134
FREEZE—DRYING
a considerably more reliable cytochemistry to be developed. Although these goals may
still be attainable in somewhat modied form Our naive ideas have had to be modied .by
contact with hard reality. Thus, as we shall outline below it is not likely that by ordinary
Ï
techniques of rapid freezing, one may freeze cells without the introduction of a process
'
which results in an apparently uninterpretable artefact—namely, to summarize it simply,
ice crystaization. In the presence of this data, webegan a search for those techniques which
would yield an ice-crystal—free preparation in the hope that the morphology resulting from
these techniques might ultimately lead us to our goal, i.e., interpretati0n in terms of living
ultrastructure. We found that control of water in the cell was vital to the production of
ice-crystal free cells, such control being obtained either by evaporation, osmotic dehydration or replacement with agents such
gly0erol or dimethyl sulfoxide. The morphology
which results, however, varies profoundly dep ending upon the specic conditions used
and thus, gives rise to a dilemma as di sturbing as that introduced by the necessity of choice
with chemical xation and perhaps even more so. On the other hand, after certain techniques
outlined below, the cells show striking resemblance in the electron microscope to similar
cells xed in glutaraldehyde so that one may accept or reject both procedures on the basis
of similar evidence from outside techniques. Thus, if the aldehyde picture reliably reproduces certain aspects of the reality, we expect a similar reliability from certain freezing
techniques outlined below. In addition—, there is considerable chance that the utilization of
Ÿ
frozen material may offer distinct advantages of ‘a cytochemical nature.
;
We have thus, in our own thinking, come full circle round. Rapid freezing has not relieved
us of the responsibility of developing criteria for the evaluation of xed ultrastructure in
-
terms of the
cells. It has, indeed, convinced us of the considerable necessity to go
outside the eld…of electron
itself
verify or reject the aesthetic pictures
which,we produce with chemicals, cells andelectron beams… By forcing these conclusions
upon us, it may further
the
applying a single technique to
vast a
problem as cell ultrastruct—ure in the living,eell, even if that technique is as powerful as. is
61èÇîr°nmi°1—“OSCOPY;
V:…
:
—_
g
We shall,
evidence for the assertions _already,made.
also
_
outline._some extremely intere8ting and useful
of glycols and dimethylsuli‘oxide
hvmg marine
hope that ;theæremarks will,attract; othersintothisdiîcùlt eld
if the,
to disprove assertions which we shall make.
.
,
_
Many of our observations on the detailed—structure of cells aftertreatment
protectiVe
are similar
(l965)on
We we‘reinitially ”attrathdÿihtdthis
and—SidmanÏ
(1958) PattenandBrown (1958)and Hancox(l957)
_;stnulu‘s after initial
,
‘
faureswas aordedbytheworkofFernandeZ-Moran(1960)
technique
—fromthat
liverrand
Wltheggsfromthesurf clamszsulasohdzsszma
'
Itseengbccae cle&räthat at leasftwo emaj
obtained by
.
1Ï‘ÎÏ:f@1ïgaràpaçfëgaSgfiand F1gu1‘e2forSp
FREEZE—DRYING
a considerably more reliable cytochemistry to be developed. Although these goals may
still be attainable in somewhat modied form Our naive ideas have had to be modied .by
contact with hard reality. Thus, as we shall outline below it is not likely that by ordinary
Ï
techniques of rapid freezing, one may freeze cells without the introduction of a process
'
which results in an apparently uninterpretable artefact—namely, to summarize it simply,
ice crystaization. In the presence of this data, webegan a search for those techniques which
would yield an ice-crystal—free preparation in the hope that the morphology resulting from
these techniques might ultimately lead us to our goal, i.e., interpretati0n in terms of living
ultrastructure. We found that control of water in the cell was vital to the production of
ice-crystal free cells, such control being obtained either by evaporation, osmotic dehydration or replacement with agents such
gly0erol or dimethyl sulfoxide. The morphology
which results, however, varies profoundly dep ending upon the specic conditions used
and thus, gives rise to a dilemma as di sturbing as that introduced by the necessity of choice
with chemical xation and perhaps even more so. On the other hand, after certain techniques
outlined below, the cells show striking resemblance in the electron microscope to similar
cells xed in glutaraldehyde so that one may accept or reject both procedures on the basis
of similar evidence from outside techniques. Thus, if the aldehyde picture reliably reproduces certain aspects of the reality, we expect a similar reliability from certain freezing
techniques outlined below. In addition—, there is considerable chance that the utilization of
Ÿ
frozen material may offer distinct advantages of ‘a cytochemical nature.
;
We have thus, in our own thinking, come full circle round. Rapid freezing has not relieved
us of the responsibility of developing criteria for the evaluation of xed ultrastructure in
-
terms of the
cells. It has, indeed, convinced us of the considerable necessity to go
outside the eld…of electron
itself
verify or reject the aesthetic pictures
which,we produce with chemicals, cells andelectron beams… By forcing these conclusions
upon us, it may further
the
applying a single technique to
vast a
problem as cell ultrastruct—ure in the living,eell, even if that technique is as powerful as. is
61èÇîr°nmi°1—“OSCOPY;
V:…
:
—_
g
We shall,
evidence for the assertions _already,made.
also
_
outline._some extremely intere8ting and useful
of glycols and dimethylsuli‘oxide
hvmg marine
hope that ;theæremarks will,attract; othersintothisdiîcùlt eld
if the,
to disprove assertions which we shall make.
.
,
_
Many of our observations on the detailed—structure of cells aftertreatment
protectiVe
are similar
(l965)on
We we‘reinitially ”attrathdÿihtdthis
and—SidmanÏ
(1958) PattenandBrown (1958)and Hancox(l957)
_;stnulu‘s after initial
,
‘
faureswas aordedbytheworkofFernandeZ-Moran(1960)
technique
—fromthat
liverrand
Wltheggsfromthesurf clamszsulasohdzsszma
'
Itseengbccae cle&räthat at leasftwo emaj
obtained by
.
1Ï‘ÎÏ:f@1ïgaràpaçfëgaSgfiand F1gu1‘e2forSp