336
10.
PARTICULATE SPECIMENS
inertness may make this possible. If this can be done, they are capable
of yielding beautiful results. Carbon films are quite brittle, however, and
they must not be handled roughly. Usually they cannot be mechanically
stripped from the specimen, at least not without a suitable backing film
for additional support.
Plastic films differ from carbon films in being tough and somewhat
elastic. Thus, sometimes films thin enough to serve as single-stage replicas
can be mechanically stripped or floated away from the specimen. (In
some circumstances, of course, one might also isolate them by dissolving
or digesting the specimen.) While very thin films often can be removed
by stripping from metal surfaces, biological applications are pretty much
limited to floating such films from the surface of glass, or freshly cleaned
mica, upon which a particulate specimen had been deposited. Under
these circumstances, the specimens can be expected to strip with the film,
and have to be removed subsequently by solution or digestion if a clean
replica is to be seen. The method has proved quite useful, however, in
dealing with macromolecules, viruses, and the like.
When one is dealing with suspensions or powders that eventually can
be removed from a replica by solution or digestion, the material can first
be deposited on a Formvar or Parlodion film which is stretched on a
copper grid. The supporting film with its particles is coated with a
moderately thick film of evaporated carbon as described later (Chapter
10.10). Then the supporting film is dissolved in an organic solvent. If the
solvent action is of sufficiently short duration enough plastic material
will remain between the grid wires and the carbon film to keep them
stuck together. Finally, the particles are removed by solution or digestion,
leaving a single-stage carbon replica behind, which will probably require
metal shadowing (between the grid wires) to reveal detail adequately.
When proteinaceous or other organic material is to be removed from
a carbon replica, vigorous oxidation can be employed. The most effective
reagent seems to be a mixture of equal parts of potassium permanganate
and potassium dichromate dissolved in about five times their volume of
concentrated sulfuric acid. Carbon coated copper grids can be floated
on this mixture without much danger of damage, but after the digestion
of the organic remains they must be washed, first in water, then in concentrated hydrochloric acid, and finally water again. After drying, the
specimen is ready for shadowing and then examination. Hot concentrated
alkali can also sometimes be used with success.
Specimens of such a geometry that very deep undercutting would occur
10.
PARTICULATE SPECIMENS
inertness may make this possible. If this can be done, they are capable
of yielding beautiful results. Carbon films are quite brittle, however, and
they must not be handled roughly. Usually they cannot be mechanically
stripped from the specimen, at least not without a suitable backing film
for additional support.
Plastic films differ from carbon films in being tough and somewhat
elastic. Thus, sometimes films thin enough to serve as single-stage replicas
can be mechanically stripped or floated away from the specimen. (In
some circumstances, of course, one might also isolate them by dissolving
or digesting the specimen.) While very thin films often can be removed
by stripping from metal surfaces, biological applications are pretty much
limited to floating such films from the surface of glass, or freshly cleaned
mica, upon which a particulate specimen had been deposited. Under
these circumstances, the specimens can be expected to strip with the film,
and have to be removed subsequently by solution or digestion if a clean
replica is to be seen. The method has proved quite useful, however, in
dealing with macromolecules, viruses, and the like.
When one is dealing with suspensions or powders that eventually can
be removed from a replica by solution or digestion, the material can first
be deposited on a Formvar or Parlodion film which is stretched on a
copper grid. The supporting film with its particles is coated with a
moderately thick film of evaporated carbon as described later (Chapter
10.10). Then the supporting film is dissolved in an organic solvent. If the
solvent action is of sufficiently short duration enough plastic material
will remain between the grid wires and the carbon film to keep them
stuck together. Finally, the particles are removed by solution or digestion,
leaving a single-stage carbon replica behind, which will probably require
metal shadowing (between the grid wires) to reveal detail adequately.
When proteinaceous or other organic material is to be removed from
a carbon replica, vigorous oxidation can be employed. The most effective
reagent seems to be a mixture of equal parts of potassium permanganate
and potassium dichromate dissolved in about five times their volume of
concentrated sulfuric acid. Carbon coated copper grids can be floated
on this mixture without much danger of damage, but after the digestion
of the organic remains they must be washed, first in water, then in concentrated hydrochloric acid, and finally water again. After drying, the
specimen is ready for shadowing and then examination. Hot concentrated
alkali can also sometimes be used with success.
Specimens of such a geometry that very deep undercutting would occur