Re: [Histonet] sample preparation/analysis to observe gas bubbles

From:Philip Oshel

Cryofixation is the only way I know to preserve this kind of 
structure. There have been discussions about this in the past, so you 
might want to poke around in the Histonet archives.
What kind of treatment? Is it something that you stimulate with say 
an electrode?
If so, there are plunge freezers designed to allow you to stimulate 
the sample while it is falling into the liquid nitrogen.
Rapidity of freezing is essential to insure vitrification of the 
water, instead of crystal formation. Although some folks maintain 
that the water doesn't vitrify, but instead forms micro-crystallites 
small enough to have no effect on structure. Others state that 
evanescent microspherules are formed, sort of neither fish nor fowl 
-- not crystalline and not glass. Either way, the frozen samples have 
to be maintained below the recrystallization temperature (around 
-80deg C or so) until after dehydraton or embedding to prevent 
crystal growth and negating all the rapid freezing goodness.
But, the 4 basic methods are high-pressure freezing, propane-jet 
freezing, slam-freezing, and plunging into cryogen. The first three 
will likely distort the air bubbles and tissue around them, however. 
Plunging works well, as long as the plunge is made very rapidly, and 
no time is spent hanging about in the cold -- well below freezing -- 
nitrogen atmosphere above the cryogen.
Many people plunge into some organic fluid like ethane which is held 
in a container in liquid nitrogen. This works, but it has a few 
issues: the cryogen is usually warmer than the LN2, so the freezing 
is not as rapid as it needs to be, and such organic cryogens are 
flammable or explosive when they warm up. Especially since, being 
near LN2 temperatures, they're enriching themselves in oxygen from 
the air (liquid air is warmer than liquid nitrogen). They can be 
handled safely, but this does require some thought.
A better way is to plunge into slush nitrogen -- LN2 near the 
freezing point, instead of near the boiling point. This is about 14 
deg C colder than LN2, so freezes samples faster, and so gives a 
greater depth of good freezing. It's also nitrogen, so there's no 
flammable gas to deal with (nor bureaucrats upset about flammable 
gases in your lab). It's easy to produce: just put a beaker full in a 
small to medium size vacuum desiccator, and pull a vacuum with a high 
capacity pump, like a dual-stage rotary pump. One used to rough out a 
large EM column usually works.


>We're exploring a treatment in which small short-lived (perhaps on 
>the order of seconds) gas bubbles may be generated in tissue.  To 
>this end, we're interested in learning the 'instantaneous' state of 
>the tissue immediately after treatment with regard to the presence 
>of any bubbles.  Currently, our experiments utilize rabbit muscle. 
>Are there any known histological sample preparation/analysis 
>procedures that have been used to preserve/observe resident gas 
>bubbles?  We have considered a flash freezing followed by staining 
>and/or electron microscopy.  However, our expertise is not in 
>histology, so we'd very much appreciate any suggestions and/or 
>details regarding possible sample preparation.  Thanks.
>/Wayne Kreider/
>/University of Washington/
>/Applied Physics Lab / CIMU/
>/106 Old Fisheries/
>Histonet mailing list

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

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