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

[Matthias Wolf]

Hi all,

I use C-flat copper 400 mesh, 1 um hole size, 2 um distance.
Glow discharge 20 sec at +20 mV (a longer time pulls my particles 
towards the carbon and 10 sec is usually the minimum to render the 
surface hydrophilic). I blot 4 ul sample manually in the cold room at 4C 
from the carbon side using Whatman Nr.40 (slow, calcium free) for 25-30 
sec. This results in reproducible ice thickness between 400-800 A. It is 
possible to get thinner ice by increasing the blot time, but the amount 
of dry grid squares will increase dramatically. The cold room is 
essential to maintain humidity near saturation throughout the seasons.

I have successfully used both C-flats and Quantifoils. However, I have 
never experienced charge-induced drift on C-flats, but often on 
Quantifoils (soaking in ethyl acetate to remove plastic residue helps, 
but not significantly). In my hands, C-flats result in thinner and more 
homogeneous ice with no charging and less cryo-crinkling than 
Quantifoils. I have reached my highest resolutions only when using C-flats.

The carbon film on the C-flats I have used so far is indeed thin, but 
just right. Yes, it is fragile and hence requires handling with care. In 
my opinion, the vitrobot is far too harsh on grids and allows no control 
  over blotting force or closing speed and its standard filter paper 
wicks too fast for adequate control by blotting time alone. I hope that 
this discussion won't prompt the manufacturer of C-flat grids to modify 
their excellent product by thickening the carbon film.

    Matthias

--
_________________________________________________
Matthias Wolf, PhD - Postdoctoral Research Fellow
Harrison Laboratory
BCMP, Harvard Medical School
Seeley G. Mudd 130
250 Longwood Ave. Boston, MA 02115
Phone: 617-432-5602, email: wolf at crystal.harvard.edu
http://crystal.harvard.edu


> On Wed, Dec 10, 2008 at 3:29 AM, Jean Watermeyer 
> <jeanwatermeyer at gmail.com <mailto:jeanwatermeyer at gmail.com>> 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 <mailto:3dem at ncmir.ucsd.edu>
>     https://mail.ncmir.ucsd.edu/mailman/listinfo/3dem
> 
> 
> 
> ------------------------------------------------------------------------
> 
> _______________________________________________
> 3dem mailing list
> 3dem at ncmir.ucsd.edu
> https://mail.ncmir.ucsd.edu/mailman/listinfo/3dem