FRET-based biosensors for the quantitative analysis of intracellular signalling, including sensors for monitoring cyclic AMP are of increasing interest. The measurement of the donor/acceptor emission ratio in tandem biosensors excited at the donor excitation wavelength is commonly used technique. A general problem, however, is that this ratio varies not only with the changes in cAMP concentration, but also with the changes of the ionic environment or other factors affecting the folding probability of the fluorophores.<br>Here we use a spectral FRET analysis on the basis of two excitation wavelengths to obtain a reliable measure of the absolute cAMP concentrations with high temporal and spatial resolution by using an "exchange protein directly activated by cAMP" (Epac). In this approach, FRET analysis is simplified and does not require additional calibration routines. The change in FRET efficiency (E) of the biosensor caused by cAMP changes was determined as DeltaE = 15%, whereas E varies between 35% at low and 20% at high [cAMP], allowing quantitative measurement of cAMP concentration in range from 150 nM to 15 microM. The method described is also suitable for other FRET based biosensors with a 1:1 donor/acceptor stoichiometry.<br>As a proof of principle, we measured the specially resolved cAMP concentration within living cells and determined the dynamic changes of cAMP levels following stimulation of the Gs-coupled serotonin 5-HT7 receptor.
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