Biology Reference
In-Depth Information
8.
Liquid nitrogen (BOC).
9.
Cressington CFE-50C (Cressington Scientific Ltd., Watford).
10.
Copper and nickel grids (400 and 600) (Agar Scientific).
11.
Grid boxes (Agar Scientific).
3. Methods
3.1. Vitreous-Ice Procedures
3.1.1. Preparation of Holey Carbon Films
1.
Prepare a solution of 0.2% formvar in chloroform.
2.
To 90 mL of the formvar solution in a 100-mL Schott bottle, add glycerol and stir
for 3 h. Increasing the glycerol from 0.01 mL and 5.0 mL, increases the hole
diameters. Glycerol at 0.1 mL produced films with holes of ~2-
µ
m diameter.
3.
Place the emulsion in an ultrasonicator bath for 30 min. Ultrasonication produces
a uniform droplet size and, consequently, carbon films with holes of a near-uni-
form diameter.
4.
Clean a glass microscope slide with a solution of household dishwashing liquid
in tap water, then rinse with ultrapure water and dry with a clean, dry cloth.
Polish the slide with 3-in-1 oil to give the slide a thin, nonstick coating on its surface.
5.
Dip the polished slide into the formvar-glycerol emulsion until 75% of its length
is submerged, and hold for 10 s. Withdraw the slide at a rate of ~1.5 cm/s to thin
the formvar film (now formed on the slide) in the chloroform vapor. Drain the
slide by standing it vertically on filter paper.
6.
When the slide is dry, mark around its surface with a pair of forceps about 1 mm
from the edge. This aids detachment of the formvar film from the slide. Immerse
the slide into a reservoir of ultrapure water, holding the slide at an angle of
approximately 20
to the water surface. The formvar film should separate from
the slide and float, intact, on the surface of the water.
°
7.
Using watchmakers forceps, place transmission electron microscope grids (typi-
cally 18) on the formvar film. Collect the formvar-coated grids from the water by
placing a strip of ordinary paper (weight ~80 g/m
2
), just wider than the film, on
top of the grids. Peel back the paper from the water with the grids and formvar
film attached to it.
8.
Air-dry the paper, with the formvar-coated grids attached, under a dustcover and
then suspend it above a reservoir of acetone for 45-60 s; the acetone vapors should
clear any pseudoholes in the formvar film.
9.
Place the formvar-coated grids, still attached to the paper, in a carbon-coating
unit. Carbon is evaporated onto the grids in the same way as that used to produce
standard carbon films
(9)
, except the film used is thicker. This requires evapora-
tion from a carbon spindle that is 2-mm long and 0.8 mm in diameter.
10.
Detach the coated grids from their paper backing with watchmakers forceps and
place them on 4 layers of 50-grade filter paper soaked in acetone (in a sealed
glass container) for several h; this dissolves away the formvar leaving just a holey
carbon film on the grids.
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