FW: Freezing tissues

From:Jim at ProSciTech <jim@proscitech.com>

It should be understood that the methods discussed are used in electron 
microscopy to achieve vitrification of ice - no crystalline structure. No harm 
in using the most rigorous methods for light microscopy - except that it is 
largely a waste of effort. Methods like the CryoJane (I have an interest in) or 
using Iso-pentane suffice. The comment by one correspondent, that she could see 
no difference between liq N2 and Iso-pentane frozen is probably right, since 
small blocks, or  dryer tissues (skin), or more rapid movement when freezing 
tissues makes a good deal of difference, and ultimately you may not be able to 
see in the light microscope small ice crystal damage.

I preferred N2 slush or cold solvent freezing over a metal- mirror slam 
(interestingly diamond has the best heat transfer rate, but not many people 
invest in diamond anvils). However, I understood that the major drawback of the 
method is, that only a very thin layer is vitrified, but that layer was 
excellent and distortion due to slamming, surprisingly is not a factor.

The iso-pentane and alternatives including nitrogen slush were discussed 
extensively on this server for a week starting on the 16 of May 2000. Its worth 
to look up the Archives for that, especially for the fundamental truth: boiling 
cools! That and all the other salient facts are in the Archives.

Incidentally, if your liq N2 does not turn into slush within a couple of 
minutes your pump is a bit under-sized. Pump volume is important, ultimate 
pressure is of little consequence. The volume of N2 slush will be larger than 
the volume of liq N2 used.
Jim Darley
ProSciTech                 Microscopy PLUS
PO Box 111, Thuringowa  QLD  4817  Australia
Ph +61 7 4774 0370  Fax:+61 7 4789 2313  service@proscitech.com
Great microscopy catalogue, 500 Links, MSDS, User Notes
ABN: 99 724 136 560                      www.proscitech.com

On Tuesday, October 03, 2000 3:08 PM, J. A. Kiernan 
[SMTP:jkiernan@julian.uwo.ca] wrote:
> Thanks Phil, for the best informal account of freezing I've ever read,
> especially the use of analogy to explain why you can't dunk objects
> in liquid nitrogen and expect them to freeze quickly. I think you
> should submit this, or a slightly edited version to Microscopy Today!
> Is it _really_ possible to apply suction to a beakerful if liquid N2 and
> have it cool to near freezing before it all boils away? Would an ordinary
> lab's public "vacuum" line do this? Also, doesn't the liquid component
> of the nitrogen slush evaporate and protect the specimen?
> You mentioned slam freezing (not very practical for most of us) but
> not freezing by simple contact with metal (such as a cryostat chuck)
> that has been cooled to liquid N2 or even solid CO2 temperature. I
> appreciate that as an electron microscopist you cannot tolerate even
> the smallest ice crystal damage.
> LM is more forgiving, at least for muscle, skin and CNS. You can't get
> away with dunking in liquid N2 or letting un-cryoprotected things freeze
> slowly in a cryostat cabinet, but visible ice crystal holes are mostly
> too small to see if you place a 2mm specimen on copper or aluminium and
> immerse in acetone that contains solid CO2 (dry ice). Freezing on a
> CO2 freezing microtome is similarly rapid and satisfactory, but you
> can't cut sections as thinly as you can with a cryostat. Tiny ice-holes
> in muscle fibres are often seen with freezing in CO2 or isopentane-liquid
> N2, often with an almost geometrical distribution, but they don't interfere
> with enzymatic or immunohistochemical staining of fibre types.
> I've left your original email in this reply because it's too good to
> delete. Someone may have missed it the first time round.
>                                  John Kiernan
>                                  London, Canada.
> _____________________________________________________________
> On Mon, 2 Oct 2000, Philip Oshel wrote:
> > There are two possibilites for plunge freezing in LN2: one is
> > "normal" liquid nitrogen -- as it comes from the dewar, the other is
> > slush nitrogen.
> >
> > 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
> > agent.
> >
> > 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
> > cryostat.
> >
> > Phil
> >
> > >I would welcome your opinions and feedback.
> > >
> > >What is the optimal method of freezing tissues with liquid nitrogen?
> > >with or without isopentane and why?
> > >
> > >thanks,
> > >Vinnie
> > >
> > >
> > >
> > >Vinnie Della Speranza
> > >Manager for Anatomic Pathology Services
> > >Medical University of South Carolina
> > >165 Ashley Avenue
> > >Suite 309
> > >Charleston, SC  29425
> > >ph:  (843) 792-6353
> > >fax: (843) 792-8974
> > >email: Dellav@musc.edu
> >
> > --
> > }}}}}}}}}}}}}}}}}{{{{{{{{{{{{{{{{{
> > Philip Oshel
> > 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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