Förster resonance energy transfer (FRET). (a) A molecule labelled with donor and acceptor chromophores (in this case a stiff polyproline peptide with Alexa Fluors 488 and 594 ). The donor fluorophore (green) can be excited specifically (blue). It can then either emit a fluorescence photon itself or transfer its excitation energy to the acceptor (red). (b) The process can be depicted in terms of a Jablonsky diagram illustrating the transitions between ground and excited states of donor (D) and acceptor (A). The rate of energy transfer, kF, depends on the distance, r, between donor and acceptor, resulting in the characteristic distance dependence of the transfer efficiency E (c). At the Förster distance, R0, E = 0.5. For currently available FRET pairs suitable for single molecule spectroscopy, R0 is in the range of 4 to 8 nm.