[3dem] Contamination problem on cryo grids

[R.I.Koning at lumc.nl]

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.

Now I can not with certainty make out what you ment with "with 2nm Carbon on top". Does that mean that you sputter 2 nm extra carbon on one side of the Quantifoil or that you float a very thin layer of carbon over the film and holes??? In any case, you seem to have put the quantifoil in the Chloroform solution AFTER you put your additional carbon.

If it is the first, make sure to sputter the carbon on both sides of the grid. If it is the second make sure not to put the film in choloroform afterward. This can cause the residual plastic to redistribute over the caron film, which will result in hydrophiic pathes whether you put the carbon yourself or not. Take notice that performing an additional washing step does not necessarily remove all plastic!! (otherwise there would be no reason to have any plastic left on the grids when you get them from Quantifoil).

What I standard do is sputter a very thin layer of carbon on BOTH sides of the Quantifoil and NOT remove any plastic. Works fine.

With best regards,
 
Roman Koning
 
R.I.Koning, Ph.D 
Leiden University Medical Center
Department of Molecular Cell Biology
Section Electron Microscopy 
Postal Zone S1-P
P.O.Box 9600, 2300 RC
Leiden, The Netherlands 
( (+31) 71 526 9296
1 (+31) 71 526 8270
* r.i.koning at lumc.nl 
" http://www.lumc.nl/1050/research/electron_microscopy.htm 

-----Original Message-----
From: 3dem-bounces at ncmir.ucsd.edu [mailto:3dem-bounces at ncmir.ucsd.edu] On Behalf Of Anselm Kusser
Sent: Friday, September 19, 2008 1:42 PM
To: 3dem at ucsd.edu
Subject: [3dem] Contamination problem on cryo grids

We are repeatedly experiencing problems with significant amount of contamination on our grids (follow the links to have a look at some example images).

http://www.cip.ifi.lmu.de/~kusser/CCD_search_01.jpg
http://www.cip.ifi.lmu.de/~kusser/CCD_search_02.jpg
http://www.cip.ifi.lmu.de/~kusser/CCD_hole_01.jpg
http://www.cip.ifi.lmu.de/~kusser/CCD_hole_02.jpg


I will try to describe the conditions we use:

buffer composition:
10 mM Hepes ph 7.8
40 mM Ammonium Sulfate
0.5 mM Mg Cl2
10 uM Zn Cl2
10 mM DTT

protein concentration:
~100µg/ml

grids:
Quantifoil precoated, Typ R3/3, Cu 300 mesh, with 2nm Carbon on top

glow discharging:
wash with Chloroform, then glow discharge 30s @ 2.2x10e-1 torr using a plasma cleaner

freezing (FEI Vitrobot, 4°C, 95% hum.):
apply 3,5µl of sample to grid, incubate 45s, blot 10s, plunge into liquid Ethane, transfer in N2 gas phase to grid box (also in gas phase) - but contaminations also occur when working in liquid N2 phase while for other poeple/samples gas phase transfer works fine (that was our first suspicion)

transfer to microscope:
transfer one single grid on a Gatan Cryo-Workstation to a Gatan Cryo-Holder, then insert into side entry stage microscope (FEI 120 kV Spirit)

Much oblidged for any suggestions.

Anselm


------------------------------------------------------------------------
Anselm Kusser
Gene Center
Department of Chemistry and Biochemistry Ludwigs-Maximilians-University of Munich Feodor-Lynen-Str. 25
81377 München
kusser at lmb.uni-muenchen.de







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