. nm; n = 19). G, H Determining wavelengths (≙ currents) at the end (orange shade) of each ΔF/F0 clamp step (≙ voltage) to deduce pseudo-I/V relation (n = 19). I, J Simultaneous OVC/electrophysiology experiment, setting ΔF/F0 clamp-steps, measuring resulting membrane potentials, to determine calibration regression (membrane potential/ΔF/F0; 5, 4, −3%: n = 7; 3, 2, 1, 0, −1%: n = 9; −2%: n = 8; −4%: n = 6; −5%: n = 5; mean ± S.E.M. (I)). K, L Voltage clamp (−24 mV) measurement of currents resulting from wavelength steps, to determine calibration regression (current/wavelength, n = 8, mean ± S.E.M. (K)). White heteroscedasticity test in (J, L, p < 0.0001). One-way ANOVA, Bonferroni correction (D, E), ***p ≤ 0.001, box plots (median, 25th–75th quartiles); open dot: mean; whiskers: 1.5× IQR. n refers to biological replicates (C–L). Source data are provided as a Source data file.
