[Histonet] Lovely set up for snap freezing

[Morken, Tim - Labvision]

Dry ice is at -78C, so you're not going to freeze ethanol with that. But the
reason to use extremely low temp freezing is to get the fastest RATE of
freezing possible to cause the minimum of ice crystal formation. LN2 can
cause some problems due to it's propensity to form a vapor layer next to the
tissue, and so decrease it's rate of freezing (hence the use of talcum
powder on tissue to "breakup" the vapor layer - whether that really happens,
I don't know). The other liquids mentioned do not form a vapor layer so may
show a higher rate of freezing than LN2 alone. Isopentane freezes around
-160C, so using it with LN2 will give a much greater rate of freezing than
any dry-ice slush (I always used a digital thermometer to check that the
isopentane was at least -150 before freezing tissue). If it's a one-off
procedure, then using dry ice slush may be OK, But if you are doing it
routinely, LN2/isopentane or using Gayle's method of floating the mold right
on the LN2 is the way to go. I've also used the CryoJane (Instrumedics, Inc)
apparatus with great results (metal "hammer" in LN2).


Tim Morken


-----Original Message-----
From: Philip Oshel [mailto:peoshel <@t> wisc.edu] 
Sent: Sunday, October 05, 2003 6:47 AM
To: Histonet <@t> Pathology.swmed.edu
Subject: RE: [Histonet] Lovely set up for snap freezing

The acetone, ethanol, isopentane, or whatever are going to be as cold 
as the refrigerant -- dry ice -- and no colder. Perhaps slightly 
warmer, depending on the set up.
Yes, this is closer to the freezing point of EtOH or acetone, but 
that doesn't matter, they're both still fluid.
The best freezing is at liquid nitrogen temperatures, either 
high-pressure freezing, which gives the greatest depth of evanescent 
spherule/microcrystaline/amorphous (vitreous) ice, depending who is 
talking, or propane-jet which is sometimes next best for depth but 
uses explosive propane under pressure, or plunging into slush 
nitrogen, which is the coldest, being at the freezing point of 
nitrogen and gives the best freezing within its depth of good 
freezing, but gives the shallowest depth of good freezing.
I've deliberately not given depths of good freezing because that is 
highly dependent on the samples, tech headaches, phase of the moon, 
the usual stuff. Propane jet might work for muscle fibers, HPF would 
except that it requires very small samples -- sized to fit on a TEM 
grid -- slush would be good for very small diameter (no, smaller) 
fibers or the outer 30, maybe 50 microns.
Equipment is expensive: HPF very, propane jet, slush relatively cheap.
None of these methods use any other vehicle: isopentane, ethane, 
EtOH, acetone, whatever. Remember: the sample thickness is tissue + 
carrier medium (buffer, or whatever not blotted off) + sample holder. 
The less non-tissue the better.
Phil

