Fig. 1: Characterization in HEK cells.

a Design principle of sDarken. b Screening result of linker variants. Bars indicate fluorescence changes upon application of serotonin. Blue bar represents mutant M34. c Expression of mutant M34 (sDarken) in HEK cells. Scale bar 20 µm. Fluorescence before and after the application of 800 nM 5-HT. Representative fluorescence trace of sensor variant M34 expressing HEK cell during the application of 800 nM 5-HT. Experiments were at least repeated three times. d (i) Representative image sequence of repetitive 5-HT application via wash in. (ii) Example fluorescence measurement, repetitive application of 5 µM 5-HT in 9 example cells in two dishes. Application of 5-HT leads to a reversible reduction of fluorescence. No baseline corrections were performed. (iii) Heatmap of all analyzed ROIs. Scale bar in the first frame applies to all following frames. Scale bar 20 µm. e Example trace of 5-HT uncaging (405 nm, laser power: 90%, bleaching period: 90 ms, interval: 50 s) evoked sDarken responses. Blue circle indicates uncaging) area. White line indicates the part of the membrane that was used for analysis. Fluorescence signal of sDarken. ΔF/F values of part of membrane (white line) over time (exemplary trial). Blue bars indicate uncaging (i.e., bleaching) intervals. Repetitive uncaging of 5-HT resulted in decreasing ΔF/F values of sDarken. f Fast 5-HT application to an outside-out patch. Representative image from an outside-out patch containing sDarken 48 h post transfection (left). Fluorescence intensity changes in the patch. The fluorescence recovery after 5-HT wash out is significantly slower than the activation. No background/baseline corrections were performed. g Dose response curve measured in response to different 5-HT concentrations. Group data n = 20 from at least 2 replicates, mean and ±SEM are shown. h Fluorescence changes after application of serotonin, related substances or neurotransmitters (if not mentioned differently, concentration 10 µM (n = 10). Box represents the 25% percentile to the 75% percentile. The line in the middle of the box represents the median. One-Way ANOVA Multiple Comparison, ***p < 0.001, 5-HT serotonin, 8-OH 8-OHDPAT, L-Tryp tryptophan, IAA 5-hydroxyindoleacetic acid, Dopa dopamine, His histidine, NE norepinephrine, GABA Glu glutamate, Ace acetylcholine, PBS phosphate buffer.