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

[Matthias Wolf]

Hi Manikandan,

I have never had problems with C-flat grids (original version, thin carbon, 400 mesh). Yes, they have thinner carbon than quantifoils, but that's one of their advantages. Similar to Terje, I glow-discharge for 20 sec at 20 mA, which is sufficient to render them hydrophilic without charging them so much that all protein sticks to the carbon. I don't clean them, but use them out of the box. Plasma cleaners like the Solarus broke all my carbon and are not necessary for this purpose.

However, I use a simple Guillotine-type plunger and blot 3.5uL sample manually in the cold room flat from the carbon side - very reproducible with a slow-wicking filter paper (e.g. 25 sec with Whatman nr.40, equilibrated at cold room humidity) and very controllable (+- 5 sec will change ice thickness). This results in consistent ice thickness for my virus samples, with most of the holes usable.  Ice thickness is on the order of 80 - 120nm (measured by drilling a narrow hole with the beam and tilting at the beginning of the session as well as by knowing the size of my virus particles, which do not overlap and are arranged in a single layer). You can get the ice even thinner, but then it's often not of equally thin (thicker towards the carbon layer). The cold room at ~4C has the advantage of quite constant ambient conditions throughout the year (with exception of barometric pressure), since the humidity in there is always close to saturation and it's the most simple solution. Of course, the addition of detergents makes things trickier. My buffer has usually nothing but 20-100mM buffer and 50-150mM salt.

Cheers,
   Matthias
 

On Aug 6, 2010, at 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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