Stephen,

Sorry buy -40C is NOT cold enough for freezing our murine tissues.  I 
believe that others have experienced this problem also, the one of getting 
freezing artifact/damaged morphology with inside cryostat temperature 
freezing.

Nothing scary about liquid nitrogen usage, although Isopentane is more of a 
worry for storage.  I was thinking more of precooling the metal device 
containing wells i.e surround the device with liquid nitrogen, then you 
have the wells at the extremely cold temperatures.  Liquid nitrogen in the 
wells would not be ideal, to be sure.

  We do this with a metal block sitting in liquid nitrogen so the metal 
block is at liquid nitrogen temperatures, then place an OCT embedded murine 
tissue (inside a Tissue Tek plastic mold) on top of the very cold metal 
block.  This permits murine tissue to freeze at a much colder temperature 
and without artifact.

I have seen your device and like its design, but if mouse tissue cannot be 
snap frozen at colder temperature than -40C, then we can't use it due to 
terrible morphology.  Cryostat freezing temperatures for murine 
cryomicrotomy simply do not do the job well enough.  It is the water ice 
crystal formation inside the tissue that presents the big problem and the 
colder the freezing temperature, the smaller the ice crystal formation - 
hence snap freezing in the true sense of the words. Precooling OCT and a 
tissue is not  feasible when you have 10 mice lined up and collecting 15 
tissues out of each mouse as fast as you can dissect and snap freeze plus 
the actual snap freezing takes only seconds or so - extremely fast.

  I would love to try your device in the way we use our metal block 
surround by liquid nitrogen as your device has a very efficient 
design.   There is no doubt it works very well for clinical  laboratories 
for rapid diagnostic work, but for our fussy murine tissues, colder 
temperatures must prevail.

There is an excellent discussion by Charles Scouten on snap freezing 
biological samples can be found at  www.myneurolab.com with details on ice 
crystal formation, etc. - what we experience with murine tissue.

Gayle Callis

   At 10:52 AM 3/7/2005, you wrote:
>HI Gail,
>
>I have not experimented with liquid nitrogen in my wells. Sounds scary!
>In order to reduce freeze artifact using my system I have a few suggestions.
>1) Cool the tissue and OCT to refriderator temp. I think this is a smart 
>idea for any
>  snap freezing technique. Why should we add the calories to go from room 
> temp to
>0 degrees C, when it takes a lot less calories to go from 1 degree to O 
>degrees C.
>2) My bars can be cooled down as low as about - 40 C and still maintain 
>the adhesive quality of the cold metal which is what allows us to be most 
>precise. Chucks can be put in
>liquid nitrogen to get them super cooled.
>
>I would love to see someone try this and tell me how their result came 
>out. I think it would freeze pretty quickly.
>
>Any takers? Got to run for a frozen.
>
>
>Stephen
>

Gayle Callis
MT,HT,HTL(ASCP)
Research Histopathology Supervisor
Veterinary Molecular Biology
Montana State University - Bozeman
PO Box 173610
Bozeman MT 59717-3610
406 994-6367 (lab with voice mail)
406 994-4303 (FAX)



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