Fluorescence Technologies for Monitoring Interactions Between Biological Molecules In Vitro - Progress in Molecular Biology and Translational Science - page 115

A

Ex donor Em donor Ex donor Em acceptor

Probe

Donor Acceptor Interaction

B

Donor Acceptor

FRET

Ex Em Ex Em

Spectral Spectral

overlap overlap

Wavelength (nm)

Figure 4.4 Förster resonance energy transfer (FRET) principle. (A) When two molecules
with appropriate donor and acceptor fluorophores interact, FRET occurs between the
donor in the excited state and the acceptor in the ground state. (B) When the donor
is excited at its absorption spectrum (blue), it typically emits at a shorter wavelength
(green) that overlaps with the absorption spectrum of the donor (yellow). Then the
resulting FRET enables the acceptor to emit fluorescence (red).

specific acceptor/donor couple to associate with interacting partners can
constitute a powerful tool to demonstrate both dynamic and in situ interac-
tions as well as conformational changes.

1.2.3 Fluorescence polarization
Fluorescence Polarization was first described in 1926 by Perrin. 14 This
specific type of polarization is based on the observation that fluorescent
molecules in solution, excited with plane-polarized light, will emit light
back in a fixed plane if the molecules remain stationary during the excita-
tion of the fluorophore; the emitted light remains “polarized.” However,
molecules rotate and tumble, and the planes in which light is emitted can
be very different from the plane used for initial excitation; the emitted light