[3dem] 3dem Digest, Vol 109, Issue 2- Contaminated cryo grids- reply to Nadav Elad

Thu Sep 1 15:25:37 PDT 2016

Hi Nadav,

Most cryo labs go through periods of contaminated grids and this can be a nightmare to track down.

If you have a contaminated grid in the scope, tilt to 60 degrees or higher. You may see if the ice is mainly on the surface. If so then the contamination may come after plunging, transferring or during pumping in the pre-pump chamber of the microscope when inserting. Check the cryo shield on the holder is well fitted and runs smoothly when opening and closing.

Although with grids, contamination can occasionally be a problem from various suppliers from batch to batch, this can be checked easily as you suggest by trying a different type, batch or supplier.

Check your liquid nitrogen supply chain. Supply needs to be "fresh" and should not sit around. Do not refill empty tanks without first drying. If a truck delivers to a large storage vessel, either the truck or the mass storage Dewar could be contaminated could be contaminated. Difficult to remedy if this is the case. Try a different supply of nitrogen if possible (does another building on campus have a different supplier?). Thoroughly dry all Dewars. We sometimes use a flow of gaseous dry nitrogen to periodically dry large LN2 tanks. Check your Dewars have no rust in the bottom otherwise chuck.

Make sure all pipette ports on the plunger are closed at all times (tape over the left one unless you have left handers) except while loading (may cause air tubulence at your grid surface in the humidity chamber. Lower the humidity to 80 or 85% on the Leica GP otherwise it creates a "storm" trying to keep the humidity high. You could out a USB humidity meter or small hygrometer into the humidity chamber to check if the reading from the machine is accurate. You could also test turning it right off.

We went from larger 5 litre bottles of ethane to 1 litre so that the turnover of bottles is faster and ethane bottles do not sit for months or years. We rotate 3 one litre bottles, each one only lasts us a month or so. The suppliers decant of a large supply (that may be quite old or contaminated) so if possible try some from a different supplier.

As already suggested, check your clip rings and clip springs. If the grid is not clipped well it can warm creating crystalline ice. If you remove a grid that showed contamination. After removal, gently touch the grid with sharp tweezer tips. If the grid moves at all, change the holder spring or clip ring. We do this a few times each year. With a Polara exchange the cartridge or ring.

At the plunger use a headband magnifier or similar and check you do not have visible "ice" or "floaties" in your ethane and/or Liquid nitrogen (safety first, watch your eyes!). Filtering liquid N2 through lint free paper is an option but is an extra step.

Check your storage grid boxes that they have no fine particles after manufacture (naturally what you see in the scope is much smaller than can be seen by eye but this is worth checking sometimes).

Frying your sample by taking multiple images to get rid of contamination ice is not to be recommended!

All the best

Ken

Ken Goldie, PhD
Facility Manager, C-CINA, Biozentrum, University Basel
Mattenstrasse 26 | D-BSSE | WRO-1058 | CH-4058 Basel | Switzerland
http://c-cina.org | Tel. +41-61-387 32 18| eMail. k.goldie at unibas.ch

Postal Address for delivery of Letters / Goods / Express Carriers:
Ken Goldie | C-CINA, University of Basel, WR0-1058 |
Warenannahme WRO-5.P.057| Mattenstrasse 24a | 4058 Basel, Switzerland

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Today's Topics:

   1. contamination in cryo samples (Nadav Elad)

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Message: 1
Date: Thu, 1 Sep 2016 09:00:11 +0000
From: Nadav Elad <nadav.elad at weizmann.ac.il>
To: "3dem at ncmir.ucsd.edu" <3dem at ncmir.ucsd.edu>
Subject: [3dem] contamination in cryo samples
Message-ID: <36EBF1ED6D4C5C44A99821B598B63132010DF61B at IBWMBX02>
Content-Type: text/plain; charset="us-ascii"

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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