X-Message-Number: 4955
Date: Tue, 10 Oct 1995 08:15:08 -0700 (PDT)
From: Joseph Strout <>
Subject: Re: variations on a straight freeze

Doug Skrecky [#4951] wrote:

>  5. Quick Freeze technique: Freeze brain at -1 C to partly dehydrate cells
>  and reduce subsequent intracellular ice crystal formation. Then dunk in
>  liquid propane cooled with dry ice. Rapid freezing limits extracellular
>  ice crystal growth. Then slowly cool further with liquid nitrogen vapour
>  to limit the formation of cracks due to increased tissue brittleness. 
>...	 
>            WORST:  1. Hamburger
>            INTERMEDIATE: 3. Microwave & 4. Freeze Substitution
>            BEST:  2. Salami & 5. Quick Freeze

Regarding "Quick Freeze": I think the freezing will only be quick for the
outer millimeter or so.  The rest of the brain will freeze quite slowly as
heat is conducted to the outside.  It seems highly doubtful that such a 
technique would be at all healthy for whole brains.

However, it would probably be better than the Salami technique.  Cutting 
unfrozen, unfixed tissue is a very difficult and messy business (I've 
tried).  Great damage is done even with very thick slices.  Cutting 
slices sufficiently thin to postfix or freeze uniformly would be nearly 
impossible.  So to cut thinner sections, you'd have to fix vascularly (or 
slowly via submersion), or else freeze the brain.  This presents us with 
a clear chicken-and-egg problem.

Working backwards, freeze substitution is an interesting idea, but again 
it will be difficult to dehydrate the center of tissue a decimeter thick.
Similar problems occur with microwaving (as we've all seen when unthawing 
a thick piece of meat).  Microwaving would fix some parts but boil 
others, scrambling parts much worse than straight freezing.  (Perhaps RF 
heating could be used to fix more uniformly?)

Freezing (your "hamburger" technique) at least has the advantage that 
motion is quickly stopped.  Parts may be disconnected, but at least 
they're in the general neighborhood of where they're supposed to be.  The 
other techniques have been developed for thin slices of tissue -- on the 
order of 0.1 mm -- and do not seem to generalize to entire brains.

,------------------------------------------------------------------.
|    Joseph J. Strout           Department of Neuroscience, UCSD   |
|               http://www-acs.ucsd.edu/~jstrout/  |
`------------------------------------------------------------------'


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