3.14.
FREEZE-DRYING
73
Hermodsson (1960) found that tissue dehydrated at —70° C had far fewer
vacuoles than similar tissues dried at —40° C. Thus, empirically, it seems
best not to let the temperature of the specimen exceed —70° C until one
is sure that drying is complete.
Hanzon and Hermodsson (1960) and Elfvin (1963) have found it
desirable to fix tissue after drying, and before infiltrating it with the
embedment. They use for this purpose a relatively long (2-5 hours)
exposure of the specimen to osmium tetroxide vapors. To accomplish
this, the warmed-up specimens are transferred from the cryostat to a
large test tube connected through a stopcock to a mechanical pump.
Crystals of osmium tetroxide are introduced into this chamber along with
the specimens, and pumping is started. After a few minutes, however, the
pump line is closed to prevent the osmium tetroxide from being pumped
out. Osmium tetroxide vapor then constitutes most of the residual
atmosphere.
Fixation with osmium tetroxide vapor helps prevent the extraction of
cytoplasmic constituents that otherwise would be maximal in undenatured frozen-dried tissue. Also, it largely prevents an artifact that
Hanzon and Hermodsson characterize as a collapse of fine structure due
to surface tension. They believe this develops particularly during impregnation procedures as the liquid interface of the plastic monomers
advances into the dry, unsupported tissue. Such artifacts may be only
slight separations of cytoplasmic membranes, but more severe damage
results in the compression of cytoplasmic material into narrow spaces. In
extreme cases the cytoplasm may be full of empty vacuoles of rather
uniform size, and the destruction may be difficult to distinguish from
damage caused by ice crystals.
The fact that osmium tetroxide fixation after the removal of all water
largely prevents tissue collapse demonstrates that this is indeed unrelated
to water crystallization, and that the infiltration of dry tissue is a hazardous process at best. This was partly recognized by Sjostrand and Baker
(1958) who considered it essential with unfixed specimens to infiltrate
methacrylate monomer under a high vacuum. To accomplish this end,
they removed the specimens from their cryostat and stored them in a
desiccator while they then froze a layer of methacrylate monomer in the
bottom of the cryostat. The specimens were put on top of this, and a
high vacuum re-established. Only then was the cryostat allowed to warm
up so that the methacrylate melted at about —120° C. At this time the
dry specimens sank down into the methacrylate melt and were impreg-
FREEZE-DRYING
73
Hermodsson (1960) found that tissue dehydrated at —70° C had far fewer
vacuoles than similar tissues dried at —40° C. Thus, empirically, it seems
best not to let the temperature of the specimen exceed —70° C until one
is sure that drying is complete.
Hanzon and Hermodsson (1960) and Elfvin (1963) have found it
desirable to fix tissue after drying, and before infiltrating it with the
embedment. They use for this purpose a relatively long (2-5 hours)
exposure of the specimen to osmium tetroxide vapors. To accomplish
this, the warmed-up specimens are transferred from the cryostat to a
large test tube connected through a stopcock to a mechanical pump.
Crystals of osmium tetroxide are introduced into this chamber along with
the specimens, and pumping is started. After a few minutes, however, the
pump line is closed to prevent the osmium tetroxide from being pumped
out. Osmium tetroxide vapor then constitutes most of the residual
atmosphere.
Fixation with osmium tetroxide vapor helps prevent the extraction of
cytoplasmic constituents that otherwise would be maximal in undenatured frozen-dried tissue. Also, it largely prevents an artifact that
Hanzon and Hermodsson characterize as a collapse of fine structure due
to surface tension. They believe this develops particularly during impregnation procedures as the liquid interface of the plastic monomers
advances into the dry, unsupported tissue. Such artifacts may be only
slight separations of cytoplasmic membranes, but more severe damage
results in the compression of cytoplasmic material into narrow spaces. In
extreme cases the cytoplasm may be full of empty vacuoles of rather
uniform size, and the destruction may be difficult to distinguish from
damage caused by ice crystals.
The fact that osmium tetroxide fixation after the removal of all water
largely prevents tissue collapse demonstrates that this is indeed unrelated
to water crystallization, and that the infiltration of dry tissue is a hazardous process at best. This was partly recognized by Sjostrand and Baker
(1958) who considered it essential with unfixed specimens to infiltrate
methacrylate monomer under a high vacuum. To accomplish this end,
they removed the specimens from their cryostat and stored them in a
desiccator while they then froze a layer of methacrylate monomer in the
bottom of the cryostat. The specimens were put on top of this, and a
high vacuum re-established. Only then was the cryostat allowed to warm
up so that the methacrylate melted at about —120° C. At this time the
dry specimens sank down into the methacrylate melt and were impreg-