Biophotonics_Concepts_to_Applications

resonance energy transfer(RET), orelectronic energy transfer(EET). The FRET
technique is used influorescence microscopy and molecular biology disciplines to
quantify molecular dynamics such as protein-protein and protein—DNA interac-
tions, to examine protein conformational changes, and for monitoring the complex
formation between two molecules.
The FRET theory can be explained by treating an excitedfluorophore as an
energetically oscillating dipole. This dipole can undergo an energy exchange with a
second nearby dipole that has a similar resonance frequency. To illustrate the FRET
process, consider the interactions between two molecules that are to be studied by
means of FRET. The molecular orientations and the FRET process are illustrated in
Fig.9.3. Here the molecule on the left contains adonorfluorophoreshown in blue,
which is capable of releasing (donating) its excited state energy to anacceptor
fluorophoreshown in red in the molecule on the right. As is described below, the
degree of FRET interaction between the twofluorophores depends strongly on their
separation distance, which typically is less than 10 nm.
The state transitions in a FRET process are illustrated in Fig.9.4. First a donor
fluorophore undergoes photoexcitation from the ground state S 0 to itsfirst excited
singlet state S 1 , as denoted by the solid blue upward arrows. The dashed downward
green arrows denote the possible fluorescence transitions of the donor. If an
acceptor molecule is within 1 to 10 nm of the donor, the donorfluorophore may
transfer the excited-state energy to the acceptorfluorophore through nonradiative
dipole–dipole coupling (see Sect.2.7). That is, the energy that is transferred
between the two molecules does not require the spontaneous emission of a photon
by the donor. The energy transfer can only take place if there is an exact energy
matching of the donorfluorescence transitions with the excitation transitions in the
acceptor shown by the dashed orange upward arrows. Following the energy
transfer, the acceptor may drop to the ground state thereby emittingfluorescent
photons, as indicated by the solid red downward arrows. The degree offluorescence
from the acceptor then can be used to measure molecular activity.
In addition to an exact energy matching of the acceptor and donorfluorescence
transitions, the absorption spectrum of the acceptor must overlap thefluorescence
emission spectrum of the donor. The absorption and emission spectra of the donor
and acceptorfluorophores are shown conceptually in Fig.9.5. The blue triangular

Molecule 1 Molecule 2

Donor fluorophore Acceptor fluorophore

Excitation Fluorescence

FRET

< 10 nm

Fig. 9.3 Molecular
orientations and basic FRET
process

264 9 Spectroscopic Methodologies