TOPOGRAPHY AND RECOGNITION IMAGING OF CELLS - Life at the Nanoscale: Atomic Force Microscopy of Live Cells - page 151

with dimensions from ~10 to ~100 nm, which were non-uniformly
distributed on the cellular surface ( Fig. 7.5b ) . The recognition eficiency
was high and remained so during subsequent rescans. The speciicity of
binding was conirmed by the addition of 5 mM EDTA (Ca 2+ free conditions)
leading to the disappearance of almost all binding events in the recognition
image ( Fig. 7.5b ' ), whereas no change in the topography image was detected
( Fig. 7.5a ' ). Figures 7.5b + and b ++ illustrate a closer look at the recognition
spots. “Hot” spots consisting of one to two large domains (50-80 nm) could
clearly be seen surrounded by smaller domains (10-20 nm) or even single-
molecule spots (typically 1-4 pixels long, 1 pixel ~4 nm) by taking into
account the size of the VE-cadherin -dimer (diameter 3 nm) and the free

orientation of PEG linker leading to the speciic binding event before/after the
binding site position. More than 600 single speciic events were recognized,
and around 6000 active

cis

cis -dimers were calculated over the scanned

area (4 μm 2 ).
The receptor-binding sites can properly be assigned to the topographical
features for heterogeneous biological samples such as chromatin. 4 Figure
7.6a illustrates the superimposition of the recognition map onto the
corresponding topographical image. This procedure allows revealing the
locations of receptors in the topographical image with high lateral resolution
and high eficiency. Interestingly, only a few VE-cadherin domains were found
directly on the top of ilaments, whereas most domains were located near
and between ilaments. The last observation indicates that at this stage of
cell maturation (day 1 or 2 after seeding), the clustering of VE-cadherin is
incomplete.

(b)

(a)

Figure 7.6. (a) Overlay of recognition map of VE-cadherin (in green) onto the
corresponding topography image. A few VE-cadherin domains are situated directly
on the top of ilaments (arrows). Colour scale (dark brown to white) is 0-12 nm.
(b) Force distribution recorded on gently ixed MyEnd surface with VE-cadherin-Fc-
coated tip in Ca 2+ -rich conditions. 23