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

Thu Sep 1 18:05:14 PDT 2016

Hi Nadav,

This has been interesting reading - we also get exactly this problem from time to time. I have a Tecnai 12, and use a home made plunging system and  Gatan 626 cryo holder. Ambient humidity in my lab is usually around 30-40%.

In our case, we have mostly cured the problem by regular drying of all nitrogen dewars , including our bulk storage, with dry nitrogen gas, and if the problem occurs later in the day, discarding any liquid nitrogen that has been sitting in the working dewar even for a few hours.

We've also found that it is most critical to have fresh dry nitrogen for loading the grid into the cryo holder, whereas we often see visible ice in the nitogen in the plunging system, with no ill effects.

All the best

Lynne

Lynne Waddington

Manager

Cryo Electron Microscopy Facility

CSIRO Manufacturing

Postal - mail: Private Bag 10, Clayton South, VIC 3169, Australia

Postal - sample delivery: Gate 3, Normanby Rd, Clayton, VIC 3168, Australia

Phone: +61 3 9545 2356

e-mail: lynne.waddington at csiro.au

-----Original Message-----
From: 3dem [mailto:3dem-bounces at ncmir.ucsd.edu] On Behalf Of Kenneth Goldie
Sent: Friday, 2 September 2016 8:26 AM
To: 3dem at ncmir.ucsd.edu
Subject: Re: [3dem] 3dem Digest, Vol 109, Issue 2- Contaminated cryo grids- reply to Nadav Elad

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<mailto: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

________________________________________

From: 3dem [3dem-bounces at ncmir.ucsd.edu] on behalf of 3dem-request at ncmir.ucsd.edu<mailto:3dem-request at ncmir.ucsd.edu> [3dem-request at ncmir.ucsd.edu]

Sent: Thursday, 1 September 2016 11:00 a.m.

To: 3dem at ncmir.ucsd.edu<mailto:3dem at ncmir.ucsd.edu>

Subject: 3dem Digest, Vol 109, Issue 2

Send 3dem mailing list submissions to

        3dem at ncmir.ucsd.edu<mailto:3dem at ncmir.ucsd.edu>

To subscribe or unsubscribe via the World Wide Web, visit

        https://mail.ncmir.ucsd.edu/mailman/listinfo/3dem

or, via email, send a message with subject or body 'help' to

        3dem-request at ncmir.ucsd.edu<mailto:3dem-request at ncmir.ucsd.edu>

You can reach the person managing the list at

        3dem-owner at ncmir.ucsd.edu<mailto:3dem-owner at ncmir.ucsd.edu>

When replying, please edit your Subject line so it is more specific than "Re: Contents of 3dem digest..."

Today's Topics:

   1. contamination in cryo samples (Nadav Elad)

----------------------------------------------------------------------

Message: 1

Date: Thu, 1 Sep 2016 09:00:11 +0000

From: Nadav Elad <nadav.elad at weizmann.ac.il<mailto:nadav.elad at weizmann.ac.il>>

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

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<mailto:nadav.elad at weizmann.ac.il%3cmailto:nadav.elad at weizmann.ac.il>>

-------------- next part --------------

An HTML attachment was scrubbed...

URL: <http://mail.ncmir.ucsd.edu/pipermail/3dem/attachments/20160901/a3665743/attachment.html>

-------------- next part --------------

A non-text attachment was scrubbed...

Name: contamination.jpg

Type: image/jpeg

Size: 147339 bytes

Desc: contamination.jpg

URL: <http://mail.ncmir.ucsd.edu/pipermail/3dem/attachments/20160901/a3665743/attachment.jpg>

------------------------------

Subject: Digest Footer

_______________________________________________

3dem mailing list

3dem at ncmir.ucsd.edu<mailto:3dem at ncmir.ucsd.edu>

https://mail.ncmir.ucsd.edu/mailman/listinfo/3dem

------------------------------

End of 3dem Digest, Vol 109, Issue 2

************************************

_______________________________________________

3dem mailing list

3dem at ncmir.ucsd.edu<mailto:3dem at ncmir.ucsd.edu>

https://mail.ncmir.ucsd.edu/mailman/listinfo/3dem
-------------- next part --------------
An HTML attachment was scrubbed...
URL: <http://mail.ncmir.ucsd.edu/pipermail/3dem/attachments/20160902/b32df408/attachment-0001.html>