X-Message-Number: 5208
Date: 18 Nov 95 00:57:29 EST
From: Mike Darwin <>
Subject: What goes there?

Richard Schroeppel wites:

>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

>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.

Richard's "outside shot" is already a well developed technology that has 
been in common use in cryobiology for over 20 years.  I have discussed not 
only its utility here, but also results we have achived with it in brains 
at BPI and 21st.  The techniuque is known as freeze subsitiution and 
invoolves dissolving ice with solvent at -79C. Lipid solubility is minimal 
at that temperature and is further minimized by the addition of osmium 
tetroxide to the solvent (we use methanol) which rapidly fixes lipid 
membranes on contact (even at -79C). Proteins and carbohydrates are 
insoluble and immobilized at that temperature.

We already have very detailed images of the grey and white matter at -79C 
in the frozen state (actually at -90 C: we have a mechanical freeezer) with 
4M and 7.4M glycerol cryoprotection.  These results have been discussed in 
previous posts.

Serial sections are now being made under different conditions in order to 
determine consevation of structure and extent of lesions in three 

Freeze fracture would be incredibly useful, but we have had technical 
problems due to the presence of cryoprotectant which make us very 
undesireable customers for repeat business; I still have one microscopist 
fuming over poisoning his scope and vacuum pumps :) or :( depending on your 
point of view.

Richard mentions dye exclusion as a method of determining plasma membrane 
integrity.  This too is a well developed technique.  We are extending on it 
in a novel way that we are in patent on.  At the moment the technique is 
being used to evaluate how well we OPEN cell membranes, but its application 
during freezing and after thawing is obvious and planned.

A small sample of our freeze-substituition work has been released to 
Merkle, Donaldson and Ettinger for personal perusal (all 4 M glycerol 
shots).  Architecture in grey and white matter is massively compressed and 
distorted by ice.  Tears and cavities formed by ice masses appear 
frequently both at 4M and (less frequently) at 7.4M glycerol.  Our level of 
resolution of cryoprotectant loaded frozen tissuer is now good: we can see 
all basic large-scale cell ultrastructure (myelin, axoplasm, cytoplasm, 
myelin layering, nuclei, cyctoplasm (organelles are too dehydrated to 
reliably identify), cell membranes, etc.

Straight frozen tissue is too dense (i.e., dehydrated) to see detailed 
structure while in the frozen state.

There is a rich literature on freeze substitution and, at least in the 
first edition of Alcor's "Cryonics Reaching For Tomorrow" there are 
pictures of freeze-substituted renal cortex (15% DMSO) at -79C in one of 
the technical appendices.  These came from Dr. Fahy's lab.

Finally, much of what we know about the mechanisms of cryoinjury (maybe 
MOST of what we know!) at the tissue level (ie., cell-to-cell level) was 
obtained from freeze-substitution studies.

Mike Darwin

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