>Interesting thread.
>I always thought you used isopentane because it had a much lower freezing
>point (-160C) compared to ethanol (-114.5C) and Acetone (-95.4C).
>Surely this would have an effect in a dry ice slurry, which would be near
>the freezing temperature of both Acetone and Ethanol and therefore not be
as
>effective as the isopentane in quenching the tissue due to the ethanol and
>acetone starting the freeze crystallisation process?
>Just a thought.
>
>Nick Kirk
>Histopathology
>Hinchingbrooke Hospital
>Huntingdon
>England
>
>-----Original Message-----
>From: histonet-admin <@t> lists.utsouthwestern.edu
>[mailto:histonet-admin <@t> lists.utsouthwestern.edu]On Behalf Of Philip
>Oshel
>Sent: 04 October 2003 23:38
>To: Histonet <@t> Pathology.swmed.edu
>Subject: Re: [Histonet] Lovely set up for snap freezing
>
>
>Gayle, Gail, & et al.,
>
>A couple of things here:
>First, why dry ice/isopentance slurry? Why not dry ice/ethanol? or
>acetone? Yes, both are flammable, but much less so (as in not
>explosive) than isopentane. I've done a fair amount of
>freeze-substitution with dry ice-ethanol or acetone, and they work
>well. And they're just as good -- or poor -- for freezing.
>Which brings up my second point, which some of you may be tired of
>hearing but ...
>You do have freezing artifacts. Just because you can't see them
>doesn't mean the artifacts aren't there and affecting your staining
>or localizations.
>The morphology may be perfectly good for light microscopy, and the
>staining results may be appropriate as well. But. More rigor needs to
>be used when working with freeze-fixation, and the exact nature of
>the purpose of the stains or other reactions must be specified.
>Otherwise the perfectly acceptable morphology may be badly misleading
>as to the real nature of the stained elements.
>There. Rant over.
>
>Phil
>
>>Gail,
>>
>>From one Gayle to another, a lovely setup, very stable and efficient.
>>
>>Curious though as to why you don't let blocks touch the dry ice?  That has
>>never been a worry for us, as they are going from much colder liquid N2
>>temps to approx -70 to -90C (dry ice) then into -55C freezer. When using
>>dry ice/isopentane slurry we put newly frozen blocks on dry ice to
>>evaporate isopentane fumes before going to -80C freezer.  I don't think
the
>>dry ice hurts anything - in fact, you would have to use it to ship frozen
>>blocks.
>>
>>Do you get some kind of defect (scratch marks, etc) from putting blocks on
>>dry ice?  We have used a solid block of dry ice to snap freeze with
plastic
>>cryomolds, OCT embedded tissue, but if the tissue is large, freezing
>>artifact is present i.e. mouse spleen.  We do use solid dry ice on
occasion
>>to snap freeze extremely tiny NALT, or nasal associated lymphoid tissue
>>from mouse - one of our toughest tissues to dissect out and maintain flat.
>>
>>We never remove the disposable plastic cryomold until just before mounting
>>block on chuck. Obviously your method is very cost effective and you reuse
>>the molds.
>>
>>Thanks for the info,
>>
>>Gayle Callis
>>
>>At 04:17 PM 10/3/2003 -0400, you wrote:
>>>Gayle,
>>>      We basically do the same thing you do.  We use a stainless steel
>square
>>>block sitting in the liquid nitrogen.  We fill the foam container with
>>>liquid nitrogen covering the s.s.block, this is left for 5-10 minutes
with
>>>adding liquid nitrogen as it goes down. Once the s.s.block is cold
enough,
>>>the level of liquid nitrogen is kept below the top of the s.s. block. You
>>>can then set your embedding boat on it. We use the Tissue Tek metal
>>>embedding boats and embedding rings with Shandon's M-1.  The metal boat
>>>chills and the M-1 freezes fast enough to keep away the freezing
artifact.
>>>The s.s. block is stationary and is easy to set the boat onto.  All the
>>>surfaces of the block are flat for cutting. It is easy to do and gives
>great
>>>blocks and tissue.  While we are collecting tissue, we pop the block out
>of
>>>the boat and put the block into a plastic bag in another foam container
>with
>>>dry ice to keep them frozen until we are done.  The blocks never touch
the
>>>dry ice and are transferred to a -55 freezer.
>>>     Your way is basically the same. I don't see anything wrong with it.
>>>
>>>    Gail Macke,HTL
>>>    Shriners Burns Hospital--Cincinnati, Ohio
>>>
>>>-----Original Message-----
>>>From: Gayle Callis [mailto:gcallis <@t> montana.edu]
>>>Sent: Friday, October 03, 2003 10:37 AM
>>>To: George Cole; Histonet <@t> lists.utsouthwestern.edu
>>>Subject: further comments on Re: [Histonet] Gayle callis' method of
>>>freezing nerve
>>>
>>>
>>>Correction, or rather clarification to these comments:
>>>
>>>George,
>>>
>>>I do NOT immerse the bottom of the crymold DIRECTLY into pure liquid
>>>nitrogen so this direct contact starts freezing the OCT/tissue/mold
bottom
>>>per your description.  The cryomold with OCT embedded tissue is placed in
>>>bottom of the EMPTY platic petri dish that has been precooled by FLOATING
>>>the dish on a layer of liquid nitrogen - basically the petri dish is
>>>canoeing on liquid N2!  The plastic dish is not as cold as direct liquid
>>>nitrogen contact, acts as a buffer, but still extremely cold to give
>>>perfect artifact free freezing.  Freezing this way does not allow for any
>>>curvature of the plastic cryomold, it stays perfectly flat. We only use
>>>Tissue Tek cryomolds as other molds have plastic that is too thick and
>>>conducts heat away slower than the thinner Tissue Tek plastic. One joy of
>>>this method is once the OCT/tissue in cryomold is sitting in cold petri
>>>dish, you can go to the next block immediatetly allowing for a large
>volume
>>>of blocks/collection session.  We do not have to hold a cryomold while it
>>>freezes. It takes a steady hand to prevent liquid nitrogen from making
>>>contact with OCT over the top of cryomold.
>>>
>>>When the OCT finishes freezing to top of mold with petri dish methods,
>>>there are no indentations, just a nice layer of OCT. The petri dish
method
>>>is not as fast a freezing as you describe although it results in artifact
>>>free tissues, and the tidge of slower freezing probably means good
>>>interface of tissue to OCT, we never get gaps unless bubbles are not
>>>removed.
>>>
>>>My experience total immersion of OCT embedded tissues into liquid N2 OR
>>>embedded in a mold results in cracked OCT, and a horrible block to
>section.
>>>However, you are holding the mold to permit freezing starting at bottom,
>>>and as said before - a steady hand helps.
>>>
>>>Our preferred method of snap freezing is dry ice/isopentane slurry, to do
>a
>>>ton of blocks at one tissue collection session.  Only when we need
>>>perfectly flat faced blocks is the petri dish canoeing on liquid nitrogen
>>>used.
>>>
>>>Gayle Callis
>>>MT,HT,HTL(ASCP)
>>>Research Histopathology Supervisor
>>>Veterinary Molecular Biology
>>>Montana State University - Bozeman
>>>PO Box 173610
>>>Bozeman MT 59717-3610
>>>
>>>
>>>
>>>At 04:56 PM 10/2/2003 -0700, you wrote:
>>>>          Histotechs, Gayle Callis&#8217; method of freezing nerves is
>>>>illustrated in the DVD&#8217;s in the Muscle and Nerve packet I&#8217;ve
>>>>been send around.  It shows a nerve in a flat plastic mold covered with
>OCT
>>>>It stresses the fact that the liquid nitrogen must touch the bottom of
>the
>>>>mold only. It must not get into the top of the mold. Freezing causes the
>>>>OCT to contract.  Freezing from the bottom like Gayle says ands the DVD
>>>>shows, keeps the contraction to only a small dent on the surface of the
>>>>frozen OCT.  No problem.  This is shown in the DVD. It&#8217;s easily
>>>>filled. If the liquid nitrogen gets into the top of the of the mold, the
>>>>contraction will take place around the nerve making sectioning difficult
>>>>indeed.  Athough DVD One band 9 shows how to patch gaps like these,
>>>>there&#8217;s no reason to do so if you freeze the tissue carefully.
>>>>georgecole <@t> ev1.net
>>>
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>>
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>--
>Philip Oshel
>Supervisor, AMFSC and BBPIC microscopy facilities
>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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-- 
Philip Oshel
Supervisor, AMFSC and BBPIC microscopy facilities
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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