Re: Freezing artifact

From:Philip Oshel


Are you dealing with specimens for light microscopy or electron 
microscopy? The answer will vary, depending on one's needs.
There are some universals:
1) Make the sample size as small as possible. Even the very best 
freeze-fixing methods have size limitations and depth of good 
fixation. This is different for light microscopy and electron 
microscopy, because of the higher resolution of the latter, but there 
are still size limits for light microscopy. Generally, the smaller 
the better, but "smaller" can mean "thinner".
2) Don't surround the with any support material or embedding 
material. This just creates an extra thickness of sample to be 
frozen, reduces the quality of freeze-fixation of the sample, and 
introduces freezing artifacts.
3) Don't use isopentane, and the like. These organics (and ethane, 
propane, etc.) are explosive, and LN2 enriches itself with oxygen 
that it freezes out of the air as one works. The combination can get 
excessively interesting. Also, the tissues can be frozen at lower 
temperatures, and so give better results, using LN2 alone IF slush 
nitrogen is used. This is because LN2 is normally "warm", near its 
boiling point. This is why tissue plunged into LN2 alone doesn't 
freeze well. The LN2 flash evaporates and forms an insulting vapor 
barrier around the specimen. Therefore the isopentane, etc.. But ... 
slush nitrogen is near LN2's *freezing* point, and is therefore 
colder, so no other cryogens, such as isopentane, are needed. This is 
much safer.
To make slush nitrogen: fill a beaker (say 600 mL or 1000 mL) about 
60 to 90 % full with LN2. Place in a glass vacuum desiccator. Hook up 
a  high-capacity (this is important) vacuum pump. A large rotary pump 
will work fine. Turn on the pump and pump away. As the pressure in 
the desiccator is reduced, LN2 evaporates at a fast rate, which cools 
the remaining LN2. Eventually, the LN2 gets cold enough to start 
freezing, and slush starts forming. Stop, vent the desiccator, and 
use the slush LN2 for freezing. This takes maybe 10 - 20 minutes, 
depending on the volume of the LN2.


Does anyone out there have any tricks on how to prevent/minimize 
freezing artefact when freezing brain biopsy tissue? We are currently 
using Isopentane/Liquid Nitrogen. Have also tried freezing tissue on 
our new Leica cryostat using the Pelletier element. Both methods have 
resulted in varying degrees of success and failure. Also, we use Gum 
Tracacanth as our freezing medium (because that's what's always been 
used here).  Any thoughts on this would be more than welcome.


Philip Oshel
Supervisor, BBPIC microscopy facility
Department of Animal Sciences
University of Wisconsin
1675 Observatory Drive
Madison,  WI  53706 - 1284
voice: (608) 263-4162
fax: (608) 262-5157 (dept. fax)

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