Biology Reference
In-Depth Information
distances of the fluorophores within the clusters are slightly larger than the
R
0
, result-
ing in poor homo-FRET efficiencies.
6.2.3
Superresolution techniques to map the nanolandscape
of the cell membrane
The diffraction limit in conventional microscopy is determined by the minimum spot
size to which a light beam can be focused with normal lens elements. In practice, the
diffraction limit implies that the minimum distance
D
x
required to independently re-
solve two distinct objects is dependent on the wavelength of the light used to observe
the specimen
and the overall objective lens system, through the numerical aperture
(NA) of the objective, as
l
D
x
l
/2
NA. With modern objectives having an NA as
high as
1.4, the resolution of conventional microscopy becomes 250-300 nm in the
case of visible light.
The landscape of the cell membrane is composed out of a vast selection of dif-
ferent proteins and lipids that organize at length scales well below the diffraction
limit of light. To inquire on the nanoscale positions of these protein and lipids or
directly visualize their organization on the cell membrane, one should employ
superresolution techniques. Nowadays, a palette of superresolution approaches are
available to the bioscience community. The various techniques such as NSOM,
STED, (F-)PALM, and stochastic optical reconstruction microscopy (STORM)
are complementary to each other in terms of specific advantages and limitations.
6.2.3.1
Far-field localization techniques (STORM/PALM)
Although the diffraction limit of light poses a restriction on the immediate number of
molecules one can see per unit area, the position of single-molecule spots separated
at distances larger than the diffraction limit of light can actually be established to a
precision of tens of nanometers. The accuracy of determining its center-of-mass
(
r
com
) depends
essentially on the number of photons
emitted through
N
1/2
, where
r
d
is the diffraction limited resolution and
N
the number
of photons (
Thompson, Larson, & Webb, 2002
). This means that in order to recon-
struct a superresolution image of a densely populated cell membrane, the challenge is
to have at each given time only a subset of molecules in the “on”-state and determine
r
com
for each molecule. This process is repeated many times such that all the calcu-
lated
r
com
are used to reconstruct a “superresolution” image. This indeed is the con-
cept of the superresolution technique called stochastic optical reconstruction
microscopy (
Rust, Bates, & Zhuang, 2006
), which is essentially based on carbocya-
nine dyes that reversibly switch between on and off states. An analogous method that
also uses image reconstruction but is based on fluorescent proteins, and therefore
directly applicable in live systems, is called (fluorescent) photoactivatable localiza-
tion microscopy (FPALM/PALM) (
Betzig et al., 2006; Hess, Girirajan, & Mason,
2006
). In here, the fluorescent proteins are engineered such that they can be switched
on by illumination with a 405 nm light source. After the few photoactivated fluores-
cent spot photobleach, a subsequent flash of the 405 nm light activates another set of
r
com
r
d
