Quantitative TCSPC FRET-FLIM Applied to Donors Exhibiting Multi-Exponential Decays: Spatio-Temporal Interaction Between Calmodulin and the Kv7.2 Potassium Channel in Living Cells

The spatio-temporal study of the dynamics of molecular interactions using FRET-FLIM is generally compromised by the large number of photons required to fit the multiple-lifetime decay of the donor population in each pixel of an image. Long acquisitions prevent interacting dynamics to be detected in an image, while the use of high excitation intensities results in artifactual measurements due to bleaching. The computation of the minimal fraction of donor molecules (mfD) undergoing FRET allows quantitative imaging of molecular interactions with either single or multi-lifetime donors such as CFP, where the complexity associated to fitting a fluorescent decay with more than two components hampers quantification with traditional least-squares fitting strategies. This novel non-fitting analysis has been recently applied to wide-field time gated FLIM systems and we now extend it to more widely available TCSPC systems.The use of mfD analysis has allowed us to study the spatio-temporal dynamics of the interaction between CFP-tagged Kv7.2 channels (donor) and YFP-labeled calmodulin (acceptor) in HEK293 living cells on a TCSPC system. We show the existence of discrete interacting domains that can be followed as a function of time (every 12-15s), where the fraction of interacting KCNQ2 with calmodulin oscillates at least between 15% and 25%.In summary, we have shown that the mfD analysis allows quantitative study of the spatio-temporal dynamics of molecular interactions on TCSPC systems. Using this non-fitting strategy we have quantitatively imaged the interacting dynamics of CFP-tagged Kv7.2 channels and YFP-labeled calmodulin in living cells.