Biology Reference
In-Depth Information
(
Goswami et al., 2008; Sharma et al., 2004
). This technique takes advantage of the
nonradiative energy transfer that occurs between an excited donor fluorophore and
an acceptor molecule separated at distances of 1-10 nm (i.e., FRET) to gather infor-
mation on how these molecules interact at the nanoscale (
Jares-Erijman & Jovin,
2003
). The efficiency of the energy transfer process is highly dependent on the dis-
tance between the donor and the acceptor molecules and other physical parameters,
such as the donor quantum yield and spectral overlap between the emission spectrum
of the donor and the absorption spectrum of the acceptor, which are included in the
donor-acceptor F¨rster radius (
R
0
)(
Rao & Mayor, 2005
). Most applications of
FRET in biological sciences monitor the energy transfer process between two differ-
ent donor and acceptor molecules. This process, usually termed hetero-FRET, is ex-
perimentally quantified by determining its efficiency from the ratio of the relative
fluorescence emission of the donor in the presence (
I
AD
) and in the absence (
I
D
)
of the acceptor (
Rao & Mayor, 2005
). However, energy transfer between identical
fluorophores, that is homo-FRET, can also occur. In this case, the ratiometric ap-
proach used to study hetero-FRET is not applicable because both the acceptor and
the donor molecules have identical spectroscopic properties. Instead, homo-FRET
is experimentally measured by quantifying how the anisotropy of the fluorescence
emission changes after fluorophores excitation. This is achieved by polarizing the
excitation light and by separating the fluorescence emission based on two 90
o
-shifted
polarization components with respect to the polarization of the excitation light. The
fluorescence anisotropy is then calculated according to the following expression
(
Bader et al., 2011
):
I
par
I
per
r
¼
(6.1)
I
par
þ
2
I
per
where
I
par
and
I
per
are ,respectively, the intensities of the fluorescence emission par-
allel and perpendicular to the polarization of the excitation light. By exciting with
polarized light, sample fluorophores that have their absorption transition moments
parallel to the orientation of the excitation light polarization are photoselected. If
the fluorophores do not rotate during their excited-state lifetime and their absorption
and emission transition moments are parallel, emission polarization has the same di-
rection to that of the excitation light and the anisotropy reaches its maximum value.
However, by a homo-FRET process, excited donors may transfer their excess energy
to nearby acceptors. As a result, fluorescence emission originates from molecules
that have not been photoselected by the polarized excitation, that is, distinctly ori-
ented from the donors. Both donor and acceptor molecules contribute for the emis-
sion polarization, lowering the fluorescence anisotropy value. However, the
rotational diffusion of the fluorophores can also critically reduce the resulting anisot-
ropy. For this reason, slowly rotating dyes such as fluorescent proteins are ideally
suited for this type of studies.
The combination of homo-FRET with fluorescence microscopy is an elegant ap-
proach to gather information of cellular processes that occur at distances smaller than
the diffraction limit. This technique is particularly suited to quantify the number of
