Biology Reference
In-Depth Information
20. Ensure chips are transferred between liquids quickly and there
is never any risk of air drying.
21. This allows any ethanol trapped in the sample time to
diffuse out.
22. This step allows the oxidized layer of chromium on the target
to be removed by pre-sputtering onto the shutter.
23. If there is any hint of brown, it suggests that the coating is
contaminated with chromium oxide. Reasons for this include
insuffi ciently initial vacuum prior to introducing argon, vac-
uum leaks, leaks in the argon pipes, air in the argon pipes after
changing the cylinder, and insuffi cient pre-sputtering onto the
shutter. If the coater has not been used for some time, chro-
mium oxide can build up on the target which will then require
longer pre-sputtering on the shutter. This can happen even
when the unit is kept under vacuum. It is good practice to
keep a chromium coating unit pumping continuously to keep
the chamber and vacuum meticulously clean.
24. Chromium fi lms on samples oxidize rapidly and samples
should be examined ideally on the same day they are coated.
There is a noticeable loss of signal over a few hours and a sig-
nifi cant loss over a few days. The deterioration of the coat can
be slowed by storing samples in a vacuum.
25. 10 kV has been found to be optimal in our Hitachi S5200
SEM but 30 kV was better in the Topcon DS130F; therefore,
it is necessary to experiment with different voltages and other
conditions to optimize for the sample, gold size, and specifi c
microscope.
26. If beam damage is observed, the current may have to be
reduced. Some analytical SEMs (such as the Hitachi SU70)
are capable of very high beam currents which would be
inappropriate.
27. It requires some experience to search and identify the struc-
ture of interest as the sample has many remnants of mem-
branes, organelles, and protein complexes that are not
necessarily of interest.
Acknowledgments
This work was supported by grants from the Biotechnology and
Biological Sciences Research Council, UK, grant number BB/
E015735/1 and BB/G011818/1. Thanks to Christine Richardson
and Helen Grindley for technical assistance.
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