[3dem] vitrified ice but not continuously, How to make it better?

[Li, Kunpeng]

Dear All,

        Recently I want to optimize the freeze conditions of a protein complex, But No matter how I can change the conditions, I cannot get the thin and continuous vitrified ice. the mass of the protein complex is almost 900 kDa. and the carbon grid is Quantifoil 1/1, glow discharge time is 1.5 minutes, the humidity in the plunger chamber is more than 80%RH,  and the ice thickness I estimated is near to 60 nm, I post the images from search mode to photo mode under CM200 with 1k*1K CCD.  I tried a low salt buffer and longer blot time at this check round. The accurate number post on the attached images.
         Do you confused with these similar problems once before?  Could you give me some clues to improve it?

          Thanks for any help.


  
-------------- next part --------------
A non-text attachment was scrubbed...
Name: vitrified ice.png
Type: image/png
Size: 938018 bytes
Desc: vitrified ice.png
URL: <http://mail.ncmir.ucsd.edu/pipermail/3dem/attachments/20150723/07c90ac1/attachment-0001.png>