[3dem] CFlat Thicker Ice in holes

[Bob Grassucci]

Hi Lige,
Are you sure that these are true holes?  We use thin carbon over our 
holey grids and this looks like what happens when the back side of the 
grid gets the sample.  There could also be a problem with the carbon 
surface or the buffer you are using but check the easy thing first.
Bob

On 11/20/2013 5:41 PM, Lige Tonggu wrote:
>
> Dear all,
>
> Since October, I frequently observe that Cryo samples prepared with 
> CFlat 2/2-4C (Lot#45207-45211) have reverse contrast, namely, the ice 
> inside the hole is thicker than the outside as it is shown in the 
> attachment while gold particles on the same grid are in the right 
> contrast.  Whether or not to coat a thin layer of carbon on CFlat does 
> not seem to make a difference. I usually deposit a layer of carbon on 
> CFlat and put the grids in desiccator for 1 week and then do 
> plasma-cleaning right before use. The CryoEM session is performed by 
> FEI Titan F20 with Eagle camera, and the images are taken with 
> underfocus around -4um. Does anyone observe similar situations? Any 
> suggestions would be greatly appreciated.
>
>
> Thank you for your help in advance!
> Sincerely,
> Lige
>
> -- 
> Lige Tonggu,Ph.D. student
> Molecular and Cellular Biology Program
> Liguo Wang's Lab
> Department of Biological Structure
> University of Washington
> http://faculty.washington.edu/lw32/
>
>
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