[Histonet] (no subject)

[Philip Oshel]

Charles,

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.

Phil

>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.
>
>http://www.myneurolab.com/global/Manuals/Tips%20and%20Techniques%20Freezing%20Artifact.pdf
>
>Drying agents are thus a good idea.
>
>
>
>
>Cordially,
>Charles W.  Scouten, Ph.D.
>myNeuroLab.com
>5918 Evergreen Blvd.
>St. Louis, MO 63134
>Ph: 314 522 0300 
>FAX  314 522 0377
>cwscouten <@t> myneurolab.com
>http://www.myneurolab.com
-- 
Philip Oshel
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)