Fig. 1: Multiparametric one-photon screening identified JEDI-1P, a voltage indicator with improved performance.

a We engineered GEVIs composed of a circularly permuted green fluorescent protein (cpGFP, green) whose brightness is modulated by voltage-induced conformational changes in the voltage-sensitive ___domain (VSD, gray and orange). b Schematic of the platform for GEVI screening under widefield one-photon illumination. c Top, Electric field stimulation (EFS) protocol. Inset: zoom-in of the 100-Hz pulse train. Bottom, representative fluorescence responses of a parental indicator, ASAP1. d Fluorescence responses to 1-ms EFS pulses and AP-like waveforms (2-ms width at half-maximum, +30 mV peak, −70 mV baseline, whole-cell voltage clamp) are highly correlated. For (d–f), n = 4 independent transfections per GEVI (EFS) or 3 (ASAP1-dpOPT), 4 (ASAP1-EGFP), 4 (ASAP1), 10 (ASAP2s), 4 (ASAP2s-H152E), and 8 (ASAP3) HEK293A cells (voltage clamp). e Fluorescence responses to EFS pulse trains and 1-s 100-mV step depolarizations (−70 mV to + 30 mV, whole-cell voltage clamp) are highly correlated. f Optical response widths to 1-ms EFS pulses and action potential (AP)-like waveforms are highly correlated. Widths were measured at 30% of the response peak. g Photostability was quantified as the area under the curve of fluorescence vs. time graphs (shaded area in this example photobleaching curve). Fluorescence was normalized to its value at t = 0. The irradiance was ~33 mW/mm². h Brightness was evaluated as the GEVI/mCherry (green/red) fluorescence ratio. Scale bar, 10 μm. i The 21 locations targeted by mutagenesis are indicated on a predicted 3D structure of ASAP2s. Green and orange residues were mutated in JEDI-1P. j Key steps of the evolutionary path leading to JEDI-1P. VSD mutations were screened under both 415R (ASAP1) and 415Q (ASAP2s) contexts. k JEDI-1P shows faster kinetics and a higher response amplitude to 1-ms EFS pulses than parental indicators (ASAP1 and ASAP2s) and screening intermediates. l GEVI multiparametric directed evolution. Values are relative to those of ASAP2s. Gray circles are screening intermediates. All panels: Error bars or shading denote the 95% confidence interval. Pearson’s r² was calculated from intercept-free unweighted linear regression of the mean values.