10.10. REPLICATION BY VACUUM EVAPORATION
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which allowed relatively easy cleaning, and also because of its stability
in the electron beam. However, with the advent of simple carbon evaporation (Bradley, 1954) the technique has been largely supplanted.
Carbon, of course, is a light element and therefore relatively electron
transparent. It is chemically quite inert which allows films to be cleaned
easily with almost any organic or inorganic solvent. Thin films are tough
and stable under electron bombardment. There is only one serious
difficulty with carbon; its films are brittle!
The brittleness of carbon practically precludes stripping replica films
from a specimen. Therefore carbon can be used only when the underlying
material can be dissolved or digested away. If a single-stage replica of
particulate material is being sought, it is often possible to deposit the
sample directly upon specimen grids previously prepared with Formvar
or Parlodion supporting films. Usually these would be metal shadowed
first, and then a carbon replica deposited on top. To insure a complete
carbon film it is best to evaporate it obliquely from three divergent
angles, or rotate the specimen in vacuo while deposition is proceeding.
Finally the plastic is dissolved away, leaving the carbon film behind and
perhaps ready for the microscope. If necessary, the organic material of the
specimen often can be removed using hot concentrated alkalis. This
treatment will not affect most preshadowed metal layers. Thus this
method has been used successfully in studies of virus particles and
macromolecules, providing images of great delicacy and resolution.
A variant method of two-stage replication, particularly useful for large
specimens with deep undercuts, starts with a thin strip of cellulose acetate
(thin film backing can be used). A grid is placed on top of this and is wet
with a drop of acetone of sufficient size to form a convex meniscus above
the grid. Some cellulose acetate is dissolved and, as the acetone evaporates
somewhat, it becomes viscous. When sufficiently thick, the specimen can
be placed on its surface, so that it indents it slightly. The acetone is then
allowed to evaporate leaving behind the hardened plastic which now
embeds the supporting grid and replicates part of the surface of the
specimen. At this point, the specimen is stripped off. The specimen impression is perhaps shadowed at this time, and in any case then coated
with evaporated carbon. This is in essence a secondary replica. The underlying cellulose acetate must be removed by judicious flushing with
acetone, which will allow the carbon film to settle down upon the grid.
A backing film of Formvar applied before the acetone flush will help protect the carbon replica during this somewhat traumatic process.
343
which allowed relatively easy cleaning, and also because of its stability
in the electron beam. However, with the advent of simple carbon evaporation (Bradley, 1954) the technique has been largely supplanted.
Carbon, of course, is a light element and therefore relatively electron
transparent. It is chemically quite inert which allows films to be cleaned
easily with almost any organic or inorganic solvent. Thin films are tough
and stable under electron bombardment. There is only one serious
difficulty with carbon; its films are brittle!
The brittleness of carbon practically precludes stripping replica films
from a specimen. Therefore carbon can be used only when the underlying
material can be dissolved or digested away. If a single-stage replica of
particulate material is being sought, it is often possible to deposit the
sample directly upon specimen grids previously prepared with Formvar
or Parlodion supporting films. Usually these would be metal shadowed
first, and then a carbon replica deposited on top. To insure a complete
carbon film it is best to evaporate it obliquely from three divergent
angles, or rotate the specimen in vacuo while deposition is proceeding.
Finally the plastic is dissolved away, leaving the carbon film behind and
perhaps ready for the microscope. If necessary, the organic material of the
specimen often can be removed using hot concentrated alkalis. This
treatment will not affect most preshadowed metal layers. Thus this
method has been used successfully in studies of virus particles and
macromolecules, providing images of great delicacy and resolution.
A variant method of two-stage replication, particularly useful for large
specimens with deep undercuts, starts with a thin strip of cellulose acetate
(thin film backing can be used). A grid is placed on top of this and is wet
with a drop of acetone of sufficient size to form a convex meniscus above
the grid. Some cellulose acetate is dissolved and, as the acetone evaporates
somewhat, it becomes viscous. When sufficiently thick, the specimen can
be placed on its surface, so that it indents it slightly. The acetone is then
allowed to evaporate leaving behind the hardened plastic which now
embeds the supporting grid and replicates part of the surface of the
specimen. At this point, the specimen is stripped off. The specimen impression is perhaps shadowed at this time, and in any case then coated
with evaporated carbon. This is in essence a secondary replica. The underlying cellulose acetate must be removed by judicious flushing with
acetone, which will allow the carbon film to settle down upon the grid.
A backing film of Formvar applied before the acetone flush will help protect the carbon replica during this somewhat traumatic process.