Supplementary Figure 7: Additional representative curves and bleed-through analysis for four- and six-parameter imaging in HeLa cells.

(a) Representative single-cell time-courses showing the changes in T-sapphire (TsR), YFP, BFP, and RFP fluorescence intensity in HeLa cells co-expressing sapphireAKAR (teal), Flamindo2 (yellow), blueCKAR (blue), and RCaMP (red) upon sequential treatment with 50 μM Fsk and 100 μM IBMX (Fsk/IBMX), 100 ng/mL PMA, and 1 μM ionomycin (iono). (b, c) Representative single-cell time-courses of the changes in TsR, YFP, BFP, and RFP fluorescence intensity in HeLa cells co-expressing Lyn-sapphireAKAR (teal), sapphireAKAR-NLS (light blue), Flamindo2 (yellow), RAB-EKARev-NLS (red), Lyn-RAB-EKARev (pink), and B-GECO1 (blue) upon sequential treatment with Fsk/IBMX, EGF, and 100 μM histamine, showing cells both without (b) and with (c) EGF-induced calcium transients. All curves are plotted as fluorescence intensity normalized with respect to time 0 (F/F₀). (d) To analyze fluorescence bleed-through, HeLa cells were individually transfected with sapphireAKAR, blueCKAR, Flamindo2, or RCaMP, and images were acquired in the TsR, BFP, YFP, and RFP channels for cells expressing each construct. Representative scatter plots show the pixel intensity in each channel as a function of the pixel intensity in the probe channel, and line traces show the pixel intensity in the probe channel alone. Obvious bleed-through of fluorescence intensity was only detected in the TsR channel in cells expressing blueCKAR, and the bleed-through correction factor was calculated by determining the slope of the TsR vs BFP pixel intensity graph for blueCKAR-expressing cells. (e) Representative single-cell traces of TsR channel intensity in a HeLa cell transfected only with blueCKAR before (dashed line) and after (solid line) BFP bleed-through correction. (f) Representative fluorescence images for each construct in each channel. Scale bars, 30 μm. Single-cell traces in a and b, c are derived from the same datasets as Figures 6e (12 independent experiments) and 6f (10 independent experiments), respectively. Experiments in d-f were repeated 5 times with similar results