Biology Reference
In-Depth Information
which may have occurred on the shields. It is often useful to switch the beam on
and allow the resulting slight temperature increase to assist with frost removal.
3.1.4. Image Acquisition (See Fig. 4)
1.
Frozen hydrated samples are extremely sensitive to beam damage, including mass
loss, upon exposure to electrons.
2.
Set the beam current at a minimum when first examining the specimen. Electron
dose on the sample needs to be carefully controlled if damage is to be prevented.
3.
Carry out searching, focusing, and basic alignment at low magnification, prefer-
ably with the assistance of a video rate image-intensifying camera.
4.
Carry out higher magnification alignment and focusing on an area adjacent to the
area of interest. Many microscopes have either beam-shift or image-shift con-
trols to simplify this task. The only electrons that should strike the area of interest
should be those used for image recording.
5.
Ideally, keep the total accumulated electron dose in the area of interest below 10
electrons Å
-2
. Frozen-hydrated samples exhibit poor contrast and under focus of
the objective lens is often necessary to increase image contrast.
6.
Only record structures lying over holes in the carbon film. Carbon-filmed areas
are used only for recording images for image assessment purposes.
7.
Notes on image analysis are in
Subheading 4.
,
Notes 1
and
2
.
See
Note 3
on
energy filtered TEM.
3.2. Mica Ice-Wedge Procedures
3.2.1. Formation of the Mica Ice Wedge
(
See
Note 5
)
1.
Cut a 75 mm
×
25 mm
×
0.15 mm sheet of mica into pieces of 15 mm
×
10 mm
×
0.015 mm with a rotary trimmer.
2. Prepare a sample of reconstituted type I collagen fibrils in phosphate buffer
(
I
= 0.2, pH 7.4) by the “warm start” procedure
(8)
.
3.
Disperse the resultant fibril gel into fibril fragments in Tris-buffered saline using
ultrasonic disintegration.
4.
Take one of the trimmed pieces of mica and cleave it. Onto the cleaved surface of
one half of the mica, put 3
L of the collagen sample and then place the other half
of the mica on top so the cleaved faces are together. Leave this “sandwich” for
30-60 s to allow the fibrils to adsorb to the mica. (
See
Note 5
.)
µ
5.
Submerge the “sandwich” in ultrapure water, separate the mica, and, holding
each piece separately with watchmakers forceps, waft them in the water for
15-30 s. Reassemble the “sandwich,” overlapping the longest edges by 2 mm,
and withdraw it from the water.
6.
Remove the excess water from the outer surfaces of the “sandwich” with filter
paper and then place it on the stage of the Cressington CFE-50C freeze-fracture/
etch device. Clamp the two halves of the mica together over the overlap and then
insert a 15 mm
0.25 mm aluminium spacer between the mica at the
opposite unclamped end, so that it overlaps the lower piece of mica by 2 mm.
×
15 mm
×
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