FIGURE 3.

Characterization of iGECInano in live human HeLa cells. (A,B) Comparison of iGECInano, iGECI, and NIR-GECO2G brightness in live HeLa cells in the absence of BV (A) and in the presence of 5 μM BV (B) measured using flow cytometry and normalized on iGECI fluorescence. The 640 nm laser was used for excitation, and a 647 nm long-pass edge filter to detect fluorescence. Fluorescence intensities were normalized to the absorption efficiencies of the indicators at 640 nm. In (A,B), n = 3 individual experiments. (C) Photobleaching curves of iGECInano, iGECI, and NIR-GECO2G in live HeLa cells excited using a 605/30 nm bandpass and imaged using a 647 nm long-pass filter. Photobleaching data were normalized to the absorption efficiencies of indicators at 605 nm. 5 μM exogenous BV was supplied 24 h before the experiment. n = 5 cells. (D) Typical Ca²⁺ transients reported by iGECInano in live HeLa cells. Ratio changes of the FRET acceptor (ex. 605 nm, em. 725/40 nm) to the donor (ex. 605 nm, em. 680/20 nm) fluorescence intensities upon treatment with 100 μM of histamine, followed by changing the media to one containing 10 μM ionomycin and 1 mM Ca²⁺ and then 2 mM EGTA.