[Histonet] sample preparation/analysis to observe gas bubbles

[Wayne Kreider]

Phil,
Thanks for the input.  Unfortunately, I haven't had much luck looking in 
the archive. 

The treatment is actually acoustic (high intensity ultrasound), so no 
electrodes are involved.  Our treatment volume that we'd ideally like to 
freeze is about 2mm x 2mm x 5mm.  However, from the little I've read 
about plunging, a guideline for the maximum sample thickness is 0.2mm.  
Using a slush as you describe would presumably help some, but I suspect 
2mm is still too thick. 

If we are able to obtain a suitably thin sample, is plunging literally 
just dropping the raw tissue into the cryogen?  Or are there some 
additional steps that should be taken in handling the sample?  Moreover, 
if we do have a larger sample, reference books suggest that it's 
necessary to use a cryo-protectant.  Is a cryo-protectant likely to 
distort the tissue, in particular any bubble-type structures?  Also, 
what time delays are typically associated with using a protectant?  Any 
suggestions about which cryo-protectant might be best suited for this 
application?  Thanks again...  I'd appreciate any thoughts you might have.
Wayne

Philip Oshel wrote:

> 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.
>
> Phil
>
>> 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
>> -- 
>>
>> /**********************/
>>
>> /Wayne Kreider/
>>
>> /University of Washington/
>>
>> /Applied Physics Lab / CIMU/
>>
>> /106 Old Fisheries/
>>
>> /206-543-1324/
>>
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>

-- 

/**********************/

/Wayne Kreider/

/University of Washington/

/Applied Physics Lab / CIMU/

/106 Old Fisheries/

/206-543-1324/