Fluorescence Technologies for Monitoring Interactions Between Biological Molecules In Vitro - Progress in Molecular Biology and Translational Science - page 114

fluorescein was one of the first environment-sensitive probes, exhibiting
pH-dependent excitation and emission wavelengths. 12,13 Although
fluorescein and its chemical analogs are still used to label specific
sites of target biomolecules, other probes have been developed with
improved fluorescence stability, quantum yield, or solvatochromism. For
example, Prodan, NBD (nitrobenzofurazan), Coumarin, Nile red, and
their derivatives constitute well-known environment-sensitive dyes that
are usually applied to sense changes in the environment proximal to the
target. 11

1.2.2 Resonance energy transfer
At the beginning of the twentieth century, Jean Perrin was the first scientist
to consider the interaction through space between molecules. 14 He pro-
posed that the excitation energy is transferred from one molecule to another
through interactions between oscillating dipoles of closely spaced molecules.
Based on this observation, Theodore F¨ rster developed the theoretical basis
of resonance energy transfer, that is, the “F¨ rster resonance energy transfer”
or FRET. 15,16 FRET generally occurs between a donor (D) molecule in the
excited state and an acceptor (A) molecule in the ground state ( Fig. 4.4A ).
The donor molecules typically emit at shorter wavelengths that overlap with
the absorption spectrum of the acceptor ( Fig. 4.4B ). Energy transfer occurs
without the appearance of a photon and is the result of long-range
dipole-dipole interactions between the donor and the acceptor. When
both molecules are fluorescent, the term “fluorescence resonance energy
transfer” is used instead, although the energy is not transferred by
fluorescence. 17 The rate of energy transfer depends on the spectral
overlap of the emission spectrum of the donor with the excitation
spectrum of the acceptor, the quantum yield of the donor, the relative
orientation of the donor and acceptor transition dipoles, and the distance
between the donor and acceptor molecules. The distance dependence
of FRET allows for measurement of the distances between donors and
acceptors. 17 Indeed, when the energy transfer efficiency ( E ) is measured,
the distance ( r ) between the two fluorophores can be calculated,
according the equation

R 0 6 R 0 6 r 6 E ¼ = þ ½ 4 : 1

where R 0 is the so-called F¨ rster distance at which the efficiency of transfer
equals 50%. This latter point is attractive with regard to the identification of
molecular interactions in vitro or in a biological context. Thus the choice of a