3.15.
FREEZE-SUBSTITUTION
77
substitution. We can be guided by the experience of freeze-drying (Chapter 3.14) and the work of Hanzon and Hermodsson (1960) who demonstrated far less artifact when freeze-drying was completed at —70° C than
when completed at —40° C. As indicated previously, they interpreted
their results as indicative of ice recrystallization even at —40° C. Thus,
surely, it is safest to use as low a temperature as is reasonable. This will
depend no doubt on the character of the tissue, the time one is willing to
wait, and, most importantly, the size of the tissue mass. What would be
acceptable in one situation would not necessarily be so in another.
Fernandez-Moran (1960) who has explored freeze-substitution for electron microscopy more fully than anyone else, has routinely used —72° C
(the temperature easily reached by dry ice mixtures) or —90° C (achieved
fairly easily with commercial refrigerator chests). He has indicated that
eventually he hopes to work with temperatures below —130° C where one
can be fairly certain that only vitreous water exists providing that it
was originally frozen sufficiently rapidly. He expects it will prove possible
to infiltrate with a plastic monomer and catalyze polymerization with
ultraviolet radiation at such low temperatures, but for the present there
are still severe technical difficulties. Bullivant (1960) has used —75° C,
and Rebhun (1961) used —80° C. It should be realized, however, that for
most purposes it presumably will prove safe to return to room temperature after the initial substitution is complete. Thus subsequent operations
including polymerization need not necessarily be performed under adverse conditions. Thus, also, the infiltration and use of any embedment
becomes practical.
The usefulness of the —40° C range (easily reached by commercial
freezers) has not been adequately explored excepting that FernandezMoran (1960) has demonstrated that methacrylate can be polymerized
easily with ultraviolet light at that temperature. When he wished to do
this, he first dehydrated tissue in acetone at —72° or —90° C. The specimen chamber then was warmed to —40° C and several changes of precooled methacrylate replaced the acetone. Low energy fluorescent ultraviolet lights were adequate to polymerize the methacrylate at this
temperature, although when efforts were made to do this at —72° C it was
found that a powerful ultraviolet source was required. It is quite possible that some tissues, or tissues which have been glycerinated, may not
show artifact if the entire substitution is carried on at about —40° C. If
so, this augments the list of potential solvents considerably. In a particularly interesting experiment which was only partly successful, Bartl
FREEZE-SUBSTITUTION
77
substitution. We can be guided by the experience of freeze-drying (Chapter 3.14) and the work of Hanzon and Hermodsson (1960) who demonstrated far less artifact when freeze-drying was completed at —70° C than
when completed at —40° C. As indicated previously, they interpreted
their results as indicative of ice recrystallization even at —40° C. Thus,
surely, it is safest to use as low a temperature as is reasonable. This will
depend no doubt on the character of the tissue, the time one is willing to
wait, and, most importantly, the size of the tissue mass. What would be
acceptable in one situation would not necessarily be so in another.
Fernandez-Moran (1960) who has explored freeze-substitution for electron microscopy more fully than anyone else, has routinely used —72° C
(the temperature easily reached by dry ice mixtures) or —90° C (achieved
fairly easily with commercial refrigerator chests). He has indicated that
eventually he hopes to work with temperatures below —130° C where one
can be fairly certain that only vitreous water exists providing that it
was originally frozen sufficiently rapidly. He expects it will prove possible
to infiltrate with a plastic monomer and catalyze polymerization with
ultraviolet radiation at such low temperatures, but for the present there
are still severe technical difficulties. Bullivant (1960) has used —75° C,
and Rebhun (1961) used —80° C. It should be realized, however, that for
most purposes it presumably will prove safe to return to room temperature after the initial substitution is complete. Thus subsequent operations
including polymerization need not necessarily be performed under adverse conditions. Thus, also, the infiltration and use of any embedment
becomes practical.
The usefulness of the —40° C range (easily reached by commercial
freezers) has not been adequately explored excepting that FernandezMoran (1960) has demonstrated that methacrylate can be polymerized
easily with ultraviolet light at that temperature. When he wished to do
this, he first dehydrated tissue in acetone at —72° or —90° C. The specimen chamber then was warmed to —40° C and several changes of precooled methacrylate replaced the acetone. Low energy fluorescent ultraviolet lights were adequate to polymerize the methacrylate at this
temperature, although when efforts were made to do this at —72° C it was
found that a powerful ultraviolet source was required. It is quite possible that some tissues, or tissues which have been glycerinated, may not
show artifact if the entire substitution is carried on at about —40° C. If
so, this augments the list of potential solvents considerably. In a particularly interesting experiment which was only partly successful, Bartl