[3dem] Breaking of C-flat carbon and vitrification

Fri Aug 6 10:49:43 PDT 2010

We had the C film broken problem with C-flat even 400 mesh grids. The company was using thin/slim bar grids to support the fragile carbon film. Those grids are very soft, and most of them were bent when were  picked out from the box.  I suggested the company to use regular Cu (25 um of bar width for 400 mesh) and thick carbon coating for us. The broken carbon area from either Vitrobot or MRC plunger dramatically decreased since we started using the new C-flat grids with regular Cu grids few months ago. The extra supporting area and stronger frame from the bars seems to hold the carbon film more firmly. Good luck,

Dan Shi

From: Bob Grassucci [mailto:rg2502 at columbia.edu]
Sent: Friday, August 06, 2010 12:58 PM
To: M.Karuppasamy at lumc.nl
Cc: 3dem at ncmir.ucsd.edu
Subject: Re: [3dem] Breaking of C-flat carbon and vitrification

Hi Manikandan,
I had some initial problems with the Mark IV giving reproducible results and it turned out that the zero blot force was never properly calibrated.   Once this was done we were able to hone in one reproducible settings with our Quantifoill grids.  I would suggest you speak to your FEI service person to make the proper adjustments.  One other question is are you sure it is the blotting that breaks your films and not the glow discharging?  In the past c-flat grids were known to be less strong than quantifoil.  They claim this is no longer the case and it may be so but I can not confirm it from personal experience.  Hope this helps.
Regards,
Bob

On 8/6/2010 12:38 PM, M.Karuppasamy at lumc.nl<mailto:M.Karuppasamy at lumc.nl> wrote:
Dear All,

Could you help us on how to get optimal & reproducible (thickness) samples for SP cryo-EM?

We use a FEI vitrobot mark IV, typically with the following blotting parameters: C-flat support, 3.5 ul sample per (30sec glow discharged) grid, 3 sec blotting time, blotting force 3, 100% humidity, RT.

Unfortunately, we frequently notice the breaking of C-flat carbon support during vitrification. Example images are attached.
Only very few meshes are left after vitrification. In those meshes, sample is only found in the middle of the mesh and generally it is still too thick (150->200nm). We played around with blotting parameters but didn't find optimal ones yet for our samples.

Thanks & regards

Manikandan K.

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