Biology Reference
In-Depth Information
15. Among all the modes of measurement provided by WSxM,
Jumping Mode plus (JM+) is optimal to image adenovirus
because very low, accurate normal forces are applied to avoid
damage of the sample by the scanning tip [
37
].
16. Adjust scanning parameters: (a) Set the
setpoint
to measure
about 0.05 V. This will determine the force that the micro-
scope applies on the sample. The applied force depends on the
sensitivity of the microscope, that it is in turn related to the
laser spot position on the cantilever and the photodiode. (b)
Set
Jump off
to a height slightly larger than the half height of
the viral particle (i.e., 45 nm for adenovirus). (c) Set
Jump
sample
to approx. twice the value of the
jump off
. (d) Set
con-
trol cycles
to a number high enough to let the feedback act (i.e.,
20). (d) To use JM+, turn on the
Stop moving if limit is reached
box and use the parameters:
limit(V)
higher than the measure-
ment set point. The
dragging factor
suggested by WSxM
usually works well. Feedback parameters depend on the piezo
actuator and cantilever.
17. Although resolution depends on the tip shape, it should be
possible to resolve the hexon trimers with the conditions
described here.
18. If needed, cells can be prepared for sectioning in their mono-
layer disposition rather than in a pellet. Autoclave circular glass
coverslides and incubate on a drop of poly-lysine (use a 100 μL
drop for a round cover slide of 12 mm diameter) for 30 min at
37 °C. Wash the coverslide with PBS and place in a culture
plate (12-well plates are recommended). The poly-lysine keeps
the cells attached to the glass throughout the subsequent pro-
cedures. Follow the steps described in Subheading
3.6.1
for
fixation and embedding, but use ethanol instead of acetone for
dehydration. For encapsulation, use gelatin instead of beem
capsules. Fill the longest half of the capsule with Epon and
place it upside down on the glass slide, which should be cov-
ered by a thin layer of Epon. Polymerize for 48 h at 60 °C. For
ultramicrotomy, the glass is removed using a rapid freeze-
thawing procedure, transferring the capsule and attached glass
quickly from liquid nitrogen to hot water several times.
19. Epon can be prepared in advance, with the exception of BDMA
that must be incorporated on the same day of use. The mixture
of resins (without BDMA) can be kept under gentle rotation
over night to mix well.
20. A new batch of fresh Epon without BDMA can be prepared at
this point and left under rotation overnight for use the follow-
ing day.
21. An alternative to the standard Epon embedding methodology
is
freeze substitution
. Freeze substituted samples are dehydrated
