Biology Reference
In-Depth Information
11.
Place the grids in a conventional TEM to check the holey carbon films are suit-
able for use. Grids are then stored in grid boxes or on dry filter paper in Petri
dishes prior to use.
3.1.2. Preparation of Samples in Vitreous Ice
1.
Prepare the CEVS (
see
Fig. 3
)
(7)
by filling the reservoirs with the same buffer
used to prepare the samples. The buffer-soaked sponges provide a large surface
area and thereby maintain the humidity of the chamber. Adjust the temperature of
the chamber to 35
°
C and allow the system to reach equilibrium.
2.
Grip holey carbon grids (
see
Subheading 3.1.
) with the modified forceps fitted
to the plunging arm of the CEVS.
3.
Fill the freezing chamber with liquid nitrogen. Add the ethane gas slowly to the
cryogen pot, where it first condenses, then starts to freeze. Condense more gas
immediately prior to sample plunging to thaw the surface of the solid ethane.
4.
Place 3
L of the sample onto the holey carbon side of the grid and remove excess
liquid by touching filter paper to both sides of the grid. This is aided by use of a
binocular microscope. If chamber humidity is properly controlled, then the
resulting thin film of sample will remain stable. The thickness of the film can be
judged by eye.
µ
5.
When a suitable sample-film thickness (~100 nm) has been achieved, the grid is
plunged into the liquid ethane. The grid is then transferred into a small container
of liquid nitrogen. Traces of ethane will freeze on the surface of the grid protect-
ing it from frosting during transfer to the microscope.
3.1.3. Electron Cryomicroscopy
1.
Fill the microscope anticontaminator Dewar flask with liquid nitrogen at least 1 h
before use.
2.
Set the accelerating voltage prior to use to stabilize the H.T. voltage.
3.
Place the cryospecimen holder under vacuum in the microscope column. Cool
the sample to the working temperature (typically -180
C). The Dewar on the
cryoholder should be pumped out periodically to maintain a vacuum adequate to
permit the specified ultimate temperature to be achieved.
°
4.
Transfer frozen grids, under liquid nitrogen, to a liquid nitrogen-cooled worksta-
tion close to the microscope. Remove the cryoholder from the microscope and
move it to the workstation. Occasionally, splash the sample holder with liquid
nitrogen to maintain the tip at low temperature.
5.
Remove the grid-retaining ring with a cold tool and transfer the frozen grid to the
holder with precooled forceps. Two pairs of forceps are often required because
the grid can become charged with static electricity, making it difficult to handle.
Ensure that the grid is sample-side up and refit the retaining ring with the cold
tool. Most cryoholders have shields that can be brought into place to protect the
grid from frosting as the holder is transferred to the microscope.
6.
Pour liquid nitrogen over the tip of the holder immediately prior to transfer.
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