Biology Reference
In-Depth Information
gun loaded with a platinum-carbon rod (95% Pt; 5% C) in the gun holder of the vac-
uum chamber at an angle of 45
and the electron gun loaded with the carbon rod at 90
.
Switch on the unit and evacuate the vacuum chamber. Cool down the freeze-fracture
unit to
150
C and wait until a vacuum of at least 10
6
mbar (usually 5
10
7
mbar
in the BF060) is reached. This process will take
2 h. It is advisable to always carry
out a trial coating prior to the actual freeze-etching and shadowing of the samples in
order to check that the evaporation devices function correctly. In case of using new
pieces of carbon rods, degassing is needed before making the first evaporation.
21.3.5.2
Mounting of samples on the specimen table
The samples need to be mounted on a specimen table before they can be introduced in
the freeze-fracture unit. We modified the specimen clamp of a specimen table with a
retaining spring for 0.3
0.8 mm gold specimen carriers so that a larger area of the
mica rectangles containing the labeled cells gets exposed (
Fig. 21.3
). Insert the lever
for the retaining clamps in the specimen table, and position it in the loading position
(
Fig. 21.3
, right). Fill a bench Dewar with liquid nitrogen and completely submerge
the specimen table loading device with the specimen table in place as well as a metal
container to hold the cryotubes with mica sandwiches (
Fig. 21.4
) and wait until the
loading device and specimen table have completely cooled down (i.e., when the liq-
uid nitrogen stops bubbling). Make sure that the specimen table and loading device
remain covered with liquid nitrogen during the complete mounting procedure. Place
a cold light source with two flexible light guides around this setup. Transfer a cryo-
vial containing the frozen mica sandwich into the metal container using insulated
rounded tweezers. Open the cryovial under liquid nitrogen using two pairs of
rounded tweezers and let the mica sandwich slip out of vial onto the sample support-
ing platform of the loading device. Submerge two pairs of fine-tipped Dumont twee-
zers into the liquid nitrogen to let them cool down. Warm tweezers may cause
crystallization of vitreous ice in and around the cells on the mica, which in turn
may damage the cells. Use one pair to hold the mica sandwich (taking care to keep
it under the liquid nitrogen), and use the other pair of tweezers to separate the two
mica halves. Identify the mica containing the labeled cells by means of the cut cor-
ner, and slide it with the cell containing side upward under the retaining clamp of the
specimen table. Discard the other half. Depending on their size, 3-4 mica rectangles
will fit on a single table, but care should be taken that the micas do not overlap. Use
another pair of fine-tipped tweezers to split and position the next sample. Alterna-
tively, thoroughly dry the same set of tweezers with a conventional hair dryer before
reusing them. Do not forget to precool tweezers before handling the micas. When all
micas have been positioned on the specimen table, immobilize the micas by turning
the lever into the locking position (
Fig. 21.3
, left) and then removing it.
21.3.5.3
Etching and replication
The specimen table with the frozen samples is quickly transferred to the precooled
temperature-controlled specimen stage within the high vacuum chamber of the
freeze-fracture unit using the loading/retrieval manipulator provided with the
