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

From:Wayne Kreider

Thanks again... all very helpful.  I think we can now plan how we'll 
have the best chance at 'catching' transient bubbles.   I'm not sure 
we'll have our techniques together for sampling and freezing the tissue 
in the very near term (this week), but I am hopeful we can do/try this.  
By the way, you mentioned that frozen samples are not useful for EM...  
how so?  Perhaps due to subsequent fixing procedures?  I'm relatively 
unfamiliar with EM although TEM had been suggested for this work.

Philip Oshel wrote:

> Wayne,
> You do have a problem ...
> 2 X 2 X 2 mm is too big for any proper freezing method. Even 
> high-pressure freezing, which can in ideal conditions give the 
> greatest depth of good freezing, only does at best 500 microns. The 
> other methods do 200 microns or less. Plunging into LN2-cooled 
> cryogens will do a few 10s of microns, no more. They're fine for light 
> microscopy (assuming migration of molecules from holed membranes or 
> desiccation isn't an issue), but not EM.
> The major source of freezing damage isn't really mechanical 
> ice-crystal growth and damage, but dehydration of cells from crystal 
> growth.
> Plunging is literally dropping the sample into the cryogen, but not 
> really. By then I mean you *rapidly* plunge it in, not just let it 
> fall. By hand, as fast as possible. This is important in order to get 
> through the layer of cold nitrogen above the cryogen (whatever it is). 
> Some commercial plungers are designed to drop the sample into the 
> cryogen from enough height to pass through the cold gas layer quickly. 
> These are used for electrophysiology -- stimulate the sample then 
> drop, or stimulate during the drop, and therefore the sample is frozen 
> X msec after stimulation.
> I can see doing this with ultrasonication, depending on the size, etc. 
> of the sonciation apparatus.
> Cryoprotectants. Um. I don't think you can use these. The tissue has 
> to soak in the cryoprotectant -- often moving up a gradient -- in 
> order to get the cryoprotectant completely infiltrated into the 
> sample. This takes an hour or more -- maybe several hours? You'd 
> either have to assume nothing happens with the bubbles in the tissue 
> while sitting around infiltrating with cryoprotectant, or you'd have 
> to get the cryoprotectant into the tissue first, then sonicate and 
> pretend the cryoprotectant doesn't change the tissue properties and 
> therefore resulting bubbles.
> I wouldn't believe either of those choices.
> I think you'll need to do the experiment, then freeze, and use pieces 
> of tissue small enough to freeze properly. But! It's not entirely bad 
> -- only one dimension has to be =<200 microns -- 100 microns, really. 
> The other dimensions could be 2 mm.
> So: 2 mm X 2 mm X 100 microns (even thinner would be better) should 
> work. Just hold the tissue by a corner, so that the cryogen has 
> maximal access to both faces of the thin side. And move the tissue 
> once in the slush LN2, don't hold it in one place -- keep fresh 
> cryogen contacting the sample.
> Let's see -- oh. Insulate the container (and vacuum desiccator) as 
> best as possible, and work quickly, since you'll only have a few 
> minutes to freeze before the slush LN2 warms up to regular 
> almost-boiling LN2.
> (I think Bal-Tec makes an apparatus for making slush LN2, and maybe 
> freezing with it, but you don't really need it, it just maybe makes 
> things easier.)
> Phil
> 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/


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