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

[Stephen Mick]

Hello All,

As a note to the community, for those who need a more rigid film we  
also offer a double-thick carbon C-flat.  Many have found that the  
thicker film adds flexibility to the sample prep process,  
particularly for 200 mesh grids. Hope this helps.

Best Regards,
Stephen


==============================
Stephen Mick, Ph.D.
Protochips, Inc.
www.protochips.com
617 Hutton St, Suite 111
Raleigh, NC, USA 27606
+001 919.341.2612 (office)
+001 919.341.2748 (fax)



On Dec 11, 2008, at 1:01 AM, Matthias Wolf wrote:

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
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