Biology Reference
In-Depth Information
its best resolution is restricted to certain dyes able to withstand repeated cycles of
excitation and depletion at extremely high intensities.
Using properly chosen fluorophores, this technique has been used to estimate pro-
tein distribution and cluster sizes on the cell membrane (
Fig. 6.2
;
Manzo et al., 2012;
Sieber et al., 2007
). To obtain information regarding the nanoscale dynamics in living
cells, STED has been combined with fluorescence correlation spectroscopy (FCS)
(
Eggeling et al., 2009
). Initially, it was shown that unlike randomly diffusing nonraft
lipids, glycosyl-phosphatidylinositol-anchored proteins (GPI-APs) and sphingolipids
are transiently trapped for about 10-20 ms in
20 nm-sized cholesterol-dependent
molecular complexes. Recently, these observations have been extended to more active
involvement of cytoskeleton-mediated interactions (
Mueller et al., 2011
).
STED can take advantage of standard confocal geometries and therefore allows
optical sectioning at the nanoscale and even provides video-rate imaging (
Westphal
et al., 2008
). However, STED requires fluorophores that can cycle many times be-
tween dark and bright states. In addition, since the two beams that reshape the ex-
citation field should be aligned properly in time and space, the optical design is
rather complex and multicolor imaging challenging.
<
6.2.3.3
Near-field techniques (NSOM)
A different concept that breaks the diffraction limit of light providing optical super-
resolution at the nanometer scale is NSOM. In NSOM, a sharp probe physically scans
the sample surface generating simultaneous optical and topographic imaging of the
sample under study. The most generally applied near-field optical probe consists of
a small aperture, typically 20-120 nm in diameter (i.e., much smaller than the wave-
length of the excitation light), at the end of a metal-coated tapered optical fiber. The
probe funnels the incident light to dimensions that are substantially below the diffrac-
tion limit. This results in a light source that has the size of the aperture. However, in
contrast to common light sources such as lightbulbs and lasers, the light emitted by the
probe is predominantly composed of evanescent rather than propagating waves. The
intensity of the evanescent light decays exponentially to insignificant levels at
100 nm away from the aperture. Effectively, the probe can excite fluorophores only
within a layer of
100 nm from the probe, that is, in the “near-field” region. The sam-
ple fluorescence can subsequently be collected by conventional optics and trans-
formed into an optical image of the sample surface in which the resolution is now
primarily dictated by the aperture dimensions rather than by the wavelength of the
light. Noteworthy, NSOM imaging can be obtained with any fluorophore and the il-
lumination geometry allows straightforward implementation of multicolor excitation.
However, like other raster-scanning techniques, NSOM imaging is inherently slow.
NSOM has been used to gather information on membrane protein organization,
protein clusters sizes, and also on membrane proteins colocalization (
de Bakker
et al., 2007, 2008
). Recently, single-molecule near-field nanoscopy has been used
to visualize the nanolandscape of G
M1
after binding by its ligand cholera toxin
(CTxB) on intact monocyte membranes (
van Zanten et al., 2010
). Pentavalent bind-
ing of CTxB to G
M1
was sufficient to initiate a minimal raft coalescence unit,
<
