‘Radical’ differences between two FLIM microscopes affect interpretation of cell signaling dynamics

Emerging evidence suggests that cell signaling outcomes depend not only on the signal strength but also on its temporal progression. Our lab employs Fluorescence Lifetime Imaging of Resonance Energy Transfer (FLIM/FRET) biosensors to study intracellular signaling dynamics. We studied activation of β1 receptors by Isoproterenol, which triggers cAMP signaling via the G protein Gαs, using two different FLIM microscopes: a widefield frequency domain FLIM (fdFLIM) setup and a fast confocal Time Correlated Single Photon Counting (TCSPC) setup. When comparing results from each FLIM setup, unexpectedly we obtained distinctively different cAMP kinetics: fdFLIM recording of cAMP in HeLa and Cos7 cells yielded transient responses, reminiscent of rapid receptor desensitization, while TCSPC recordings exhibited sustained responses lasting over 30 minutes. We initially suspected phototoxicity due to the intense light locally in the laser focus spot in confocal microscopy to interfere with normal termination of signal transduction and set out to map photosensitive steps in the signaling cascade in detail. We find no evidence for light-sensitivity in either generation or breakdown of cAMP, but rather, our findings show that the kinetic differences are due to selective degradation of β1 agonists on the fdFLIM setup. Agonist degradation appeared due to the commercial FluoroBrite medium, even though this has been specifically advertised to lower phototoxicity and reduce autofluorescence. Mass spectrometry identified Folic acid, an undisclosed constituent of FluoroBrite, as the culprit leading to artifacts in fdFLIM measurements. In all, our study underscores the impact of subtle phototoxicity effects on experimental outcome, and it shows that in this case confocal TCSPC provides the more reliable data needed to study response kinetics. This work also emphasizes the it is crucial that scientific vendors fully disclose chemical formulations. ### Competing Interest Statement F.E.O. declares competing financial interests as co-founder and shareholder of UbiQ Bio BV.