Biology Reference
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in their subsequent phase, the LE phase will be lower in height. Indeed, by
speciically labelling the LE phase, a perfect correlation was found between
topographical and luorescence signals.
47
To extend these indings, Hollars
and Dunn used tapping-mode feedback NSOM in air to additionally obtain
compliance information of the lipid monolayer.
48
Because the carbohydrate
chains of the lipids from the LC phase are highly saturated, they pack in an
ordered fashion as compared with the lipids from the LE phase. As expected,
the LC phase was found less compliant than the LE phase.
As such Hollars
and Dunn demonstrated the strength of NSOM as compared with luorescence
or scanning probe techniques on their own.
In a more recent work supporting the formation of lipid rafts on model
membranes, small amounts of labelled GM1 revealed that GM1 is not
homogeneously distributed throughout the LC phase. Instead, they were seen
to constitute their own 100-200 nm sized domains.
48
In fact, upon closer
examination, the labelled GM1 distribution appeared to be more complex. To
better characterize the GM1 behaviour, GM1 lipids were labelled with Bodipy.
This luorophore displays a redshift in the emission spectra when present in
higher concentrations because of excimer formation, thus being able to probe
the local lipid density.
49
Because of the strong tendency of GM1 to partition
in gel or liquid-ordered phases, high-concentration GM1 was found in the
LC phase, showing the redshifted emission, even while using low deposition
pressures.
50
Nevertheless, a rather large fraction of single Bodipy-GM1 was still
found randomly distributed in the LE phase. Upon increasing the deposition
pressure towards expected cell membrane pressures, the LC domain phases
became smaller, and the labelled GM1 appeared to preferentially partition
into the LC phase.
51
The use of NSOM to investigate monolayers has also been extended towards
bilayers
51
52
and protein containing lipid layers in dry or buffer conditions.
41,53-
55
The addition of proteins to such lipid phase-segregated model systems
will be an important step in understanding how lipid-based interaction can
inluence protein distribution. Subsequently, monitoring the dynamics would
then provide a more complete spatio-temporal map of proteins and lipids
in a lipid bilayer. Work in this direction has been performed using AFM in
combination with luorescence correlation spectroscopy (FCS).
The recent
proof-of-principle indication that dynamical studies can be also performed
with NSOM
56
opens up an exciting ield that combines high-resolution
imaging with ultrafast dynamics. Indeed, the advantage of performing FCS
on conined volumes has been recently demonstrated on living cells.
57
The
incorporation of this approach in NSOM would also provide, in addition to
surface sensitivity, topography and resolution, temporal information.
58,59
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