Re: CryoJane & Gentle Jane

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From:Robert Schoonhoven <>
To:"J. A. Kiernan" <>
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I agree completely with John, further more, the quote from Insturmedics,
Inc. is both misleading and scientifically irresponsible.  At best their
method MIGHT give a few MICRONS of amorphic ice, but that would have to
be proved to me using a cryo-ultramicrotome and Cryo-TEM.  The statement
below leads one to believe that their method will freeze an entire
specimen "amorphously".  This is just not possible with the size of
tissues submitted for Frozen Sectioning.  The BEST methods using either
liquid nitrogen or helium and a highly polished silver block (the "metal
mirror" method) will only give about 20 microns of amorphous ice using
very small samples (less than 1 mm).  I believe that I covered this in a
post about a month or so ago and see no sense in repeating it.

While their method may produce a frozen sample free of visable ice
crystals by light microscopy, not all that difficult to do, I am cetain
that at the Cryo-TEM level there will indeed be visable ice crystals. 
Also if a block is forzen "in 8 to 10 seconds" the freezing rate,
measured in thousands of degrees per second would be such that ice
crystals would be visable at the light microscope level a millimeter or
two below the surface of the tissue.  

While I do believe that their method will give you a frozen section that
will not have any visible (by LM) ice crystals I feel that their
statement below is erroneous and misleading.  

"J. A. Kiernan" wrote:
> On Tue, 4 Apr 2000, Instrumedics, Inc. wrote:
> > All the  "Janes" are confusing!  Clarification!
> >
> > ... tissue and CryoGel or other embedding medium. The block is frozen in
> > 8-10 seconds. The temperature of the heat extractor,-196deg. C, and the
> > thermal exchange derived from its highly polished chrome finish, produces
> > "amorphous" ice rather than "crystalline" ice. minimizing ice crystal
> > artifact. The morphology of the tissue is preserved!
>   You aren't going to get amorphous (i.e. vitreous) solid water by
>   this method, especially if it takes 8 seconds to freeze the
>   middle of the block, though the ice crystals may be too small
>   to make visible holes in the tissue. For vitrification you need
>   liquid helium temperature. See Bald, WB (1983) Optimizing the
>   cooling block for the quick freeze method. J Microsc 131: 11-23;
>   also Chapters 2 & 3 in Pearse's "Histochemistry," 4th edn, Vol 1.
>  John A. Kiernan,
>  Department of Anatomy & Cell Biology,
>  The University of Western Ontario,
>  LONDON,  Canada  N6A 5C1

best regards,
Robert Schoonhoven
Laboratory of Molecular Carcinogenesis and Mutagenesis
Dept. of Environmental Sciences and Engineering
University of North Carolina
Chapel Hill, NC 27599
office 919-966-6343
   Lab 919-966-6140
   Fax 919-966-6123 

Don't go around saying the world owes you a living; the world owes you
nothing; it was here first. 
Mark Twain [Samuel Langhornne Clemens] (1835-1910)

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