[3dem] contamination in cryo samples

Wim Hagen hagen at embl.de
Thu Sep 1 07:14:53 PDT 2016


Hi Nadav,

If the holder is too warm due bad clip-ring clamping, things are too warm, but I would then expect a time dependence, first you get “leopard pattern” which then migrates into cubic spots. Are the spots cubic?  This process always happens but it is faster when too warm, here’s an example of a badly clipped Polara cartridge after one night in the column:

   

Could also be your humidifier is "raining too course”, maybe try a wet sponge in the Leica chamber instead of the humidifier.

Is your cryo-box cold and stable? Take images every minute in LM mode with the  box visible, cool down the cold trap, you should see movement only over the first 30 minutes, then it needs to remain stable. If it keeps going back and forth between certain positions, it starts touching something, so it warms up, stops touching, cools down again etc.

An example of 30 min from our properly working Tecnai 12 cryo-box:



Best

Wim

> On Sep 1, 2016, at 11:00 AM, Nadav Elad <nadav.elad at weizmann.ac.il> wrote:
> 
> Dear all
>  
> We are having a serious contamination problem in our cryo preparations and would be grateful for any suggestion. Although this issue has been discussed in the past in this forum, the solutions raised do not seem to work. The attached image shows the contamination on a bad day. I am referring mainly to the lighter, smaller particles (10-20 nm) which are the most persistent. They seem to be above or below the ice layer and are the first to evaporate under the electron beam. The concentration of contaminants is variable. 
>  
> We are using a Leica EM GP plunger for freezing, and Tecnai T12 and F20 microscopes with Gatan side entry holders. We have tried to replace the ethane cylinder and are in the process of ordering a third one, cleaning the glow discharger machines, different cryo holders, different cryo-transferring units and grids of varying sources. There seems to be no leaks in the compustages of the microscopes.
>  
> Thank you for your help
>  
> Nadav
>  
> --
> Nadav Elad, Ph.D.
> Electron Microscopy Unit
> Weizmann Institute of Science
> Rehovot, Israel
> Tel: +972 (0) 8 934 2115
> Mobile:  +972 (0) 52 3909 420
> Email:  nadav.elad at weizmann.ac.il <mailto:nadav.elad at weizmann.ac.il>
>  
> <contamination.jpg>_______________________________________________
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