Learning from fluorescence decay dynamics: A better read out from the spectroscopic ruler

The spectroscopic ruler is a noninvasive technique exploiting fluorescence resonance energy transfer (FRET) to determine intramolecular distances on the nm scale in proteins and other bio-molecules [1,2]. It is widely applied in the life sciences where it is used in vitro and in vivo to probe the separation between fluorescent markers attached to the molecules under investigation. Because FRET scales strongly with the separation distance and orientation between the optical dipole moments of the donor and acceptor molecules, the accuracy of the technique is limited when the molecules undergoes thermal fluctuations under physiological conditions and the orientation of the optical dipoles is not static.