Biology Reference
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To overcome issues associated with cell elasticity and lateral diffusion of
receptors, a ixation procedure can be applied similar to immunochemistry
experiences. The ixation procedure usually makes the soft biological objects
stiffer, and as a consequence, it generally gives access to high lateral resolution
in AFM images as was observed with proteins (GroES).
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However, the common
ixation of cells in buffer solution at room temperature causes the smoothing
of the cell surface with the loss of most ilamentous features, which were seen
in AFM pictures of living cells.
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The nucleus also most probably becomes
visible because of the membrane collapse during dehydration caused by
the ixation procedure. When the unpuriied solution of glutaraldehyde is
used, the undesirable formation of globular large features on the cell surface
(e.g., polymers of glutaraldehyde) can also be detected. A method has been
found to gently ix the cells with a solution of glutaraldehyde containing
monomers (EM grade) similar to the procedure described by Oberleithner
and co-workers.
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The prepared stock solution of glutaraldehyde (~200 μL,
5% in Hank's balanced salt solution [HBSS]) was added and gently mixed
with the culture medium (~2 mL), and the cells were then incubated at 37
°C for 1-2 hours. Such a method is likely to prevent unexpected osmotic and
temperature changes in the cell culture medium. As a result, the cell volume
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which makes further AFM investigations possible at a subcellular level.
(b)
(a)
Figure 7.4.
(a) AFM topography image of gently ixed MyEnd cells. Colour scale
(dark brown to white) is 0-400 nm. (b) Schematic of dynamic recognition imaging to
visualize VE-cadherin on MyEnd cell surface.
AFM functional imaging was performed with magnetically coated AFM
tips that were decorated with a recombinant VE-cadherin-Fc
cis
-dimer
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