From: Eugene Leitl (eugene.leitl@lrz.uni-muenchen.de)
Date: Tue Nov 10 1998 - 09:28:00 MST
Peter Passaro writes:
> They do nasty things like tearing cell membranes. If
> you freeze an object very quickly the crystals are
> probably kept in a less organized state and of a
> diameter which is not particularly dangerous to
> cells.
The types of damage to biological caused by
temperatures below their optimum are manifold,
and still largely unknown. Proteins denaturate,
as a result some superstructures are damaged or
fragment, lipid bilayers undergo phase transitions,
as the result integral membrane protein structure
and/or their distribution get disturbed. Further
down the slow thermal descent (only tiny specimens
can be cooled rapidly) extracellular ice growth
begins, increasing the cytosol concentration
(which in itself is denaturating to many proteins).
Randomly oriented ice crystals anisotropically
deform tissue by pushing it along their main
growth axis. If ice is removed by freeze-substitution
(washed out with, say, isopropanol at dry ice
temperature), the resulting tissue strongly resembles
an irregular mesh, with about 100 um holes iirc. Though
difficult to assess without watching the dynamics
process/using cell markers, the whole tissue looks
scrambled (randomly, nonlinearly distorted). If
frozen tissue is warmed, ice crystals melt suddenly,
resulting shrunken cells full of highly concentrated
cytosol to be surrounded by solute of low ionic
strength -- causing cells to rupture due to osmotic
shock, resulting membrane debris closing up into
vesicles. You think you see lots of intact structures,
but a lot of them are just artefacts. (Notice that
most of these points, grave as they are, are largely
irrelevant to the gros of the patients, where
factors like system-wide damages due to the original
disease/age, hour-long normothermic ischaemic
damage (go neuro, people, the periphery is junked,
anyway), clotting and spotty perfusion, mechanical
damague due to brain swelling etc. dominate
overwhelmingly).
Of course we do not need to warm the patient for
reconstruction, in fact the only way to do a mapping
at molecular scale is to do it in solid state, using
the structure as blueprints only. Since the scan is
destructive, you essentially rebuild the structure
anew minus the suspension damage (using damn
smart filters), whether physically, or as model
in machina (at least slicewise,as you will need
for applying smart filtering, anyway). However,
you are still facing a severe, very probably
irreversible information loss due to the
'turbulent' deformations (irreversible mapping:
a given distorted structure could have been
originated by a large number of structures, which
are all more or less equally probable).
To avoid such information lossage we can use
vitrification, fixation (with a complex perfusate/protocol
which would have to be developed) and subsequent
storage at low temperatures or uploading in vivo
('gradual uploading'), which has the major advantage
of supplying lots of low-level operation signatures
for what is an essentially indefinite duration (which,
at the top level, you can even tweak or correlate
with sensomotoric information). If the duration
(hours to decades) and resolution (say, a
sample/um^3) of the sampling is good enough,
you essentially don't need to make a molecular
map anymore.
Unfortunately, you need devices made with the
mechanosynthetic paradigm, which is not yet validated.
ciao,
'gene
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