Biomedical Engineering Reference
In-Depth Information
(called donor D) to another nearby absorbing (but not necessarily emitting)
molecule (called acceptor A). Thus, there is a concerted quenching of D and
activation of A fluorescence (Fig. 10.10). For this reason, the acronym FRET
is often used to designate
fluorescence
resonance energy transfer. The process
involves the resonant coupling of emission and absorption dipoles and is thus
nonradiative. That is, it competes with other radiative (fluorescence) and
nonradiative pathways for deactivation, resulting in a decrease of the donor
lifetime. The energy transfer rate from the donor to the acceptor decreases
with the sixth power of the distance and thus is apparent only at distances
shorter than 10 nm [32]. At the critical distance where 50% of the donor
energy is transferred to an acceptor, the Forster radius, the donor emission
and fluorescent lifetime are each reduced by 50%, and sensitized emission
(acceptor emission specifically under donor excitation) is increased.
Because of its utility in reporting nanometer-scale interactions, FRET has
become an important tool in cell biology [33-38]. FRET in cell biology is used
commonly to verify whether labeled proteins physically interact: by measur-
ing the FRET
e
ciency
, distances on the nanometer scale (a scale within the
globular radii of proteins) can thus be estimated using a light microscope. By
measuring these effects, FRET microscopic imaging can verify close molecular
associations between colocalized donor and acceptor-labeled fusion proteins
that are far beyond the resolution of fluorescent microscopy. Obvious di
cul-
ties in intensity-based FRET measurements in cells is that the concentrations
of the donor and acceptor are variable and unknown, the emission band on
the donor extends into the absorption and emission band of the acceptor,
and the absorption band of the acceptor commonly extends into the absorp-
tion band of the donor. A further complication is that only a fraction of the
Fig. 10.10.
The principle of FRET and dependence of FRET e
ciency on donor-
acceptor distance
