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

Bob Grassucci rg2502 at columbia.edu
Fri Aug 6 09:58:10 PDT 2010

  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.

On 8/6/2010 12:38 PM, 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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