Biology Reference
In-Depth Information
BAF060 unit. Try to keep distance and time of transfer as short as possible, in order
to minimize condensation and ice crystal formation on the samples. When using
freeze-fracture units without an air lock (such as the Balzers BAF 400T), vacuum
of the vacuum chamber will have to be broken right before introducing sample
and vacuum will then have to be reestablished before continuing the procedure. Po-
sition the cold shutter carrying the knife above the specimen table (
Fig. 21.2
, right)
and raise the temperature of the specimen table up to
80
C, while the temperature
150
C. This permits sublimation of water molecules to
form in the surface of the cells with the shutter functioning as a cold trap. Proceed
with etching for 12 min to sublime surface ice.
The specimens are then shadowed with a 2 nm thick layer of atomic platinum
from the Pt/C-loaded electron evaporation beam gun. This physically fragile
electron-dense replica is strengthened by a uniform 20 nm thick electron-translucent
carbon layer using the C-loaded gun. The thickness of the deposited layers is mea-
sured with a quartz crystal film thickness monitor included in the freeze-fracture unit.
The specimen table can then be removed from the unit using the loading/retrieval
device after which one should immediately proceed with cleaning and mounting
of the replicas (see the succeeding text). To perform more than one round of replica
generation, it is convenient to have a clean and dry replacement specimen table
ready. In general, the carbon gun can be used twice before it needs to be reloaded.
The Pt/C gun can be used several times before it is exhausted, but you must check the
position of the Pt with respect to the tungsten filament after each session.
of the shutter remains at
21.3.6
Cleaning and mounting of replicas
Remove specimen table from the freeze-fracture unit. Immediately upon retrieval,
shadowed micas are removed from the specimen table, and while holding them with
tweezers at the end that was positioned under the holding clamps of the cold table and
with the coated surface facing upward, they are very slowly submerged at a 30
-45
angle in a well of a porcelain spotting dish filled with undiluted domestic bleach
(
Fig. 21.5
, left). The replica should come off the mica piece at the surface of bleach
solution and float. It is normal that the replica breaks in smaller pieces upon floating.
However, if it disintegrates in barely visible pieces, the sample cannot be processed
any further and has been lost (also see
section 21.5.9
, replica floating). Replica pieces
are incubated at room temperature overnight floating on bleach for digestion of
organic material. To remove attached organic material, floating replica pieces are
washed two times with triple-distilled H
2
O by carefully transferring them onto the
surface of water-filled wells using a molten tip Pasteur pipette (
Fig. 21.5
, center).
Perform a third wash for 1 h. Pick up single-layer replica pieces with bare 400 mesh
hexagonal copper grids directly from the water surface. Any trace of water is
carefully removed with pointed strips of filter paper applied to the edge of the grid
and grids are placed on tissue paper for desiccation. Watch out to pick up unfolded
replicas as doubling will impair transparency of the sample during TEM observation.
