[3dem] Problems blotting the liquid ethane

[Yifan Cheng]

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


Eduardo Sanz-Garcia wrote:
>
>
> Bill Tivol wrote:
>>
>> Dear Eduardo,
>> I don't blot the liquid ethane at all. When I remove the tweezer from 
>> the ethane and dip it into LN2, the ethane freezes.  The ethane that 
>> remains on the grid protects against ice crystals landing on the grid 
>> during the transfer to the grid box, and the ethane is gone by the 
>> time the grid is loaded into the scope. 
> Do you have any guest of what is the highest temperature during the 
> transfer?
> I have an hypothesis: I do the whole process so near to liquid N2 
> temperatures that there is not chance for the solid ethane on the grid 
> to sublime.
> Is that possible?
> If the ethane is still solid when holder is inserted in the microscope 
> how long would it take to sublime?
>> Furthermore, ethane is non-polar, so it would not be expected to wick 
>> into the filter paper, which is cellulose and, therefore, polar.
> This is not true. Filter paper can blot non-polar substances, 
> including liquid ethane.
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-- 
************************************************************
Yifan Cheng, Ph.D.
University of California San Francisco
Department of Biochemistry and Biophysics
Mission Bay, Genentech Hall
600 16th Street, Room S312B
San Francisco, CA 94158-2517, USA
Phone: 1-415-514-9707 (office), 1-415-514-9708 (lab)
Fax:   1-415-514-4145
email: ycheng at biochem.ucsf.edu
website: http://cryoem.ucsf.edu
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