Extended Data Fig. 4: Characterization of QuasAr6a-Citrine and QuasAr6b-Citrine in HEK293T cells (Related to Fig. 3).
From: Video-based pooled screening yields improved far-red genetically encoded voltage indicators
a. Arch-channel (exc: 635 nm, em: 670–746 nm) fluorescence images of QuasAr6a-Citrine and QuasAr6b-Citrine expressed in HEK cells (n > 20 cells for each construct). b. Relative brightness per molecule of Archon1-Citrine (n = 10 cells), QuasAr6a-Citrine (n = 7 cells), and QuasAr6b-Citrine (n = 10 cells) measured as a ratio of whole-cell FArch to FCitrine. n.s. not significant, p > 0.05; ***p : 0.0001~ 0.001 (two-sided Wilcoxon rank-sum test). The brightness per molecule was calculated as the ratio of Arch-channel fluorescence (exc. 635 nm; 420 W/cm2) to Citrine-channel fluorescence (exc. 488 nm; 0.1 W/cm2). c. Voltage sensitivity measured by concurrent voltage clamp and fluorescence in HEK cells. Left: Fractional fluorescence change vs. membrane voltage; shading: S.D. Right: Voltage sensitivity (ΔF/F per 100 mV: Archon1-Citrine, n = 4 cells; QuasAr6a-Citrine, n = 5 cells; QuasAr6b-Citrine, n = 6 cells). n.s. not significant, p > 0.05; **p: 0.01 ~ 0.05 (two-sided Wilcoxon rank-sum test). Error bars mean ± S.D. d. Voltage step-response kinetics measured by recording the average fluorescence change during a 100-ms voltage step from −70 mV to +30 mV (Archon1-Citrine, n = 6 cells; QuasAr6a-Citrine, n = 7 cells; QuasAr6b-Citrine, n = 7 cells); shading: SEM. Measurements were performed at 30 °C and a frame rate of 2,443 Hz. e. Summary of the step-response kinetic data at 30 °C, fitted with a biexponential model. Compared with Archon1, QuasAr6b showed significant improvement in both activation and deactivation kinetics. **p: 0.01 ~ 0.05 (two-sided Wilcoxon rank-sum test). f. Photobleaching by 635 nm laser (420 W/cm2) over 10 min (n = 2 cells for each construct). All constructs showed < 40% photobleaching over 10 min. S.D. g. Voltage clamp measurement of HEK cells expressing QuasAr6a or QuasAr6b showed negligible photocurrents under either 488 nm, 635 nm or combined illumination at either −70 mV or 0 mV holding potentials (488 nm: 124 W/cm2; 635 nm: 1500 W/cm2). All photocurrents were less than the variability in baseline holding current and were < 10 pA (in most cases < 2 pA). The onsets of red or blue illumination are indicated with dashed lines and numbered sequentially. h. Summary of the photocurrent measurement in g. All values are mean ± S.D. Transient changes in the holding current were calculated as the differences of the mean holding currents during the 20-ms epochs before and after the light was turned on. Red-on: average of 1′ and 3′; Blue-on: average of 4′ and 6′; Red after blue: 5′; Blue after red: 2’.
