72
3.
FIXATION
been frozen-dried and subsequently embedded have had their lipids
removed by the solvent action of the plastic monomers. The membrane
systems of the cells thus are ghostly in appearance, visible mainly insofar
as they are outlined by other structures.
Freeze-drying, of course, requires fairly costly apparatus which in part
has to be custom fabricated. The principles of freeze-drying (fundamentally molecular distillation), employing modern diffusion pumps and
suitable cold traps, are well understood by physicists and engineers
(Bell, 1956). Symposia on freeze-drying, edited by Harris (1954) and by
Meryman (1960), discuss some of this background.
Modern freeze-drying equipment, adaptable for electron microscopy,
embodies to a considerable extent principles outlined by Glick and
Malmstrom (1952). Sjostrand and Baker (1958) used equipment patterned
on this, as did Elfvin (1963). A particularly elegant and fundamentally
simple freeze-drying apparatus that is a variant of this has been described
in considerable detail by Hanzon and Hermodsson (1960). Grunbaum
and Wellings (1960) also have described in detail an apparatus that perhaps could be more easily assembled than the others, but presumably
would not be as efficient in that fairly long, narrow-bore connections were
inevitable in its design.
All of these various authors have relied upon molecular distillation to
achieve the drying. The specimen ordinarily has been kept at approximately —72° C (the temperature of a dry ice-alcohol bath) during dehydration. A cold trap chilled to about —196° C with liquid nitrogen (or
liquid air) has been placed as close to the specimen as possible. In the
presence of a good vacuum, water then evaporates from the specimen area
and condenses on the cold trap. In the design of Hanzon and Hermodsson
the entire outer wall of the drying chamber served as the cold trap, and
the chamber was connected to a diffusion pump with a short, wide-bore
tube giving the system particularly great efficiency.
Tissue that has been inadequately frozen and dried appears vacuolated.
This may be so severe as to almost destroy morphological order. It is
evident that the damage may be due to "ice artifact," that the cavities at
one time contained ice crystals. It was natural originally to assume that
the principal hazard occurred during the initial freezing process. But
subsequent work suggests that warming tissue above about —40°C while
ice is still present may be at least an equal hazard, for a slow recrystallization can occur which may produce damagingly large crystals even though
the temperature is maintained much below 0°C. Indeed, Hanzon and
3.
FIXATION
been frozen-dried and subsequently embedded have had their lipids
removed by the solvent action of the plastic monomers. The membrane
systems of the cells thus are ghostly in appearance, visible mainly insofar
as they are outlined by other structures.
Freeze-drying, of course, requires fairly costly apparatus which in part
has to be custom fabricated. The principles of freeze-drying (fundamentally molecular distillation), employing modern diffusion pumps and
suitable cold traps, are well understood by physicists and engineers
(Bell, 1956). Symposia on freeze-drying, edited by Harris (1954) and by
Meryman (1960), discuss some of this background.
Modern freeze-drying equipment, adaptable for electron microscopy,
embodies to a considerable extent principles outlined by Glick and
Malmstrom (1952). Sjostrand and Baker (1958) used equipment patterned
on this, as did Elfvin (1963). A particularly elegant and fundamentally
simple freeze-drying apparatus that is a variant of this has been described
in considerable detail by Hanzon and Hermodsson (1960). Grunbaum
and Wellings (1960) also have described in detail an apparatus that perhaps could be more easily assembled than the others, but presumably
would not be as efficient in that fairly long, narrow-bore connections were
inevitable in its design.
All of these various authors have relied upon molecular distillation to
achieve the drying. The specimen ordinarily has been kept at approximately —72° C (the temperature of a dry ice-alcohol bath) during dehydration. A cold trap chilled to about —196° C with liquid nitrogen (or
liquid air) has been placed as close to the specimen as possible. In the
presence of a good vacuum, water then evaporates from the specimen area
and condenses on the cold trap. In the design of Hanzon and Hermodsson
the entire outer wall of the drying chamber served as the cold trap, and
the chamber was connected to a diffusion pump with a short, wide-bore
tube giving the system particularly great efficiency.
Tissue that has been inadequately frozen and dried appears vacuolated.
This may be so severe as to almost destroy morphological order. It is
evident that the damage may be due to "ice artifact," that the cavities at
one time contained ice crystals. It was natural originally to assume that
the principal hazard occurred during the initial freezing process. But
subsequent work suggests that warming tissue above about —40°C while
ice is still present may be at least an equal hazard, for a slow recrystallization can occur which may produce damagingly large crystals even though
the temperature is maintained much below 0°C. Indeed, Hanzon and