Figure 2. Directed evolution of an archaerhodopsin-derived genetically encoded voltage indicator.

a. Genetic composition of the library cells. Spiking HEK cells containing a candidate GEVI mutant, channelrhodopsin actuator, mEos4a phototag, and Na_V1.5 and K_ir2.1 to imbue electrical excitability. Here TS is the Trafficking Sequence from K_ir2.1⁶², ER2 is the endoplasmic reticulum export motif from K_ir2.0 ^{63, 64}, and P2A is a self-cleaving peptide. B. Optogenetically triggered spike of the spiking HEK cell, recorded via whole-cell patch clamp (exc. At 488 nm, 4.4 mW/mm², see also Extended Data Fig. 2a). c. Threshold response of spiking HEK cells to 10-ms increasing optogenetic stimulus strengths, visualized with the voltage-sensitive dye BeRST1 (exc. 635 nm). D. Sample preparation for pooled screening. Library cells were mixed with electrically excitable but non-fluorescent and optically inert spacer cells in approximately a 1:10 ratio. E. Examples of fluorescence traces extracted from individual sources in a pooled library screen (Arch fluorescence channel). Precisely timed spikes were evoked by blue light stimulation (blue ticks, exc. 490 nm, 10 ms). ΔF_Arch is calculated as the average baseline-to-peak difference in Arch-channel fluorescence and F_{0, Arch} is the average baseline fluorescence. For the details of image segmentation, see Extended Data Fig. 3c. f. Scatter plot of $\mathrm{\Delta }{F}_{Arch}/\sqrt{F_{0,Arch}}$ vs. F_{0, Arch} for all the automatically segmented ROIs in a 2.3-mm × 2.3-mm FOV. The quantity was used as a measure of shot-noise-limited SNR. Selection threshold: 50^th percentile for F_{0, Arch}; 75^th percentile for $\mathrm{\Delta }{F}_{Arch}/\sqrt{F_{0,Arch}}$. g. Representative FACS data showing three distinct populations: photoconverted library cells; unselected library cells; spacer cells (Green channel: exc. 488 nm; Red channel: exc. 561 nm). h. Three rounds of iterative enrichment shifted the population phenotype. i. Manhattan plot showing the mutation frequency at each nucleotide in starting and post-screening libraries. j. Logarithmic plot of the starting mutation frequency vs. the fold of change. In this library, there were 970 missense mutations and 405 silent mutations.