Biology Reference
In-Depth Information
2.
Some manufacturers glue cantilevers to a base. Test whether the glue is soluble
in methanol before preforming crosslinking.
3.
Cantilever with different spring constant, with and without coating for better laser
reflection, as well as variety of sizes, shapes and material of tips are available.
Principally, contact-mode silicon nitride cantilevers with spring constant
0.01-0.1 N/m, coated with gold on the top side for better reflection of the laser,
with tips of 10-50-nm diameter should be used. For smaller molecules, smaller
and sharper tips are recommended in order to match the size of molecule with a
tip. This assures that only one molecule can be accommodated on the tip and thus
measurements of adhesion forces between individual molecular pairs.
4.
Different brands of turbo vacuum evaporator are available. Follow the manu-
facturer's instruction for proper coating of gold.
5.
To keep gold more firmly attached to mica and silicon nitride, a 10-30-nm chro-
mium layer can be deposited before gold coating
(1)
.
6.
Other crosslinking buffers such as 0.1
M
bicarbonate pH 8.0 or 50-100 m
M
phos-
phate buffer pH 7.0-9.0 can be also used.
7.
Different types of crosslinking procedures are possible with a variety of commer-
cially available bifunctional crosslinkers for carboxyl amino and sulfhydryl
groups
(1
,
3)
. It is essential to form a selfassembled monolayer to shield long-
range electrostatic forces between gold-coated surfaces (
see
Fig. 2
).
8.
Care should be taken when manipulating cantilevers. Tips should never be
touched and not dried during crosslinking of adhesion molecules and mounting
to AFM.
9.
Each AFM manufacturer has slightly different computer software procedures for
calibrating scanners, adjusting the photodetector, optimizing the signal of the
reflected laser and for the contact mode approach, acquisition of force-distance
curves, and direct determination of the spring constant of the cantilevers. Manu-
facturer instructions should be carefully read to avoid crashing the tip on the
surface and to assure quantitative measurements.
10.
Different brands and models of AFMs have different designs of liquid cells. Some
are more complicated and require patient following of the procedure to avoid
bubble formation. The simplest liquid cell operates in a droplet of solution. In
this case, exchange is easy and bubble formation can be avoided, however, cau-
tion should be taken for evaporation after extensively long work, which can lead
to changes of concentration. Thus, more frequent exchanges of solution are nec-
essary. Prevent heating of the small volume of liquid by strong-illumination
optical-microscope devices available with some AFM brands.
Acknowledgments
This work was supported by the private funds of Dr. G. N. Misevic, Conseil
Régional Nord-Pas de Calais, CNRS, Marcel Mérieux Foundation, Swiss
National Science Foundation, and Cancer League Basel.
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