Fig. 3: PEAQ biosensing in live cells.
a \({({{\Delta }}{{{{F}}}}/{{{{{F}}}}}_{0})}_{{{{{{{{\rm{hv}}}}}}}}}\) titration curves of individual HeLa cells (n = 60 cells) transfected with GCaMP6s-Q in comparison to the in vitro GCaMP6s-Q titration curve, measured using the same settings (black). The cell traces have been color-coded according to cell brightness at Ca2+ saturation as shown in panel b. c Fluorescence time trace of a GCaMP6s-Q-expressing HeLa cell after stimulation with 25 μM histamine, recorded by performing a photochromism cycle every two seconds. The off-switching light also serves as the excitation light for imaging. d Fluorescence image of HeLa cells expressing GCaMP6s-Q representative of 20 successful independent repetitions of the experiment. The expansion and white arrow mark the cell for which the PEAQ analysis is shown in panel d. e \({({{\Delta }}{{{{F}}}}/{{{{{F}}}}}_{0})}_{{{{{{{{\rm{hv}}}}}}}}}\) calibration curve at different \(\left[{{{{{{{{\rm{Ca}}}}}}}}}^{2+}\right]\), measured in vitro using the same instrumental settings as for cell imaging. The extreme 5% regions (red shading) are considered to be out of the working range (green shading). The inset to panel e shows a schematic overview of the methodology. f Fluorescence (black), \({({{\Delta }}{{{{F}}}}/{{{{{F}}}}}_{0})}_{{{{{{{{\rm{hv}}}}}}}}}\) (red), and absolute \(\left[{{{{{{{{\rm{Ca}}}}}}}}}^{2+}\right]\) (green, right panel) traces of the cell indicated with the white arrow in panel d after stimulation with 25 μM histamine. Red markers represent \(\left[{{{{{{{{\rm{Ca}}}}}}}}}^{2+}\right]\) calculated from \({({{\Delta }}{{{{F}}}}/{{{{{F}}}}}_{0})}_{{{{{{{{\rm{hv}}}}}}}}}\) values in the extreme 5% ranges of the calibration curve. The image sequence shows the change in fluorescence throughout a subset of the experiment. Specific time points are highlighted in the graphs and images (orange arrows). Additional cell trajectories acquired in the same experiment are shown in Supplementary Fig. S13.
