Re: Slam freezing.
|From:||Philip Oshel <firstname.lastname@example.org>|
Thanks! A nice procedure. Slam-freezing can produce some nice
results, and can be the only way to get well-frozen biopsies (with
one of the slam-freezing "guns"). But. The layer of well-frozen
tissue is not much thicker than that obtained by plunge-freezing into
slush nitrogen, and the slamming does create compression artifacts,
possibly even disruption. Have you had the opportunity to compare
your slam-frozen samples to propane-jet or high-pressure frozen
Rereading your post, are you slamming the tissue into the copper
block by hand? One quick "HaiYAH!" and whack! ? If so, this isn't the
slamming I meant. I was thinking of the slammer devices that propel
the tissue at a rapid rate into the cooled block. This causes
compression artifacts. If you are doing this by hand, then no matter
how fast you are, you're not "slamming", but rapidly freezing by
contact to a cooled metal block. Similar to plunge freezing, without
the Leidenfrost problem, but not as efficient as plunging into slush
nitrogen. You will get -- as your images show -- excellent freezing
of the outer layer of the tissue, but this won't extend much below
10, maybe 20 microns (some tissues do better, but not a whole lot).
You do do something that is critical to getting the best morphology
from freeze-fixed tissue: freeze-substitution. Do you have a special
recipe for this?
Just been reading the posting by Phil and the answer by John.
Slam freezing, couldn't be easier and all with home made equipment.
Solid copper rod, 2x3 inches. Get your workshop to polish one
end. Then sit with fine paper and polish to a near mirror finish and
finish with metal polish. A friendly EM Unit is handy, they usually
have polish for the Wehnelt cylinders. Then attach the polished block
to an insulated rod or if you can find one, a racket that you can
wind up and down.
Bamboo sticks, barbecue type. Buy a pair of Sorbothane
insoles then with a punch cut out pieces with a similar diameter to
the bamboo sticks. Glue the Sorbothane cores to the bamboo sticks.
Millipore filters, or similar, cut into small pieces.
1.) Fill a wide mouth dewer, almost to the top, with LN2.
2.) Place the copper rod into the dewer and leave to cool.
Topping with LN2 if necessary.
3.) Dissect out the tissue and cut into small pieces under a
suitable Ringer solution.
4.) Quickly, place a piece of tissue onto a moistened filter
then on top of the Sorbothane.
5.) Even more quickly, raise the copper block just out of the
nitrogen, then with one even movement slam the tissue against the
6.) Transfer to a pot of LN2 and cut of the Sorbothane core.
The frozen tissue can now be sectioned or freeze substituted
and embedded for sectioning.
1.) Keep the level of LN2 high thus avoiding pre-cooling and freezing.
2.) Only bring the copper block just out of the LN2 and only
before you are going to slam. Any longer than necessary and frosting
will occur, a real bummer. You must have a polished surface.
I've used this technique and published the results for many
years. Once you perfect the skill it's really easy. Using this
technique my lab has produced some stunning electron micrographs from
freeze substituted tissue. I've even got one on the wall at the back
of my computer, now is that not sad.
Dr. Ian Montgomery,
West Medical Building,
University of Glasgow,
Tel: 0141 339 8855. Extn:6602.
Fax: 0141 330 2923
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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