The nonradiative energy transfer by a resonance dipol-dipol interaction between two fluorophores is a sensitive tool to study inter-dye distances and was used in many works to determine structures of biomolecules such as proteins and nucleic acids. Due to energy transfer, donor lifetime is decreased and acceptor fluorescence intensity increased. Additionally, fluorescence emission becomes depolarized. The efficiency, E, of the energy transfer follows the equation
where RDA is the distance between the fluorophores and R0 is the Förster radius. For the donor-acceptor pair Rhodamine Green (RhGr) and Cy5, R0 is approximately 52 Å. It varies slightly between different molecular systems due to different overlap integrals of the emission and excitation spectra of the RhGr and Cy5, respectively. For the first experiments, a molecule is chosen to have a FRET-efficiency close to 100%, as seen by steady-state experiments (scheme in Fig. 1).