3.15.
FREEZE-SUBSTITUTION
75
were picked up with hairs mounted in handles, and tacked down upon
screens. This writer has picked up ribbons of dry sections by using a
simple micromanipulator mounted on the knife holder platform of a
Porter-Blum microtome. A sharpened applicator stick with its point
dipped in grease served to attach the first few sections as they were cut,
and then the ribbon was held suspended as further sections were added
to it. A specimen grid could be brought underneath such a ribbon suspended in space. One might suppose that more efficient ways could be
devised for obtaining dry sections.
Successful frozen-dried sections are not the immediate cure for all
problems. Indeed, materials prepared in this way have such low intrinsic
density that unstained frozen-dried sections must be relatively thick in
order to have sufficient contrast to make a picture that is at all satisfactory.
The thickness of sections then precludes high resolution micrographs.
Stains, of course, ordinarily are applied from aqueous solutions which
we would like to avoid in handling frozen-dried material. So a new
technology, achieving effective staining from vapors, will have to be
developed before such sections will be generally useful. Chromyl chloride
probably could be used for such a purpose, although I know of no such
application so far. Normally it is applied as a vapor in a dry atmosphere
(Chapter 7.8). Its vapors may be extremely poisonous, so that great
precautions must be observed in handling it. Thus it is at the present
time that unstained, frozen-dried sections are useful mainly for demonstrating the reality of cytoplasmic features that we can study much more
effectively after chemical fixation. For the future, of course, the way is
now prepared for the development of histochemical applications.
3.15.
Freeze-Substitution
Freeze-substitution differs from freeze-drying in several important
respects. By means of suitably chosen intermediates, a piece of tissue can
be dehydrated and infiltrated with an embedding medium at a low
temperature without there ever being a gas phase present. Instead of
requiring a highly efficient pumping system, one needs only suitable cold
baths and boxes. It offers possible advantages of exposing tissue to
fixation and staining agents during the dehydration or
infiltration
process. It may even prove possible to incorporate photosensitive material
into tissue blocks and so make feasible in situ autoradiography (Fer-
FREEZE-SUBSTITUTION
75
were picked up with hairs mounted in handles, and tacked down upon
screens. This writer has picked up ribbons of dry sections by using a
simple micromanipulator mounted on the knife holder platform of a
Porter-Blum microtome. A sharpened applicator stick with its point
dipped in grease served to attach the first few sections as they were cut,
and then the ribbon was held suspended as further sections were added
to it. A specimen grid could be brought underneath such a ribbon suspended in space. One might suppose that more efficient ways could be
devised for obtaining dry sections.
Successful frozen-dried sections are not the immediate cure for all
problems. Indeed, materials prepared in this way have such low intrinsic
density that unstained frozen-dried sections must be relatively thick in
order to have sufficient contrast to make a picture that is at all satisfactory.
The thickness of sections then precludes high resolution micrographs.
Stains, of course, ordinarily are applied from aqueous solutions which
we would like to avoid in handling frozen-dried material. So a new
technology, achieving effective staining from vapors, will have to be
developed before such sections will be generally useful. Chromyl chloride
probably could be used for such a purpose, although I know of no such
application so far. Normally it is applied as a vapor in a dry atmosphere
(Chapter 7.8). Its vapors may be extremely poisonous, so that great
precautions must be observed in handling it. Thus it is at the present
time that unstained, frozen-dried sections are useful mainly for demonstrating the reality of cytoplasmic features that we can study much more
effectively after chemical fixation. For the future, of course, the way is
now prepared for the development of histochemical applications.
3.15.
Freeze-Substitution
Freeze-substitution differs from freeze-drying in several important
respects. By means of suitably chosen intermediates, a piece of tissue can
be dehydrated and infiltrated with an embedding medium at a low
temperature without there ever being a gas phase present. Instead of
requiring a highly efficient pumping system, one needs only suitable cold
baths and boxes. It offers possible advantages of exposing tissue to
fixation and staining agents during the dehydration or
infiltration
process. It may even prove possible to incorporate photosensitive material
into tissue blocks and so make feasible in situ autoradiography (Fer-