Performance of K-GECO1 in HeLa cells and cultured dissociated neurons. a Representative fluorescence time-course traces for HeLa cells expressing K-GECO1 with pharmacologically induced Ca²⁺ changes. b Imaging of spontaneous Ca²⁺ oscillations in dissociated neurons expressing K-GECO1. Inset: Fluorescence image of dissociated neurons expressing K-GECO1 (scale bar, 30 μm). c Average responses for one action potential for K-GECO1 compared with other red GECIs (the same color code is used in panels c–h). d Responses of ten action potentials of red GECIs. e–h Comparison of K-GECO1 and other red GECIs as a function of number of action potentials. e Response amplitude, ΔF/F₀. f Signal-to-noise ratio (SNR). g Half decay time. h Half rise time. For (e–h), n = 56 wells, 827 neurons for K-GECO1; n = 66 wells, 1029 neurons for R-GECO1; n = 38 wells, 682 neurons for jRGECO1a; n = 105 wells, 2420 neurons for jRCaMP1a; n = 94 wells, 2995 neurons for jRCaMP1b. Supporting numeric data are provided in Additional file 9. GECI genetically encoded Ca²⁺ indicator, SNR signal-to-noise ratio

In vivo imaging of K-GECO in zebrafish Rohon–Beard cells. a Schematic setup of the experiment. b Image of Rohon–Beard cells expressing K-GECO1 with region of interest (ROI) indicating cytoplasm. c K-GECO1 Ca²⁺ response to pulse stimuli in the cytosol. d K-GECO1 Ca²⁺ response to pulse stimuli in the nucleus. e Fluorescence fold change of K-GECO1 and f jRGECO1a under various numbers of pulses. g Half decay time of K-GECO1 and h jRGECO1a under various numbers of pulses. Supporting numeric data are provided in Additional file 12

K-GECO1 expression patterns in zebrafish Rohon–Beard (RB) cells. a Schematic view of the image window. b Representative images of K-GECO1 expression in RB cells. c Representative images of jRGECO1a (with NES) expression in RB cells. (TIF 1782 kb)