[3dem] C-flat grids cryo-EM with manual blotting

[Terje Dokland]

We have used C-flat grids and manual blotting for some time, until we  
recently got our Vitrobot. Like you, we find that if we glow  
discharge the grids, we cannot get thin ice. The problem seems to be  
that the water goes through the holes, and since we blot only from  
one side we get thick ice forming on the other side. In the Vitrobot,  
we don't have this problem, since the grids are blotted from both  
sides. Perhaps it would work better if you blot from manually from  
both sides. We have never tried that (too complicated).

If we don't glow discharge and blot for about 2-3 sec we get some  
thin ice, but the thickness is very variable from one area to the  
other and you tend to get huge gradients of ice thickness ("lumps" of  
ice of which only the edge is usable) as you have also observed, but  
that's how we have been operating, until we got the Vitrobot.

On the other issue that you mention, we find that the film on the 200  
mesh grids is too fragile and is almost always completely broken by  
the time we get it in the microscope. The 400 mesh grids are more  
robost and retain most of the film, however.

Terje


On Dec 10, 2008, at 3:29 AM, Jean Watermeyer wrote:

> Hi all
>
> Is there anyone on this list who has been doing cryo-EM with C-flat  
> grids, using manual blotting, and glow-discharging?
>
> We have been trying to optimise our blotting for the thinnest  
> possible ice and have found several problems:
>
> - if the blotting is too rough or the plunging too deep, all of the  
> carbon comes off the grid
> - if we glow-discharge for 10s at 20 mA, blotting for ~8 s at 50%  
> humidity, we get ice that is much too thick
> - without glow-discharging, we get too steep a gradient of ice  
> thickness
>
> We'd really appreciate hearing from anyone who has managed to get  
> very thin ice with these grids using manual blotting.
>
> Sincerely
> Jean Watermeyer
> _______________________________________________
> 3dem mailing list
> 3dem at ncmir.ucsd.edu
> https://mail.ncmir.ucsd.edu/mailman/listinfo/3dem