76
3.
FIXATION
nandez-Moran, 1960). It promises to be more useful than freeze-drying
for histochemistry since it offers much greater flexibility.
The history of freeze-substitution, as well as its methods and principles
as these apply to conventional microscopy, have been discussed by Feder
and Sidman (1958). Fernandez-Moran (1960) has particularly explored its
potentialities for electron microscopy. Bullivant (1960) and Rebhun
(1961) have applied the method to specific problems. The success of the
method depends in essence upon ice being soluble in many solvents at
temperatures far below its melting point. If the amount of ice is minute,
and the volume of cold anhydrous solvent large, escaping water molecules
do not appreciably dilute the latter. In effect there is never a free water
phase present to damage the tissue during the substitution.
Freeze-substitution, of course, begins with a minute block of tissue
frozen so rapidly that hopefully no ice crystals of appreciable size are
formed. It often would be possible to glycerinate the tissue first and
further reduce the hazard of ice crystal formation. The freezing of such
tissue samples is discussed in a previous section (Chapter 3.13).
Frozen tissue is then moved into the precooled solvent bath. There is
no agreement as to what solvent is best. Indeed, one must consider the
desired end result, for instance whether or not "fixation" is desired, or
whether one is striving to leave intact the native proteins. The basic
requirements of the solvent are that it be miscible with water, and also
remain fluid at the temperature at which the exchange is to occur.
Acetone, absolute methyl and ethyl alcohols, and methyl cellosolve
(monomethyl ether of ethylene glycol) undoubtedly have been the most
commonly employed solvents. But, substances such as propylene oxide,
dioxane, dimethyl formamide, isobutyric acid, and hydroxypropyl methacrylate are possible alternates. All of these compounds have freezing
points below —40° C, with several below —70° C. However, I have no
knowledge of how soluble ice may be in most of these solvents at low
temperatures, and indeed it is unlikely that such information would be
available. However, van Haareveld and Cowell (1964) have studied the
solubility of water in cold acetone. They have found that at —50°C a
14% solution about represents saturation, and at —80°C a 2-3% solution does. Also NaCl is importantly soluble at —50°C, but is essentially
insoluble at —80°C. In the present developmental stage of freeze-substitution, the interested investigator will have to explore for himself, and
decide what best suits his purposes.
There is no general agreement as to the best temperature for freeze-
3.
FIXATION
nandez-Moran, 1960). It promises to be more useful than freeze-drying
for histochemistry since it offers much greater flexibility.
The history of freeze-substitution, as well as its methods and principles
as these apply to conventional microscopy, have been discussed by Feder
and Sidman (1958). Fernandez-Moran (1960) has particularly explored its
potentialities for electron microscopy. Bullivant (1960) and Rebhun
(1961) have applied the method to specific problems. The success of the
method depends in essence upon ice being soluble in many solvents at
temperatures far below its melting point. If the amount of ice is minute,
and the volume of cold anhydrous solvent large, escaping water molecules
do not appreciably dilute the latter. In effect there is never a free water
phase present to damage the tissue during the substitution.
Freeze-substitution, of course, begins with a minute block of tissue
frozen so rapidly that hopefully no ice crystals of appreciable size are
formed. It often would be possible to glycerinate the tissue first and
further reduce the hazard of ice crystal formation. The freezing of such
tissue samples is discussed in a previous section (Chapter 3.13).
Frozen tissue is then moved into the precooled solvent bath. There is
no agreement as to what solvent is best. Indeed, one must consider the
desired end result, for instance whether or not "fixation" is desired, or
whether one is striving to leave intact the native proteins. The basic
requirements of the solvent are that it be miscible with water, and also
remain fluid at the temperature at which the exchange is to occur.
Acetone, absolute methyl and ethyl alcohols, and methyl cellosolve
(monomethyl ether of ethylene glycol) undoubtedly have been the most
commonly employed solvents. But, substances such as propylene oxide,
dioxane, dimethyl formamide, isobutyric acid, and hydroxypropyl methacrylate are possible alternates. All of these compounds have freezing
points below —40° C, with several below —70° C. However, I have no
knowledge of how soluble ice may be in most of these solvents at low
temperatures, and indeed it is unlikely that such information would be
available. However, van Haareveld and Cowell (1964) have studied the
solubility of water in cold acetone. They have found that at —50°C a
14% solution about represents saturation, and at —80°C a 2-3% solution does. Also NaCl is importantly soluble at —50°C, but is essentially
insoluble at —80°C. In the present developmental stage of freeze-substitution, the interested investigator will have to explore for himself, and
decide what best suits his purposes.
There is no general agreement as to the best temperature for freeze-