lNI‘ERPRETAÏION or ŒTR‘ASTRUCTURE
145
reagents in the cross linking process and thus might allow for selective stabilization of
“cellular components based on knowledge gained from biochemically isolated systems.
In relation to the above
also be mentioned that criticism of the freeze—
substitution, as opposed to freeze-drÿing techniques, has been made on the basis that in
freeze—substitution, incomplete dehydration of frozen cells may result in thawing during
warmmg to room temperature,
giving rise to reconstitution Of et structure
similar to that seen aftet.chemlcal
1964). Feder
made a thorough study of the problem of dehydration and found
to be com—
plete in about 3-4 days at — 79 °C (see also van Harreveld et al., 1965—F ernandez—Moran,
1961). In our own work, we leave our tissue in the substituting uid for at least two weeks at
Which time that which is in substitutihg uid_containing osmium turns brown in color
(Rebhun, 1965) indicating that reaction with osmium has occurred. This is difcult to
imagine unless the osmium has penetrated the tissue with the‘Sub'stituhg uid. Thus, it is
diîcult to believe that dehydration has not occurred prior to
’
impression has been created (Sjostrand, 1964,
that
always
gives “cells which appear strikinglÿ
artefacts” "(sjæüàa,
were
this so, many a dismal day —at,the
could…have .bee#avoided in the.past
seven years. Indeed, as we hope has been
_iceäcrystals» are na—
voidable (although we di‘er with Sjostrand
mme-crystal artefact)by ourtechniques
if extreme care is taken to avoid water
leagues have give up freeze-substitution.qpreciseÏygbecauseroc—crystal artefact was their usual
fare. In fact, they were too careful techniCians. Had1they beenïlessÿassiduôus"mdetails, they
could also have contbuted to
of cells for
electron microscopy.
,
_
;
"_
‘
Finally, it should
out that had
‘iiiédhîpletély,dehÿdrated cells
actually occurred, and xationtaken place?by the
at the time of thawing” (Sjostrand,l 19.64),
that due to
acetone not a water solution of_ osmiuia—$ used mthe freeze—thawstudiesof Baker. (1962)
_
This manifestly is not the
,
All in all, I feel that We can lay,to restîheseCüClSmSOf
and ill-thought out. There is a real pfôbleni‘ôf‘What_di‘èences thereàfe
.
substituted and freeze-dried
with protective agent
freeze—drying apparatus.
some ”real andusefulmformaüoncan
"
result from a study of… this kind
in“
the interpretation of
are likely
Again, the point of view Which ;we feel
one
-
technique is “better” than another, but to discover What
from
each technique which is useful for the interpretationth
'
‘
'
,
145
reagents in the cross linking process and thus might allow for selective stabilization of
“cellular components based on knowledge gained from biochemically isolated systems.
In relation to the above
also be mentioned that criticism of the freeze—
substitution, as opposed to freeze-drÿing techniques, has been made on the basis that in
freeze—substitution, incomplete dehydration of frozen cells may result in thawing during
warmmg to room temperature,
giving rise to reconstitution Of et structure
similar to that seen aftet.chemlcal
1964). Feder
made a thorough study of the problem of dehydration and found
to be com—
plete in about 3-4 days at — 79 °C (see also van Harreveld et al., 1965—F ernandez—Moran,
1961). In our own work, we leave our tissue in the substituting uid for at least two weeks at
Which time that which is in substitutihg uid_containing osmium turns brown in color
(Rebhun, 1965) indicating that reaction with osmium has occurred. This is difcult to
imagine unless the osmium has penetrated the tissue with the‘Sub'stituhg uid. Thus, it is
diîcult to believe that dehydration has not occurred prior to
’
impression has been created (Sjostrand, 1964,
that
always
gives “cells which appear strikinglÿ
artefacts” "(sjæüàa,
were
this so, many a dismal day —at,the
could…have .bee#avoided in the.past
seven years. Indeed, as we hope has been
_iceäcrystals» are na—
voidable (although we di‘er with Sjostrand
mme-crystal artefact)by ourtechniques
if extreme care is taken to avoid water
leagues have give up freeze-substitution.qpreciseÏygbecauseroc—crystal artefact was their usual
fare. In fact, they were too careful techniCians. Had1they beenïlessÿassiduôus"mdetails, they
could also have contbuted to
of cells for
electron microscopy.
,
_
;
"_
‘
Finally, it should
out that had
‘iiiédhîpletély,dehÿdrated cells
actually occurred, and xationtaken place?by the
at the time of thawing” (Sjostrand,l 19.64),
that due to
acetone not a water solution of_ osmiuia—$ used mthe freeze—thawstudiesof Baker. (1962)
_
This manifestly is not the
,
All in all, I feel that We can lay,to restîheseCüClSmSOf
and ill-thought out. There is a real pfôbleni‘ôf‘What_di‘èences thereàfe
.
substituted and freeze-dried
with protective agent
freeze—drying apparatus.
some ”real andusefulmformaüoncan
"
result from a study of… this kind
in“
the interpretation of
are likely
Again, the point of view Which ;we feel
one
-
technique is “better” than another, but to discover What
from
each technique which is useful for the interpretationth
'
‘
'
,