[3dem] Contamination problem on cryo grids

[Shi, Dan (NIH/NCI) [C]]

In most case, there are few dirty or contamination spots each grid
square, one always can find nice area to collect data. If the
contaminated spots are dense enough to affect the data collection, one
has to minimize the contamination for recording images. The dirty or
contaminated spots may come from contaminated carbon film, the chemicals
from buffer and/or even from the protein you put in. I would check the
grids by EM with following steps to identify the 'contamination' source:
carbon coated grids with and without glow discharge; if they are clean,
frozen-hydrated grid of pure water; if no contamination, freezing a grid
with buffer only. Once you know which caused the problem, you may find a
way to decrease the contamination as people suggested.
I'm not sure hydrophobic surface could cause this problem, because I
have used strong hydrophobic carbon film and did not see contamination
like that. Those dark areas seem to contain some high Z elements other
than H and O, you can use diffraction mode to see if those areas have DP
of ice.

Regards,

Dan Shi
LCB, NCI 

-----Original Message-----
From: Norm Olson [mailto:nholson at ucsd.edu] 
Sent: Monday, September 29, 2008 1:22 PM
To: 3dem at ncmir.ucsd.edu
Subject: Fwd: RE: [3dem] Contamination problem on cryo grids

>
>Dear Anselm,
>
>This looks very much like a hydrophobicity problem. The support is 
>not hydrophilic enough, preventing the water to spread nicely over 
>the film and the holes.
>
>I encountered this as a typical Quantifoil problem, since I (and 
>others) noted that there is residual plastic.
>

-- 
I have been reading this thread with a great deal of interest.  It 
seems like everyone has their own way things should be done but I 
have not seen any studies, I could have missed them, where controlled 
tests were performed.  Carbon films have always had somewhat of a 
"religion" around them but with the manufactured holey carbons it 
seems like it has gotten worse.  As an example, Quantifoils need to 
be treated to remove the manufacturing plastic, to reduce 
beam-induced charging, to reduce contamination, or to make the films 
more hydrophilic.  Is any of that correct? Maybe all of it is 
correct, who knows.  On the other hand, how many people are there out 
there who don't treat them in solvents at all?  Others I have talked 
with say that the C-flats give more of an even ice thickness because 
they don't have plastic but they still do charge which seems to 
contradict others who say that the plastic causes beam-induced 
charging.  Some people have told me that it is necessary to go 
through several different solvents, others have their one favorite 
solvent.

Just my observations from the bleacher seats....

Norm

______________________________________________________________
Norm Olson
Cryoelectron Microscopy Facilities Manager
1510 Bonner Hall
Department of Chemistry & Biochemistry, MC-0378
University of California San Diego
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nholson at ucsd.edu
http://cryoem.ucsd.edu
Cell:  (858)220-2183
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