[3dem] Gatan 914 Holder Woes

[Bob Grassucci]

Cindi,
It sounds like you eliminated the grid making itself so what remains id 
the transfer and in column contamination build up.
It appears you are getting the type of contamination that appears inside 
your scope when there is insufficient anti contamination.  Are your 
cryo-shields inserted and cold?  I imagine since you are using a high 
tilt holder you are doing tomography and therefore want to get as much 
clearance as possible at high tilt.  You may be compromising on 
anti-contaminator efficiency.  You could use them for about 20 minutes 
to get the majority of the contamination after transfer.  This also 
happens sometimes with the ct3500 style of cryo transfer holder if you 
open the transfer shield too soon after putting it into the column.  
This happens because only the area immediately around the sample remains 
cold in the microscope and the rest of it warms to room temperature 
releasing any ice that came along for the ride.  Waiting about 20 
minutes after the transfer is usually sufficient for the majority of  
contaminating ice to get trapped by the anti-contaminator blades. Give 
these suggestions a try and good luck.
Regards,
Bob

Cindi Schwartz wrote:
> Hello List,
>
> We are having problems with plunge-frozen cryo grids only when using 
> our 914 holder. Basically, we are getting what I like to call, the 
> cauliflower effect. Sometimes it's very subtle and sometimes it's 
> blatant. Here are the facts:
>
> 1. This is happening to multiple people with multiple sample types
> 2. This happens when using the vitrobot or by hand-blotting
> 3. Grids from the same freezing session viewed with a Gatan 626 holder 
> do not have bad ice
> 4. Temperature measurements of the 914 holder during all phases of 
> loading the holder and inserting it into the microscope never get 
> above -170C
> 5. Grids have this regardless of which of 2 tanks of ethane we use
>
> Any ideas of what may be causing this would be greatly appreciated.
>
> The pictures were too big to send to the list (although quite small by 
> my standards). They are posted on our ftp site:
>
> ftp://bio3d.colorado.edu/914
>
> Thanks,
> Cindi
>
>
>
> **********************************
> Cindi L. Schwartz
> University of Colorado, Boulder
> Dept. MCDB
> BL3DEMC
> O: 303-492-7980
> F: 303-735-0770
> **********************************
>
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