A novel approach combining real-time imaging and the patch-clamp technique to calibrate FRET-based reporters for cAMP in their cellular microenvironment.

Fluorescence resonance energy transfer (FRET)-based reporters are invaluable tools to study spatiotemporal aspects of cyclic adenosine monophosphate (cAMP) signaling and compartmentalization in living cells. These sensors allow estimation of relative changes of cAMP levels in real-time and intact cells. However, one of their major shortcomings is that they do not easily allow direct measurement of cAMP concentrations. This is mainly due to the fact that the methods to calibrate these sensors in their physiological microenvironment are not generally available. All published approaches to calibrate FRET-based reporters rely at least in part on data derived under nonphysiological conditions. Here, we present a protocol to calibrate FRET reporters completely "in cell." We introduce a combination of FRET imaging of cAMP and the whole-cell patch-clamp techniques to microinfuse or dilute intracellular cAMP to known concentrations. This method represents a general tool to accurately estimate intracellular cAMP concentrations by allocating concentration values to FRET ratio changes.