Biomedical Engineering Reference
In-Depth Information
Lipid membranes form extremely flat surfaces, and membrane proteins or lipid domains
often are visible in AFM as sub-nanometre height features, so it is generally desired that the
substrate be atomically flat. In order to form a bilayer the substrate should be hydrophilic,
so freshly cleaved mica is typically the best substrate to use, although silicon [646] or
template-stripped gold [647] may be used, particularly when a specific surface chemistry is
required. Langmuir-Blodgett (LB) film deposition is carried out by first forming a layer of
the lipids on a water surface, and passing the substrate through the air-water interface
[648]. This has some advantages in terms of control of the pressure of the bilayer, and the
ability to form half-bilayers or mixed bilayers. On the other hand, vesicle fusion is an
extremely simple process. A solution of vesicles is pipetted onto a mica surface and
typically left for 20-40 minutes for fusion to take place, before rinsing with buffer solution.
The vesicles collapse on the surface, leaving a well-organized bilayer, with typically some
remnant vesicles which are washed away [646, 649]. This preparation process is quick and
simple to carry out, although it lacks some of the control of the LB film technique. An
advantage for imaging under liquid is that the sample need never dry out during the
preparation process. Imaging of bilayers can be carried out in contact or oscillating
modes, (either non-contact AFM or IC-AFM) [649], [125] and in air or, more commonly,
in liquid [650].
In either case, when bilayers are prepared, the sample is often referred to as a supported
lipid bilayer (SLB). This is to make explicit the fact that these bilayers are not in vesicle
form, but on a surface; they will therefore have some interaction with the substrate surface.
In fact it's known that the interaction with the surface changes somewhat the properties of
the bilayer compared to a vesicular bilayer. An example showing height differences in
the bottom bilayer of a multi-bilayer stack due to this surface interaction is shown in
Figure 7.22. Studies of bilayer structure can include measurement of phase separation
in mixed lipid systems, which is an important process due to the involvement of lipid rafts
in many biological processes [129, 651]. Phase separation can be studied by a number of
AFM techniques. Due to the incredibly high height resolution of AFM, discrimination of
phases with a few
˚
height difference can be carried out directly [129]. In addition,
friction contrast (measured by LFM) [652-654], or phase imaging [471, 655] can help to
differentiate phases of very similar heights. Examples of this are shown in Figure 7.22. If a
nanoindentation-type experiment is carried out on lipid bilayer with a flexible probe, then
at a certain threshold force, a 'breakthrough' can be observed in the force-distance curve
[129, 656]. This can be used as a measure of the coherence of the lipid film, and its
thickness may be measured from the curve. Practically, this measurement can be used to
prove the existence of the films, as lipid bilayers on mica can be uniform to the point of
being featureless [308].
AFM is also ideal to study the interaction of peptides or proteins with membranes [657].
Some peptides are known to disrupt and damage lipid membranes, whilst other are drug
candidates that need to be able to cross membranes. The changes in lipid membranes are
typically disruptions with dimensions on the order of a few nanometres, so the action of
these materials is usually studied indirectly. With AFM their action can be observed
directly, typically manifesting as appearance of small 'holes' or other morphological
changes in the SLBs [658-661].
Probably the most important application of studies of SLBs is the study of protein
incorporation in membranes. By AFM this is rather simple, and can be performed by
