[3dem] Ice contamination

Shi, Dan (NIH/NCI) [E] shid at mail.nih.gov
Wed Sep 28 06:24:28 PDT 2011


Hi Liz and Bob,
You may try to use diffraction mode (spotsize 10 or 11, illuminating area 5 - 10 um, camera length 200 mm - 800 mm) taking few diffraction patterns. If the contaminations are ice nano-crystals, you should be able to see diffraction ring(s) around 0.36 nm. Normally, the crystalline planes diffract e-beam away to generate contrast for ice or any form of crystals, unless salts or protein having heavier atoms. If the contaminations are not crystals, you may need check oil or other large molecule contaminations from the chamber or humidifier. 
Bob's suggestion should work if the contamination is ice, in that case, FEI should improve the humidifier or you could try to raise chamber's temperature. Good luck,

Dan Shi, PhD
LCB, NCI/CCR
50 South Dr, Bldg 50/4306 
Bethesda, MD 20892

-----Original Message-----
From: reinhard rachel [mailto:reinhard.rachel at biologie.uni-regensburg.de] 
Sent: Wednesday, September 28, 2011 4:36 AM
To: 3dem at ncmir.ucsd.edu
Subject: [3dem] Ice contamination

Hi Liz, hi all, 
first, I checked briefly - these blobs are about 50 to 70 nm in diameter (right?), and appear round in projection: are they round in the 3D reconstruction, Liz? or flat? 
Second, the contrast of the blobs is lower than the Gold, but considerable, clearly higher than the ice in the holes of the carbon film, and they are even clearly visible in the area where the carbon film exists. As the bacterial cells (Caulobacter?) are about 500 to 600 nm in diameter, the frozen ice layer is about of this thickness, at least. - The contrast of the blobs is larger than the surrounding ice: I would not expect this if they are aqueous / buffer, and embedded in the frozen ice layer (Liz writes: "... appear to be in the ice"). Where does the contrast come from? What is their composition? Do you have access to a TEM with a filter (in-column; post-column), in order to get some insight into this? 
Are these (membrane) vesicles shed from the cells? --> did you check the cells / the medium if these blobs are present in the sample already (e.g. classical GA fixation - no OsO4 -, then directly applying onto a carbon-coated grid, air-drying, Pt/C shadowing or UAc staining)? Did you check the buffer alone?
kind regards,
Reinhard
 
> 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.
>
>> 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.
>>


-- 
Prof. Dr. Reinhard Rachel
University of Regensburg
Centre for EM / Anatomy
Faculty of Biology & Preclin. Med.
Universitaetsstr. 31
D-93053 Regensburg - Germany
tel +49 941 943 2837, 1720
fax +49 941 943 2868
mail reinhard.rachel at biologie.uni-regensburg.de
office: VKL 3.1.29 

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