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interactions plotting the largest force obtained in each of 16 × 16 small
areas over 3 μm × 3 μm regions. The mapping results, shown in
Fig. 12.1b
,
reveal that the mannan distribution on the cell surface was not uniform at
all but highly varied over the cell surface. The authors reproduced the local
variations of the relative surface density of mannan based on the force curve
measurements on six different positions on a yeast cell surface, repeating
the measurements twice on all spots every 10 minutes to conirm the
reproducibility of the mapping results. Mapping results after longer time
intervals showed gradual changes in the local mannan density partly because
of the drift on the scanning area under the AFM probe and/or damages that
would have taken place to the immobilized concanavalin A on the AFM probe.
Two other convenient methods of yeast cell immobilization were
developed. Gad
et al.
37
conined yeast cells by half embedding them in a thin
layer of agarose gel so that the cells would not be rolled about under the
imaging force of the AFM probe. Under imaging with a bare probe, most
cells were proved to be alive from the growth of their height in the time
lapse AFM images. Another method of immobilizing yeast cells proposed
by Kasas
et al.
38
is based on the use of porous membranes, which is also
suitable for manipulations of cell surfaces with AFM.
12.3 MAPPING OF VITRONECTIN AND PROSTAGLANDIN
RECEPTORS
The radius of a typical AFM probe at the very tip is in the order of
10-50 nm. A sharp tip with small radius is good for high resolution
mapping of membrane proteins over a wide area of the cell surface. We
sometimes need a probe with a larger diameter to increase the contact area
with the cell surface. Kim
et al.
39
used a colloidal probe of 5 μm in diameter
to increase the area to be scanned by the force volume mode of the AFM.
With this probe, the mean indentation depth was about 165 nm and the
contact area was calculated to be 2.6 μm
2
. To obtain the contact area (
S
),
they assumed the Hertz contact model and used the following equation.
40,41
∫
0
T
= 2
π
sin
θ
θ
,
S
R
2
d
(12.1)
where
T
= sin
1
(
d
/
R
)
1/2
,
R
and
d
are the probe radius and the depth of
indentation, respectively.
Mapping of membrane proteins using a colloidal probe cannot measure
the unbinding force of a single ligand-protein pair. Therefore, Kim
used
the integrated area in the force-extension curve calling it the separation
work in the unit of J.
Figure 12.2
displays the result of mapping over 4 × 4
et al.
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