Collective Effects Influencing Fluorescence Emission - Advanced Fluorescence Reporters in Chemistry and Biology - page 119

efficiencies are determined by the interplay of the rate constants of all transforma-
tions of the excited states, starting from that of the initially excited donor [ 54 ]. The
overlap integral J for the transfer is larger if the donor emission is at shorter and
acceptor absorption is at longer wavelengths than in the opposite case (see Fig. 2 ),
which makes the transfer strongly directional. By selecting dyes with optimal
spectral overlap, one can excite fluorescence at short wavelengths and in a transfer
process, obtain emission at much longer wavelengths. Such a system can operate as
an efficient “ wavelength converter ”.
Wavelength converting can be very useful. FRET can occur in a cascade manner
in a sequence of dyes, in which an energy acceptor can serve as the donor to another
dye with lower excitation and emission energy. One can select the primary donor
with the excitation spectrum that ideally fits the used light source and the final
acceptor with the position of the emission maximum at the desired optimal wave-
length for the assay. This wavelength can be chosen throughout the visible range,
down to the near-infrared region. Moreover, a two-photonic excitation can be used.
If this is not achievable with a single donor-acceptor pair, the “intermediate” dyes
serving as both donors and acceptors may be needed to fill the gap in the energy
transfer chain. Thus, by exciting a single donor at a single wavelength, one can
achieve the broadest range of emission colors.
A transfer, in which several different dyes provide the chain of transfer events
for achieving a very significant shift in emission wavelength, is called a “ cascade
energy transfer ”[ 55 ], as depicted in Fig. 5 .
Recent literature contains many examples of the construction of “cascades” [ 56 ].
Usually they are made by the covalent linking of monomer dyes, which allows strict
control of their stoichiometry. The pyrene-Bodipy molecular dyads and triads are
examples [ 57 ]. Efficient energy flow was reported in a purpose-built cascade
molecule bearing three distinct chromophores attached to the terminal acceptor
[ 58 ]. A combinatorial approach with the selection of the best hits can be applied
using the assembly of fluorescent oligonucleotide analogs [ 59 ].

D

A 1 A 2 A 3

S 1

FRET

FRET

h n a

h n 1

h n 2

FRET

h n 3 h n 4

S 0

green orange

red

yellow

Fig. 5 Cascade systems with directed excited-state energy transfer. On each step, the emission
spectrum shifts to longer wavelengths