X-Message-Number: 5193
Date: Thu, 16 Nov 1995 16:17:56 MST
From: "Richard Schroeppel" <>
Subject: damage assessment & ice microscopy

One of our constant arguments is "How much freezing damage ...?"

We should be investigating ways to examine frozen tissue,
without thawing it first.  The only technique I know of is
freeze fracture; I've no clue as to its applicability.

There are various heroic techniques for examining a few atoms
of a surface in a vacuum; these are probably unsuitable for
our needs, since they don't look at enough material, and we're
not ready to use atomic level data anyway.

One possibility would be to develop a good method of slicing
a block of frozen tissue without melting the surface we are
trying to examine.  The top millimeter or so could be examined
with an ordinary microscope.  Obvious candidates are lasers,
sharp knives, and corrosives; whether they could produce an
interpretable surface is questionable, but worth looking into.
We might also see what techniques have been used by other
scientists who were reluctant to thaw their subjects - ice-man,
mammoths, ice-cores.

Another possibility would be to prestain the test animal (before
freezing) - as an example, if we knew that fluorescein didn't enter
cells, we could give the animal fluorescein, and see what the
distribution looked like after the freezing protocol.  Conceivably
this could be combined with NMR to develop high resolution data.

An outside shot would be to look for a sequence of fluid substitutions
that preserved tissue & cell structure.  It's already been mentioned
that ice dissolves readily in methanol.  The problem to be overcome
is to find a sequence of substitutions that leaves some tissue to
examine.

As always, a bad preservation result doesn't mean a lot, while a
good preservation result would validate the freezing protocol.

Development of methods for examining frozen tissue would probably
have other scientific uses besides cryonics.

Rich Schroeppel   


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