Biology Reference
In-Depth Information
(amorphous) ice ( Fig. 1 ). The vitreous ice technique has been described in
detail elsewhere ( see [4] for review), and a complete description is beyond the
scope of this chapter. In brief, molecules rapidly frozen in a thin (10-100 nm)
ice layer can be imaged unstained in a suitable transmission instrument oper-
ated in low-dose mode (1-10 electrons Å -2 ). The technique preserves the native
structure of the specimen to near-atomic resolution. Images recorded under
these conditions have a low signal-to-noise ratio and generally require image
processing and averaging procedures to extract high-resolution information.
The second technique is aimed at acquiring structural information about the
surface of hydrated collagen fibrils. This consists of high resolution shadowing
of freeze-etched fibrils with retained bound water ( Fig. 2 ). Raspanti et al. (5)
have compared different methods for studying the surfaces of collagen fibrils
and have found that the highest resolution information is obtained by freeze-
etching and rotary shadowing of a freeze-fractured tendon sample. To examine
collagen fibrils in suspension, originating either from in vitro collagen fibril
assembly systems or tissue, we have developed a freeze-etching and shadow-
ing (mica ice-wedge) procedure to cryopreserve collagen fibrils in a hydrated
state. The mica ice wedge procedure is related to the quick-freeze deep-etch
method described by John Heuser (for review see ref. [6] ).
2. Materials
2.1. Vitreous-Ice Procedure
1.
Chloroform (BDH, London, UK).
2.
Formvar (BDH).
3.
Copper and nickel grids (200 and 400) (Agar Scientific, Stansted, UK).
4.
6-mm carbon rods (Agar Scientific).
5.
Nanotech coating unit.
6.
Ultrapure water (Purite Stillplus HP).
7.
Glass slides (Blue star).
8.
Glycerol (BDH).
9.
Fine forceps (Agar Scientific).
10.
Acetone (BDH).
11.
50-Grade filter paper (Whatman, Clifton, NJ).
12.
Ultrasonicator bath (Agar Scientific).
13.
3-in-1 oil (E. R. Howard Ltd., Stowmarket).
14.
Dispersed collagen fibrils from bovine skin (7) .
15.
Ethane (BOC, Trafford Park, Manchester, UK).
16.
Controlled Environment Vitrification System (CEVS, provided by Professor Ishi
Talmon, ishi@tx.technion.ac.il: http://www.technion.ac.il/technion/chem-eng/
talmon/) (see also ref. [7] ).
2.2. Mica Ice-Wedge Procedure
1.
Mica sheets 75 mm
×
25 mm
×
0.15 mm thick (Agar Scientific).
2.
Rotary cutter (Myers precision cutter).
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