Collective Effects Influencing Fluorescence Emission - Advanced Fluorescence Reporters in Chemistry and Biology - page 114

Donor Acceptor

Overlap

λ D λ A

λ A λ D

Wavelength, nm

Fig. 2 Absorption ( solid lines ) and fluorescence ( dashed lines ) spectra of two fluorophores
exhibiting FRET. The light-absorbing and emitting at shorter wavelengths fluorophore (donor)
can transfer its excitation energy to another fluorophore (acceptor) absorbing and emitting at
longer wavelengths. For efficient transfer, the absorption spectrum of the acceptor should overlap
the emission spectrum of the donor ( shaded area ). At close donor-acceptor distance, the excitation
of the donor results in emission of the acceptor, and if the distance is large, the donor itself will
emit. This produces distance-dependent switching between two emission bands

The theory predicts a 1/ R 6 dependence of energy transfer rate on their separation
distance, R , which is very steep. Deviation from this dependence is frequently
observed for extended conjugated dye molecules, metal [ 38 ], and semiconductor
[ 39 ] nanoparticles, the sizes of which are comparable with R , but the validity of this
theory and 1/ R 6 dependence are confirmed in the studies of small dye molecules
interacting at significant distances.
It is important that FRET is mechanistically reversible and that dyes with similar
excitation and emission spectra may exchange their excited-state energies. At high
local dye concentrations, energy can travel within the population of these dyes and
be directed to the dyes with longer wavelengths of absorption and emission.
Kinetically, FRET competes with other pathways of deactivation of the donor
excited state, and the acceptor acquires the property to emit light with the lifetime
of the donor. This is because the energy transfer is a stochastic process that
develops during the donor fluorescence lifetime. These properties will be discussed
in more detail in the following sections of this chapter.

4 Site-Selective Red-Edge Effects

Light of definite energy and polarization has a selective power to exclusively excite
dye molecules whose electronic transition energy and orientation match these
parameters. Thus, if a dye is excited by polarized light, its emission will also be
highly polarized. Depolarization occurs only when the time correlation of these
selectively excited species is lost due to their rotation or participation in some