Re: freezing muscle in liquid propane- lon reply

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From:Robert Schoonhoven <>
To:Sue Danielson <>
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I have had a lot of experience with using liquid propane for freezing
tissues for cryo TEM.  I have also (in the past), done a lot of muscle
work using Isopentane.  If you are working at the light microscopy level
propane will not improve your results if the sample sizes are the same. 
If the sample size is larger (for the propane freezing), you will only
have marginally better results towards the center of the biopsy.

There is of course more to than that.  There are 3 main phases to frozen
water and each is dependent on not just the freezing temperature but
(primarily) the thermal exchange rate.   These are hexagonal, cubic and
amorphous.  Amorphous 'ice' is water that is frozen so rapidly that NO
ice crystals have formed.

Hexagonal is formed at temps. of 0oC to about -80oC with thermal
transfer rates below 10,000oC.  The lower the temperature and the faster
the transfer rate the smaller the crystals formed.  At the extreme end
the crystals are not visible with the light microscope.

For cubic ice the temp. should be at -80oC or lower and the thermal
transfer rate should be better than 10,000oC/sec.. these crystals are
not visible at the light microscopic level and even at the low TEM range
of magnifications.

For amorphous ice temp. -140oC or more with thermal transfer rates at or
better than 80,000oC/ achieve that, rapid immersion into liquid
propane at -190 and plunge freezing at.... 2.5 - 3 m/sec = freezing rate
of  90,000oC/sec (see papers by Costello and Corliss, late '70s early
'80s)   The metal mirror method using a copper or silver mirror and LN2
will have a thermal transfer rate of about 100,000oC/sec ! But only for
a few microns.  The metal mirror method will give you about 20 microns
of amorphously vitrified specimen (in the TEM that is).  There are (or
were) also propane jet cryofixation devices for which claims in excess
of 20 microns were made.

It should also be noted that of both hexagonal and cubic ice crystal
formation are dependent on the temperature and thimble transfer rate,
the lower the temp. and higher the thermal transfer rate the smaller the

Having said all that, by increasing the temperature of a well frozen
specimen to cryostat temp. will indeed grow larger and, if if the temp.
becomes warm enough to cause ice crystals large enough to damage the
cells than there is nothing that can be done to repair the damage.  
At -25--30 oC they generally remain small enough not to be visible with
the light microscope.  Once the larger crystals have formed they do not
shrink or change from one type of ice to another if exposed to colder
temperatures.  There is quite a lot in the literature

Without the proper equipment (supplied by Leica, Balzars and others)
liquid propane and ethane are EXTREMELY DANGEROUS!  Even with the proper
equipment accidents can and have happened.  I would strongly advise you
not to try using propane freezing in a standard histology laboratory
setting.  There was much written in the E.M. literature in the '80's and
some home made devices were described.  If you really, really want to do
this then you could look them up.  I will not describe them.

So much for the meandering mind of an old man,  I've gone on for
paragraphs and not answered your question (and won't).

Sue Danielson wrote:
> Hello histonetters,
> We would like to give it a go and try to freeze some of our larger muscle
> specimens in liquid propane (as opposed to our normal technique using
> isopentane).
> Having no prior experience with propane (except on the BBQ grill), I am
> wondering what others have experienced when using this technique.
> Particularly, what is the best way to get the propane out of the tank and
> into our cup without it flashing to the gas phase immediately.  I believe
> this stuff is at a pretty high psi.  Basically, I have the tank, some
> stainless steel tubing, a manual control valve but no phase separator.  Hmmm...
> Any suggestions/past experiences shared are appreciated!
> Thanks,
> Susan Danielson, MS
> Neuromuscular Lab Coordinator
> Medical College of Wisconsin
> ph:  414.259.3836
> fax: 414.454.7905

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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