Biophotonics_Concepts_to_Applications

area indicates the overlap between the donor emission and the acceptor excitation
spectra.
The efficiency E of the energy transfer is given by the Förster relation

E¼

R^60

R^60 þR^6

ð 9 : 1 Þ

where the Förster distance R 0 is thefluorophore separation at which 50 % of the
energy is transferred and R is the actual distance between thefluorophores. Thus,
because the energy transfer efficiency is inversely proportional to the sixth power of
the distance between the donor and the acceptor, the FRET process is extremely
sensitive to small changes in the separations of the donor and acceptorfluorophores
(see Example9.2and Prob. 9.3). In FRET applications the experimental separation
distances typically are on the order of the Förster distance.

S 0

S 1

Donor

Acceptor

Energy-matched

donor/acceptor

transitions

Acceptor

excitation

transitions

Non-radiative

transitions

Acceptor

fluorescence

transitions

Donor

excitation

transitions

Donor

fluorescence

transitions

Fig. 9.4 State transitions in a FRET process

Normalized intensity

Wavelength (arbitrary units)

Donor

absorption

Donor

emission

Acceptor

absorption

Acceptor

emission

Donor/acceptor

spectral overlap

Fig. 9.5 Absorption and emission spectra of the donor and acceptorfluorophores

9.2 FRET/FLIM 265