TOPOGRAPHY AND RECOGNITION IMAGING OF CELLS - Life at the Nanoscale: Atomic Force Microscopy of Live Cells - page 147

microscopy (for more details see Chapter 9 ) 14,15 or stimulated emission
depletion microscopy. 16 The lateral resolution in these studies ranged from a
few tens of nanometres 13-16 to ~200 nm because of the diffraction phenomenon
also known as Abbe limits. In addition, no information about topography can
be attained. At present, AFM offers an exceptional solution for obtaining
topography images with nanoscale resolution and single molecular interaction
forces on different biological specimens such as proteins, DNA, membranes,
cells, etc., under physiological or near-physiological conditions and without
the need for scrupulous sample preparation or labelling. 17 Hence, the spatial
nano-mapping of molecular recognition sites can be obtained by performing
AFM adhesion force mapping using the force-volume technique, which
represents the molecular recognition imaging using force spectroscopy 18-21
(for more details see Chapter 12 ). On the other hand, dynamic recognition
mapping (TREC) is faster and enables better lateral resolution than adhesion
force mapping. 1,2,4 Because of the continuous progress in the technical aspects
of the AFM and “smart” tip functionalization procedures, the investigations
of receptor-ligand interactions on living cells at the single-molecule level
have become achievable. Because cells represent systems of more complex
composition, organization and processing in space and time than proteins,
the next goal is the application of TREC to cellular membranes that contain
different functional domains enriched in sphingolipids, cholesterol and
speciic transmembrane proteins. 22

7.4.1 Nano-Mapping of Vascular Endothelial-Cadherin on
Endothelial Cells

The irst TREC studies on cells were conducted on microvascular endothelial
cells from mouse myocardium (MyEnd) to locally identify vascular endothelial
(VE)-cadherin binding sites and correlate their position with membrane
topographical features ( Fig. 7.3 ) . 23 VE-cadherin belongs to the widespread
and functionally important family of calcium-dependent cell adhesion
molecules, cadherins (this name arises from the approximate contraction
of “Calcium dependent ”), which are single-pass

transmembrane glycoproteins known to be crucial for calcium-dependent,
homotypic (or homophilic) cell-cell adhesion 24 and are also essential for the
morphogenesis of tissues and the maintenance of tissue function. In the case
of vascular endothelium, the adhesion between cells has to be strong enough
to resist the hydrodynamic forces created by blood low (shear stress of up to
10 Pa) or blood pressure (wall distension). VE-cadherin is strictly located at
intercellular junctions of essentially all types of endothelium. This molecule
not only regulates adhesive intercellular endothelial junctions (e.g., adherent

ADHER ent prote IN