OBSERVING THE NANOSCALE ORGANIZATION OF MODEL BIOLOGICAL MEMBRANES BY ATOMIC FORCE MICROSCOPY - Life at the Nanoscale: Atomic Force Microscopy of Live Cells - page 2

In this complex context, artiicial membranes have been extensively used
to mimic membrane organization, using either free-standing membranes like
liposomes or planar and supported model membranes. 4 Giant unilamellar
vesicles (GUVs) are very useful to study dynamic events and have been
widely used to explore lipid domain formation using single-molecule optical
microscopy. 5 However, this approach is restricted by diffraction-limited
resolution and is therefore not suitable to probe membrane on the mesoscopic
scale. Membranes supported on a solid support (supported lipid bilayer,
or SLB) are very useful and robust systems that are compatible with most
biophysical techniques, including luorescence microscopy, ellipsometry
and atomic force microscopy (AFM). The advantage of AFM, compared with
other techniques, is the possibility to image, in real time, the topography of
samples with nanometer lateral resolution. AFM, which consists in raster
scanning of a sample surface with a sharp tip at the end of a soft cantilever,
has been largely used for probing the two-dimensional (2D) organization of
model membranes and for elucidating the mechanisms underlying lateral
segregation of membrane constituents, especially membrane microdomain
formation (for recent reviews see Refs. 6-8). Structural information of
membrane proteins incorporated into SLBs with a subnanometer lateral
resolution can also be obtained under conditions where proteins are tightly
packed. 9,10
In this chapter, we describe the main strategies to prepare SLBs that are
suitable for AFM analysis. After a brief methodological description of AFM
imaging in liquid, we review major advances in the exploration of the topology
of SLBs, focusing on the study of membrane microdomains and of membrane
proteins. Progress in nanobiotechnology and recent technical developments
that have improved the time and lateral resolution of AFM are also covered.
1.2 PREPARATION OF ARTIFICIAL SUPPORTED LIPID
MEMBRANES
Artiicial membranes are generally prepared on chemically inert, hydrophilic
and flat solid supports, such as mica, highly oriented pyrolitic graphite, glass,
silicon and gold. Different methods have been developed to prepare SLBs, but
the most popular technique, irst described by McConnel's group, 11 remains
the formation of supported membranes by fusion of large unilamellar lipid
vesicles (LUVs) on a solid surface. LUVs are generally prepared via sonication or
extrusion, and the vesicle solution is then added on top of the support. Vesicles
then adsorb on the substrate before rupturing ( Fig. 1.1 ) . The composition of
the buffer bathing the substrate has to be inely tuned for allowing optimal