Biology Reference
In-Depth Information
molecules within clusters, having been successfully used to estimate the number of
protein molecules in cell membrane oligomers (
Bader, Hofman, Voortman, van
Bergen en Henegouwen, & Gerritsen, 2009
). From steady-state fluorescence anisot-
ropy images, the number of fluorophores in a cluster is given by
Bader et al. (2009)
and
Runnels and Scarlata (1995)
:
þ ot
1
þ
N
ot
þ
1
r
et
N
ð Þot
1
þ
N
ot
1
r
¼
r
mono
(6.2)
where
r
mono
and
r
et
are the anisotropy values of a single directly excited molecule and
of an excited molecule by a homo-FRET process,
ot
accounts for the efficiency of
ot¼
E
/
E
1, and
N
is the number of fluor-
the energy transfer and can be defined as
ophores.
r
et
and
have to be determined in order to extract a quantitative value of
the steady-state anisotropy measurement. Nonetheless, the calculation of the number
of protein molecules per cluster can be simplified if time-resolved anisotropy images
are performed. In this case, multiple homo-FRET processes occur within the protein
cluster in the few nanoseconds that follow excitation. As a result, the anisotropy is
leveled at its limiting value
r
inf
and every fluorophore has the same probability to
emit a photon. The number of molecules in the cluster can thus be determined di-
rectly from
r
inf
, according to
Bader et al. (2009
):
ot
r
mono
1
r
et
N
1
r
inf
¼
N
þ
(6.3)
N
The size of the clusters and their spatial distribution can be estimated using the
loss of anisotropy due to controlled photobleaching of the fluorophores. Sequential
photobleaching of the fluorophores leads to a gradual loss of the fluorescence anisot-
ropy since the probability of a homo-FRET process diminishes with increasing
photobleaching time. Proper modeling of the resulting anisotropy curve can be used
to estimate the size of the cluster (
Sharma et al., 2004
). Moreover, hetero-FRET can
be used at varying concentrations of donor/acceptor molecules as a complementary
method to quantify cluster size.
Although time-resolved homo-FRET microscopy is a powerful tool to study the
cell membrane organization and protein clustering, some cares should be taken when
using it. Due to the length scale of the interactions involved (
1-10 nm), homo-
FRET can also occur in samples presenting a high density of molecules displaying
a purely Brownian distribution. This, however, should not be interpreted as molec-
ular clustering. A study of how the anisotropy changes when reducing the concen-
tration of molecules might allow distinguishing between actual clustering of
molecules and a high density of molecules with a Brownian distribution and inter-
particle distances close to their characteristic
R
0
. True clustering of molecules can be
identified if the scatterplot of fluorescence intensity versus anisotropy displays a con-
stant anisotropy value independent on the molecule density of the sample (
Varma &
Mayor, 1998
). In contrast, if the anisotropy value anticorrelates with the fluorescence
intensity of the sample, homo-FRET can be explained by a high density of dye
molecules. This technique might also underestimate clustering when interparticle
