Figure 2. Characterization of Archons in cultured cells.

(a) Representative fluorescent images of Archon1 (left, excitation (λ_ex) with 637 nm laser light, emission (λ_em) at 664LP) and GFP (right, λ_ex = 475/34BP from an LED and λ_em = 527/50BP) channels in a cultured mouse hippocampal neuron (n = 32 cells from 5 independent transfections). Scale bar: 10 μm. (b) Relative fluorescence of QuasAr2, Archer1, Archon1, and Archon2 in cultured neurons (n = 18, 16, 23, and 23 cells respectively, from 4 independent transfections each, from one culture; λ_ex = 637nm laser light at 800 mW/mm² and λ_em = 664LP for Fig. 2c–g; ***P < 0.0001, Kruskal–Wallis analysis of variance followed by post-hoc Steel-Dwass test on each pair; see Supplementary Table 5 for full statistics for Fig. 2). Box plots with notches are used (see caption of Fig. 1d for description). Open circles represent outliers, data points which are less than 25^th percentile or greater than 75^th percentile by more than 1.5 times the interquartile range. (c) Representative fluorescence response of Archon1 in a cultured neuron, to a 100 mV change delivered in voltage-clamp. τ_fast and τ_slow indicate time constants with the fluorescence trace fit according to $\frac{\mathrm{\Delta }F}{F}(\mathrm{t})={\text{Ae}}^{-\mathrm{t}/\mathrm{\tau }\text{fast}}+{\text{Be}}^{-\mathrm{t}/\mathrm{\tau }\text{slow}}$, with the % indicating A/(A+B). Image acquisition rate: 3.2 kHz. (d) Representative fluorescence traces of Archon1 in response to a series of voltage steps in voltage-clamp mode. Image acquisition rate: 2.3 kHz. (e) Population data corresponding to the experiment of d (n = 8 neurons from 3 cultures). Data was normalized so that −70 mV was set to 0 ΔF/F. (f) Single-trial optical recording of Archon1 fluorescence responses (magenta) during spontaneous activity, with concurrent current clamp trace (black), for a cultured hippocampal neuron. Peak marked with arrow is zoomed-in in (g). Image acquisition rate: 2.3 kHz. (g) Zoomed-in view of peak marked with arrow in (f), scaled to match peaks. (h) Quantification of electrical and optical full width at half maximum (FWHM; dashed lines connect data points from same neuron), ΔF/F, and signal-to-noise ratio (SNR), per action potential (AP) across all recordings (n = 160 APs from 7 neurons from 5 cultures). *P = 0.0156, Wilcoxon signed-rank test. (i) Photobleaching curves of Ace, QuasAr2, Archer1, Archon1 and Archon2 under continuous illumination (n= 5, 7, 5, 9, and 7 neurons from 1, 1, 1, 2, and 2 cultures, respectively; 475/34BP from an LED at 13 mW/mm² for Ace2N-4aa-mNeon, 637nm laser light at 2.2W/mm² for QuasAr2 and Archer1, 637nm laser light at 800mW/mm² for Archon1 and Archon2; light intensity was adjusted to have the same initial signal-to-noise ratio (SNR) of action potentials, e.g. 25±8, 26±12, 26±10, 26±10 and 28±7 for Quasar2, Archer1, Archon1, Archon2 and Ace2N-4aa-mNeon respectively; image acquisition rate: 333Hz); *P = 0.0184 for Archon1 and Archon2, Archon1 and QuasAr2, and Archon2 and QuasAr2; *P = 0.0456 for Archon1 and Archer1, Archon1 and Ace, and Archon2 and Ace; Kruskal–Wallis analysis of variance of bleaching time followed by post-hoc Steel-Dwass test on each pair).