[3dem] Problems blotting the liquid ethane

[Bob Grassucci]

Hi Everyone,
    Just my 2 cents.  Ben mentions grease/oil on the bottom of the 
ethane tank.  It usually appears a small high contrast approximately 
circular in  appearance.  It can be eliminated or minimized by either 
using high purity ethane (5.0) or avoiding completely emptying your tank 
(since the nasty stuff is generally on the bottom) and definitely try 
not to agitate the tank for about an hour before using it for plunging.  
If you want to see some photos of what the ethane contaminants look like 
you can find them in our article . Nature Protocol.2007;2(12):3239-3246 
Figure 2a.
    Regards,
    Bob

Benoit Zuber wrote:
> Hi everyone,
> I have experienced exactly what Yifan describes. When I plunge-freeze 
> a grid, if this grid has a large droplet of solid ethane and if I 
> transfer it straight away in the microscope, then even after being 
> pumped by the turbo for 45 sec, the remaining ethane always causes a 
> vacuum crash. Moreover, when the droplet of solid ethane is too thick 
> I cannot well position the clip ring to hold the grid.
>
> Therefore, I usually blot the ethane just after freezing the grid by 
> raising the grid just above the level of liquid nitrogen and touching 
> it gently with a whatman blotting paper. I take care not to absorb any 
> liquid nitrogen with the blotting paper, so that as soon as ethane 
> melts I can see it being sucked by the paper. From the moment I take 
> the grid out of nitrogen and the moment ethane melts, it takes roughly 
> ten seconds. I make sure no liquid remain on the grid (after 2-3 sec 
> of blotting), and then plunge it back in liquid nitrogen.
>
> By doing so, I have no more problems of vacuum crash or difficulties 
> to clip the ring; plus my grids are still vitreous.
>
>
> Now regarding, the contamination. It is certainly not ethane, because 
> as others mentioned earlier it evaporates in the column. However it 
> could be some grease/oil contamination coming from a nearly empty 
> ethane gas cylinder.
>
> Regards,
>
> Ben
>
> Yifan Cheng wrote:
>> Hi Eduardo,
>>
>> Here is what I think: Frozen ethane on grids will not evaporate 
>> during the transfer or very very slowly. You wouldn't be able to 
>> remove a large drop of frozen ethane even if you pump the holder for 
>> more than 60 sec before insertion, because the vacuum by rotary pump 
>> or turbo pump is not high enough. Frozen ethane will evaporate very 
>> fast, in a couple of seconds, at higher vacuum such as by oil 
>> diffusion pump or at column vacuum.  If you have a large drop of 
>> ethane frozen on grid, what will happen is that the column vacuum 
>> will crash after insertion of holder. This is not because of a bad 
>> operation, but because of a sudden drop of vacuum caused by 
>> vaporization of frozen ethane. Of course, your vacuum will fully 
>> re-cover after a few minutes, no big deal. (If you use JEOL Helium 
>> microscope, column vacuum crash will not happen, because the transfer 
>> arm is pumped by oil diffusion pump.) But in any case, if your grid 
>> looks dirty in the electron beam, it is not because of frozen ethane 
>> but something else.
>>
>> If you keep you grids in the lipid N2 tank for some days, frozen 
>> ethane drop often fall off from grids. It's better to blot off the 
>> liquid ethane if you want to look at your sample right away and don't 
>> want to crash the vacuum of your column after every transfer. I blot 
>> off ethane for every grids I freeze.
>>
>> Yifan
>>
>>
>
>


-- 
********************************

-Robert Grassucci-
Howard Hughes Medical Institute
Wadsworth Center Empire State Plaza
Albany, NY 12201-0509
bobg at wadsworth.org
Phone: (518)474-5821
Fax: (518)486-2191




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