Re: Freezing tissue
|From:||Philip Oshel <firstname.lastname@example.org>|
There are two possibilites for plunge freezing in LN2: one is
"normal" liquid nitrogen -- as it comes from the dewar, the other is
If using LN2, then something like isopentane, ethane, or propane --
ordinary cooking propane will do -- *must* be used. The caveat is
that using these liquid gases is a serious fire and explosion hazard,
especially since liquid oxygen forms at liquid nitrogen temperatures,
and dissolves into the liquid hydrocarbon. These gases can be used
safely -- I have done so -- but they take care and understanding what
you're doing, and a safe place to dispose of the cryogen. Safe,
keeping in mind that the isopentene, etc., is enriched in dissolved
oxygen and heavier than air so it likes to move along floors, etc.,
also, is a reasonably effective explosive.
A simpler and much safer way is to use slush nitrogen. Slush nitrogen
is produced by placing beaker (or whatever) full of liquid nitrogen
in a vacuum chamber and then pulling a one atmosphere vacuum with a
good -- meaning high-capacity -- rotary vacuum pump or the like. Pump
on the LN2 for 10 or 30 minutes until it starts to form a slush. This
slush can be used for plunge freezing without isopentene or any other
Plain LN2 can't be used for plunge freezing because of the
Leidenfrost effect. What happens when water is dropped on a very hot
skillet. Inside of immediately boiling, the water drop survives for a
while, skittering around on the skillet. The heat of the skillet
flash-evaporates a layer of water vapor, which then insulates the
drop and keeps it from boiling. The same thing happens when plunging
tissue into LN2. The relatively hot tissue flash-evaporates some LN2,
creating an insulating layer of nitrogen gas around the specimen.
This both slows down the rate of freezing and creates a longer
temperature gradient over which heat must leave the specimen. This
gives more time for ice crystals to form and grow, creating more ice
damage. This does not happen with isopentene, ethane, etc.
Then there are the slam-freezing methods, where the tissue is quick
frozen by slamming (literally) it onto a polish metal block held at
LN2 temperature. This is rapid and avoids the Leidenfrost effect, but
there is the obvious potential for tissue damage. The slamming is
done with some force, it's not a gentle act.
Having said all that, plunging into LN2 would give better freezing
than just sticking a specimen to a piece of metal and setting it in a
>I would welcome your opinions and feedback.
>What is the optimal method of freezing tissues with liquid nitrogen?
>with or without isopentane and why?
>Vinnie Della Speranza
>Manager for Anatomic Pathology Services
>Medical University of South Carolina
>165 Ashley Avenue
>Charleston, SC 29425
>ph: (843) 792-6353
>fax: (843) 792-8974
AMFSC and BBPIC
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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