[3dem] Problems blotting the liquid ethane

[Benoit Zuber]

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
>
>


-- 
Benoît Zuber
MRC Laboratory of Molecular Biology
Hills Road
Cambridge
CB2 0QH
United Kingdom
+44 1223 402209
bzuber at mrc-lmb.cam.ac.uk