Biology Reference
In-Depth Information
This is achieved by adjusting the current and voltage to the guns and the duration
of shadowing. Film thickness is determined by quartz-crystal-film-thickness
monitors built into the Cressington.
3.
With the stage of the Cressington set at -189
C and the microtome still shielding
the sample, degas the platinum and carbon guns. The guns are degassed at a preset
current and voltage (lower than that used for evaporation), for a preset duration.
°
4.
Start the stage rotating at approximately 60 rpm and move the microtome arm
clear of the sample, and then start evaporating from the platinum/carbon gun.
Check to be sure there is an appropriate thickness of platinum with the thickness
monitor and then back it with carbon from the carbon gun (also to an appropriate
thickness) to complete the replica.
5.
When the replica is complete, stop the stage rotating and attach the transfer rod to
it. Withdraw the stage from the vacuum chamber through the airlock. Detach the
mica with the replica on it from the stage and allow it to reach room temperature
under cover, in preparation for replica collection.
3.2.4. Collection of the Replica
1.
Once the mica and attached replica are at room temperature, pick up the mica at
the edge of one of its shorter sides with watchmakers forceps.
2.
Slide the mica into a reservoir of ultrapure water at an angle of approximately
20
. The replica should detach from the mica and float on the surface of the
water. (
See
Note 7
.)
°
3.
Looking at the replica, a thin band appears shiny (where the thinnest part of the
wedge was originally). This is where freeze-drying has occurred and most of the
water has sublimed off the mica. Next to the shiny band is a wider region of duller
replica where there has been no freeze-drying of the sample (as the ice in the
wedge was much thicker here) and an ice layer still persists on the mica surface. The
region between these two bands is where the replica needs to be collected in order to
get fibrils that have been exposed by etching, but retain much of their water.
4.
Grip the edge of a transmission electron microscope grid with watchmakers for-
ceps and submerge it in the reservoir of ultrapure water, away from the replica.
Bring the grid underneath the appropriate part of the replica and raise it slowly,
to attach the selected region.
5.
Bring the grid clear of the water and place it carefully on grade-50 filter paper to
remove any water from it, being careful not to damage the replica.
6.
Once the replica has been collected on several grids, and they are dry, they can be
stored in a grid box or on filter paper in a Petri dish before viewing in a conven-
tional TEM.
3.2.5. Electron Microscopy
1.
Examine the platinum/carbon replicas of the collagen fibrils in a transmission
electron microscope operated at an accelerating voltage in the region of 100 kV.
2.
Images can be collected on normal photographic film or recorded digitally on a
CCD camera attached to the microscope.
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