[3dem] contamination in cryo samples

benoit.zuber at ana.unibe.ch benoit.zuber at ana.unibe.ch
Thu Sep 1 23:58:14 PDT 2016


It could also come at the moment you insert the grid in the microscope, and even inside the microscope, through a tiny leak. If without cooling down the anticontaminator you cannot reach an IGP value of 6 (on F20) when a holder is inserted but you can reach it when no holder is in, it speaks for a leak. Even if you reach IGP value within specification (e.g. 14), this might be creating this ice contamination. You heat your holder to -90 until the contamination is gone (it should go away if it is water). Then let it cool down again and wait. If you see it reappear then you have a tiny leak. We had this problem and an intervention on the compustage solved it.
Further possible issues that we also experienced : The anticontaminator had been taken out of position… When we noticed it and brought it back in position (unfortunately after several weeks of head scratching !) the leopard skin disappeared !

Benoit

De : 3dem <3dem-bounces at ncmir.ucsd.edu> au nom de "Shi, Dan" <shid at janelia.hhmi.org>
Date : jeudi, 1 septembre 2016 21:52
À : Nadav Elad <nadav.elad at weizmann.ac.il>, "3dem at ncmir.ucsd.edu" <3dem at ncmir.ucsd.edu>
Objet : Re: [3dem] contamination in cryo samples

Hi Nadav,
The contaminations could be either ice or impurity of ethane, if it’s from ethane,  you may heat the holder tip to –150 C to see if the particles evaporate, if not, they are ice.
Such nanometer granule shape of ice contamination seems to be formed in cold air first and deposited onto the cold grid, warmer holder would tend to crystallize original amorphous ice forming the ‘leopard –cubic pattern’ of  contamination as Win described. Minor vacuum leaking into microscope should generate evenly distributed ice coverage and not nucleated ice particles, because the column is at RT.

There are two places could generate such contamination, most likely, the contamination is from the Leica EM plunger’s grid transfer chamber, the positive pressure from LN2 evaporation is not enough to keep humid air off the chamber; the second place may be Gatan loading station, if you open the lid to mount the clip ring, wet air can sneak in to contaminate your grids. You may compare with Vitrobot and/or mount a grid with the plastic lid on all the time.
Good luck,
Dan

From: 3dem <3dem-bounces at ncmir.ucsd.edu<mailto:3dem-bounces at ncmir.ucsd.edu>> on behalf of Nadav Elad <nadav.elad at weizmann.ac.il<mailto:nadav.elad at weizmann.ac.il>>
Date: Thursday, September 1, 2016 at 5:00 AM
To: "3dem at ncmir.ucsd.edu<mailto:3dem at ncmir.ucsd.edu>" <3dem at ncmir.ucsd.edu<mailto:3dem at ncmir.ucsd.edu>>
Subject: [3dem] contamination in cryo samples

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>

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