[3dem] Re: Ice contamination

Wed Sep 28 01:54:14 PDT 2011

Hi Mrs Wright,

to comment the previous answer of Mr Grassucci:

As I said I have the same blobbies, looking exactly like this, but they are in diameter roughly 30-40 nm, and I am using a vitrobot mark IV with humidity chamber set to 100%. As I also said when I am preparing grids in a row, I experience this only at the end of my grid series preparation. Thus, the first grids had the same conditons regarding the humidifier box and didn't show up such bloobies. So I dont think that it has anything to do with spray contamination of the humidifier box. I assume that it looks more like contamination by plunge-freezing through liquid ethane or kind of post-contamination when transferring it to the grid box through the liquid nitrogen atmosphere. But it is still not clear to me what is really causing such a ice formation. 

Best regards,
C. Geiss

----------------ursprüngliche Nachricht----------------- 
Von: "Bob Grassucci" rg2502 at columbia.edu 
An: 3dem at ncmir.ucsd.edu, "Elizabeth Wright" erwrigh at emory.edu 
Datum: Tue, 27 Sep 2011 18:56:13 -0400 
----------------------------------------------------------

>
>Hi Liz,
>Definitely not ethane. The contrast of blobs is too low. It could be coming from the 
>humidifier in the Vitrobot. Try turning off the humidifier when you are making the grids and see if 
>they disappear. I am thinking they are getting deposited from the spray while plunging.
>Bob
>
>On 9/27/2011 6:46 PM, Wright, Elizabeth R. wrote: 
>>
>>
>>
>>
>>Dear All,
>>
>> 
>>
>>For those of you patient enough to look at a larger image, please find it located at the 
>>following link:
>>
>>[http://dl.dropbox.com/u/23095990/Caulobacter_2sept2011.tif
>>
>> 
>>
>>The contamination is the small globular looking material distributed throughout 
>>the ice. There are 10 nm gold fiducials and a bacterial cell. To our eyes, when we generate 
>>the 3D reconstructions, the 'blobs' appear to be in the ice, albeit at or close to the 
>>surface. It does not look like frost or particles that have landed on the surface of a frozen 
>>specimen.
>>
>> 
>>
>>Thank you all for your help.
>>
>> 
>>
>>Sincerely,
>>
>> 
>>
>>Liz
>>
>>
>>
>> 
>>
>>
>>From: Elizabeth Wright erwrigh at emory.edu erwrigh at emory.edu 
>>Date: Tue, 27 Sep 2011 16:38:47 -0400
>>To: 3dem at ncmir.ucsd.edu 3dem at ncmir.ucsd.edu 
>>Subject: Ice contamination
>>
>> 
>>
>>
>>
>>
>>
>>Dear 3D EM friends,
>>
>> 
>>
>>We are experiencing a problem with ice contamination within the ice of our 
>>plunge-frozen samples (frozen with an FEI Vitrobot). We have purchased new hoses, regulators, 
>>ethane (99.999% pure) and liquid nitrogen tanks, attempted chloroform or methanol 
>>treatment of the grids, glow discharged the grids with different units, and still seem to be 
>>unable to remove this contaminant. It is not specimen dependent as we use different 
>>buffering systems for the samples. It is also not microscope dependent, we have seen it when 
>>using a variety of instruments.
>>
>> 
>>
>>Has anyone observed this before (image below)? Has anyone found a way to remove it? Any 
>>thoughts or suggestions would be greatly appreciated. If the image does not come through, 
>>please let me know.
>>
>> 
>>
>>Thank you all for your help.
>>
>> 
>>
>>Sincerely,
>>
>> 
>>
>>Liz
>>
>> 
>>
>> 
>>
>>
>>
>> 
>>
>>
>>----
>>
>>Elizabeth R. Wright, PhD
>>Assistant Professor
>>Emory University
>>erwrigh at emory.edu erwrigh at emory.edu 
>>Ph: (404) 727-4665
>>
>>
>>
>>
>>
>>
>>
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>>http://dl.dropbox.com/u/23095990/Caulobacter_2sept2011.tif]
>>
>> 
>>
>>
>>-- 
>>*********************************
>>Howard Hughes Medical Institute
>>Columbia University Medical Center
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-- 

Msc. Christian Geiss
Universitaet Frankfurt FMLS 

Frangakis Gruppe 

Max-von-Laue-Strasse 15 

D - 60438 Frankfurt am Main 

Phone: +49-(0)69-79842597 

Fax: +49-(0)69-79846467 

geiss at biophysik.org 

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