RE: [Histonet] storage of cryosections
My experience is all with freezing whole rodent brains. I have seen severe swiss cheese artifact, mild freezing arifact, and tissue that looked all good to me under light microscopy. I don't know what happens at EM levels, or with other tissues, but it is possible to immersion freeze (or powedered dry ice) whole rodent brain, approximately 1 cubic centimeter (thinner in one dimension, larger in the other two), examine under light microscopy, and see no detectable freezing artifact.
I have tried, but not yet found any reference to the evanescent spherulites as water, only other polymers, on the web. The refereence I sited in my linked article did not mention such a structure.
Charles W. Scouten, Ph.D.
5918 Evergreen Blvd.
St. Louis, MO 63134
Ph: 314 522 0300
FAX 314 522 0377
From: firstname.lastname@example.org [mailto:email@example.com] On Behalf Of Philip Oshel
Sent: Wednesday, December 15, 2004 11:33 AM
Subject: RE: [Histonet] storage of cryosections
A couple of points, mostly for the fun of the discussion:
There is another form of frozen water, in the temperature/pressure realms biologists are concerned with: evanescent spherulites. Some people argue that vitreous water is never obtained, except in *very* small volumes (microliters) at *very* high freezing rates (>10,000 deg C/sec), but rather that evanescent spherulites are formed.
Practically speaking, the results are the same.
I agree about the cubic ice. I think cubic ice is formed during freezing with a high-pressure freezing (HPF) apparatus, but that's speculation from pressure/temperature tables. I haven't meant anyone who really knows what's happening during high-pressure freezing.
(Most are surprised when it's mentioned that a pressure spike necessarily means a temperature spike before the cooling.) There is one point in your note with which I disagree:
"...and some ice crystals will form in the interior of any piece of tissue over 10mm from the cold source."
Under ideal conditions, with the best freezing method -- HPF or rapid plunging into slush nitrogen -- crystal-free freezing will occur only to a depth of ~500 nm at best.
It might perhaps be possible to get freezing rapid enough that crystals are not easily seen by light microscopy at greater distances from the freezing surface, but there will definitely be crystals and freezing artifacts 10 mm from the surface of the specimen.
>I agree that initial freezing is most important, but that freezing
>artifact can develop in storage because the ice will reform and
>crystalize. See the following link for a thorough discussion.
>Drying agents are thus a good idea.
>Charles W. Scouten, Ph.D.
>5918 Evergreen Blvd.
>St. Louis, MO 63134
>Ph: 314 522 0300 FAX 314 522 0377
Supervisor, BBPIC microscopy facility
Department of Animal Sciences
University of Wisconsin
1675 Observatory Drive
Madison, WI 53706
voice: (608) 263-4162
fax: (608) 262-5157 (dept. fax)
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