Some surprising and important news from Nature Biotechnology about a common technique in cellular imagining, fluorescence resonance energy transfer, or FRET. Specifically, it looks like ATP/Mg can significantly alter the FRET signal, which has commonly been used for looking at Ca, voltage, and various other binding interactions in neurons:
Given these findings, we predict that fluctuations in free or Mg2+-bound ATP will affect the signal output of most—if not all—CFP-YFP–based FRET indicators.
The authors continue with the following recommendations:
Currently, not much is known about effects of intracellular molecular crowding or the occurrence of local fluctuations in ATP concentration in distinct cellular locales, including the unstirred molecular layers in the vicinity of membranes8, 9. We think, however, that the dynamic concentration(s) of compartmentalized local free Mg-ATP will almost certainly overlap the 1–20 mM range, or be even higher. Thus, our observations may have important implications for microscopy studies of a broad range of local signaling events or metabolite changes in a broad range of cells. Findings should be interpreted with caution if genetically encoded CFP-YFP–based biosensors are used in cells with large and sudden fluctuations in local or global ATP concentration and appropriate controls with bound and nonbound ligands and CFP and YFP placed at appropriate spacing should be used at all times.
Source: ATP and FRET—a cautionary note
Marieke Willemse, et al. (Feb. 2007)