Fig. 5: Intensity-independence of PEAQ biosensing.

a Raw fluorescence traces obtained on purified GCaMP6s-Q protein at 39 μM free Ca²⁺ with varying powers of cyan irradiation. Full data including other Ca²⁺ concentrations is shown in Supplementary Fig. S20. b Raw fluorescence trace (black) of a representative GCaMP6s-Q-expressing Hela cell at high Ca²⁺ concentration during photoswitching. Off-switching was achieved via either 10 acquisitions with 47 mW cyan excitation (I_hν), or 20 acquisitions with 24 mW cyan excitation (0.51 I_hν). Green dashed lines show the exponential fits of the off-switching decays. The thick orange bands indicate the 10 data points used for fitting. c Ca²⁺ titration of GCaMP6s-Q expressed in a representative HeLa cell. \({({{\Delta }}{{{{F}}}}/{{{{{F}}}}}_{0})}_{{{{{{{{\rm{hv}}}}}}}}}\) was calculated using either F_end measured after ten acquisitions, or an F_end parameter obtained from the plateau of an exponential fit. `Uncorrected' indicates calibration curves that were obtained without exponential fitting. For other curves the F_end signal was determined using the exponential fit-based procedure.