78
3.
FIXATION
(1962) completed substitution in glycol methacrylate at —60° C, as well
as at —40° C, and then directly polymerized this at low temperature so
that the plastic monomer was the only solvent to which the specimen was
exposed.
Unfixed frozen-substituted tissue presents most of the same difficulties
that similar frozen-dried tissue possesses. Its relative contrast in the electron microscope is very low, and substances may be leached from sections
as they are floated away from the knife edge while being cut. Thus, efforts
have been and will be made to fix tissue while it is being dehydrated.
There is much better opportunity for doing this during freeze-substitution than after freeze-drying. Osmium tetroxide can be added to chilled
samples of most if not all of the above list of organic compounds, without
having notable reaction or decomposition. Thus, presumably, the osmium
tetroxide would be available to fix the tissue at the instant it was exposed
by the solution of the ice.
In our own laboratory we have dissolved appreciable quantities of
formaldehyde in some substitution solvents. This was accomplished by
adding an excess of paraformaldehyde powder to the solvent, and heating
gently to depolymerize and dissolve as much formaldehyde as possible.
The solution was then cooled, leaving in it a reserve of undissolved
powder. Although I have no knowledge of exactly how much formaldehyde has been put into solution in this way, one's nose is an adequate
sensor of substantial quantities. Other aldehydes, with higher boiling
points, and, therefore, available as pure liquids, would be easier to use
in so far as they prove to be effective fixatives. We have begun promising experiments with crotonaldehyde which Sabatini et al. (1963) have
already indicated to be an excellent fixative. This aldehyde is a liquid at
ordinary temperatures, and does not freeze until — 69°C.
It should be realized also that electron stains can be introduced during
freeze-substitution. Fernandez-Moran reported the use of uranyl acetate,
lanthanum nitrate, platinic chloride, and apparently employed still other
unspecified organo-metallic compounds. In a particularly interesting
experiment, van Haareveld and Cowell (1964) added silver nitrate to
their cold acetone, and the subsequent deposition of silver chloride
crystals demarked the original distribution of the chloride ion.
The design of an apparatus which permits fluid transfer in a cold
specimen chamber without water vapor contamination deserves some
thought. Fernandez-Moran found it convenient to use separatory funnels equipped with Teflon stopcocks as containers for bulk fluids. They
3.
FIXATION
(1962) completed substitution in glycol methacrylate at —60° C, as well
as at —40° C, and then directly polymerized this at low temperature so
that the plastic monomer was the only solvent to which the specimen was
exposed.
Unfixed frozen-substituted tissue presents most of the same difficulties
that similar frozen-dried tissue possesses. Its relative contrast in the electron microscope is very low, and substances may be leached from sections
as they are floated away from the knife edge while being cut. Thus, efforts
have been and will be made to fix tissue while it is being dehydrated.
There is much better opportunity for doing this during freeze-substitution than after freeze-drying. Osmium tetroxide can be added to chilled
samples of most if not all of the above list of organic compounds, without
having notable reaction or decomposition. Thus, presumably, the osmium
tetroxide would be available to fix the tissue at the instant it was exposed
by the solution of the ice.
In our own laboratory we have dissolved appreciable quantities of
formaldehyde in some substitution solvents. This was accomplished by
adding an excess of paraformaldehyde powder to the solvent, and heating
gently to depolymerize and dissolve as much formaldehyde as possible.
The solution was then cooled, leaving in it a reserve of undissolved
powder. Although I have no knowledge of exactly how much formaldehyde has been put into solution in this way, one's nose is an adequate
sensor of substantial quantities. Other aldehydes, with higher boiling
points, and, therefore, available as pure liquids, would be easier to use
in so far as they prove to be effective fixatives. We have begun promising experiments with crotonaldehyde which Sabatini et al. (1963) have
already indicated to be an excellent fixative. This aldehyde is a liquid at
ordinary temperatures, and does not freeze until — 69°C.
It should be realized also that electron stains can be introduced during
freeze-substitution. Fernandez-Moran reported the use of uranyl acetate,
lanthanum nitrate, platinic chloride, and apparently employed still other
unspecified organo-metallic compounds. In a particularly interesting
experiment, van Haareveld and Cowell (1964) added silver nitrate to
their cold acetone, and the subsequent deposition of silver chloride
crystals demarked the original distribution of the chloride ion.
The design of an apparatus which permits fluid transfer in a cold
specimen chamber without water vapor contamination deserves some
thought. Fernandez-Moran found it convenient to use separatory funnels equipped with Teflon stopcocks as containers for bulk fluids. They