Re: Freezing tissue

From:Philip Oshel <>


Pardon me if I act embarassed. I'll have to contact myself about 
submitting the note to M.T., but I don't know if I'll get around to 
it ... 8-)

>Is it _really_ possible to apply suction to a beakerful if liquid N2 and
>have it cool to near freezing before it all boils away? Would an ordinary
>lab's public "vacuum" line do this? Also, doesn't the liquid component
>of the nitrogen slush evaporate and protect the specimen?

Yes, I've done it. The trick is to have a high pumping capacity 
(read: fast) rough pump, like a good rotary pump, scroll pump, or the 
like. An ordinary lab vacuum line ... hmmm ... maybe. Depends on how 
good the house vacuum is. It's not so much how low the pump can pull 
the vacuum, they'll all go down to 10^ -1 torr or so, but how fast 
the pump is. It has to pump away a lot of nitrogen gas (1 liter of 
LN2 makes something like 800 L of N2 gas I think -- someone correct 
me if I'm wrong).

The liquid doesn't insulate the specimen -- this is the fun part -- 
for the same reason the ethane (etc.) doesn't insulate the specimen. 
The freezing done by slush nitrogen is actually being done by the 
liquid part of the slush in intimate contact with the specimen. This 
liquid no longer evaporates and so can't insulate by the Leidenfrost 
effect because it is now not near its boiling point. Ordinary LN2 is 
just below its boiling point, therefore it easily flash evaporates. 
LN2 in slush nitrogen is near its *freezing* point, and so doesn't 
flash evaporate. Therefore, there's no Leidenfrost effect when it 
contacts a relatively hot specimen.

>You mentioned slam freezing (not very practical for most of us) but
>not freezing by simple contact with metal (such as a cryostat chuck)
>that has been cooled to liquid N2 or even solid CO2 temperature. I
>appreciate that as an electron microscopist you cannot tolerate even
>the smallest ice crystal damage.
>LM is more forgiving, at least for muscle, skin and CNS. You can't get
>away with dunking in liquid N2 or letting un-cryoprotected things freeze
>slowly in a cryostat cabinet, but visible ice crystal holes are mostly
>too small to see if you place a 2mm specimen on copper or aluminium and
>immerse in acetone that contains solid CO2 (dry ice). Freezing on a
>CO2 freezing microtome is similarly rapid and satisfactory, but you
>can't cut sections as thinly as you can with a cryostat. Tiny ice-holes
>in muscle fibres are often seen with freezing in CO2 or isopentane-liquid
>N2, often with an almost geometrical distribution, but they don't interfere
>with enzymatic or immunohistochemical staining of fibre types.

This is of course true. I am not totally convinced about the lessened 
need for the best quality freezing for LM, though. For morphology, 
obviously you're right. But for immunostudies, I wonder. My question 
is: given that the ice crystals disrupt membrane structure, both by 
mechanical damage, and more so by dehydration, how much are cellular 
components mobilized by this damage? That is, if an antigen is 
normally confined to the E.R., how likely is it to be found in the 
cytoplasm after freezing for LM-level morphology, instead of confined 
to the E.R.? Or, if a protein is normally on the cytoplasmic face of 
the plasmalemma, how likely is it to be found on the extracellular 
face after freezing for LM? These are important questions for 
EM-level freezing, also.

The point being, LM morphology is much more forgiving in terms of 
freezing rates and ice crystal damage, but is LM immunowhatever? I 
don't know, but I suspect that that's an important issue.

Philip Oshel
Dept. of Animal Health and Biomedical Sciences
University of Wisconsin
1656 Linden Drive
Madison,  WI  53706-1581
voice: (608) 263-4162
fax: (608) 262-7420 (dept. fax)

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