[3dem] [EXTERNAL] Re: contamination...leopard?

[Grassucci, Robert]

Regarding pump down times for side entry holders.  We have used up to 120 s for pump down but you can play with it to find the sweet spot.  If it is too long you could get devitrified ice but that is unlikely at 120 S.

Regarding checking the grid clamping simply used fine tweezers to try and move the clamped grid.  It should be tightly secured and shred if moved.  If it moves relatively freely then either your clamp ring needs to be changed (worn out) or the spring that holds the ring in the holder needs to be replaced.

As Wim said the cooling of the cryo shield can be checked by going to low mag and watching it move as it cools.  This was what the video he attached shows.  Unfortunately they do not have a read out of the shield temperature as they had in the early cryo shields.

Good luck.
Bob

From: 3dem [mailto:3dem-bounces at ncmir.ucsd.edu] On Behalf Of jb
Sent: Wednesday, January 5, 2022 9:17 AM
To: 3dem at ncmir.ucsd.edu
Subject: [EXTERNAL] Re: [3dem] contamination...leopard?

Hello together,
what would be the best or maximum time to pump the airlock at the Tecnai Spirit Scope??
I changed allready from 30 seconds to 45 seconds.
Vacuum values look good.
We suffer a lot from contamination and different ice formations since quite some time... every grid looks different.
How can we check if the clip ring at the Gatan holder 626 is perfectly adjusted?
I mean this is the place where the grid has to have the perfect contact a heat bridge to the holder.
How can we check cryo-box or cryo-shield?
Kind regards
Jörg


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

Jörg Bürger



Charité - Universitätsmedizin Berlin

Institut für Medizinische Physik und Biophysik

Charitéplatz 1, CCO - Virchowweg 6

10117 Berlin



Max Planck Institut für molekulare Genetik

Microscopy and Cryo-EM microscopy group

Ihnestr. 63-73

14195 Berlin



tel. 030-8413-1644/1278

fax. 030-8413-1385



email. buerger at molgen.mpg.de<mailto:buerger at molgen.mpg.de>



On 05.01.2022 14:51, Wim Hagen wrote:

In the very early days it was even suggested to use ethane to "coat the sample” as a measure of preventing transfer contamination:









Best,

Wim



On 5. Jan 2022, at 14:45, Reinhard Rachel <Reinhard.Rachel at biologie.uni-regensburg.de><mailto:Reinhard.Rachel at biologie.uni-regensburg.de> wrote:



I see your point, Wim.



On systems with side-entry cryo-holders, the pump-time for holder insertion



is limited and pressure is not measured by the vacuum system.



it is measured on our 2100F.

I doubt that 20 sec is a good time for cryo-transfer. Vacuum always needs

time!

the sample temperature should be kept low enough for quite some time (120 /

180 sec, at least).

but the vacuum improves quite considerable when going from short pump times

(20sec) up to 180 sec. or even longer.

this is at least the case for the air-lock/gonio of our 2100F (holder with two

O-rings ...).

thus:

- tilting to -30 or -35 degrees is fully sufficient

- pumping up to 120/180 sec (or even longer?) is fine, and the sample looks

fine, too.



If a grid

contains a lot of frozen ethane (from a fast transfer from ethane into

liquid

nitrogen on the plunge freezer), this ethane will sublimate in vacuum: too

much ethane and/or short pump time -> column vacuum crashes as the ion

getter

pumps see a lot of ethane coming in when the holder is inserted.



this is the point - thank you, Wim.

thus, I think Tobias & Co may try longer times for pre-pumping in the

air-lock?

I do not know this type of air-lock in the Halo -  but it might work.



kind regards,

Reinhard



--

Prof. Dr. Reinhard Rachel

University of Regensburg

Centre for EM / Anatomy

Faculty of Biology & Preclin. Med.

Universitaetsstrasse 31

D-93053 Regensburg - Germany

tel +49 941 943 -2837, -1666

mail reinhard.rachel at biologie.uni-regensburg.de<mailto:reinhard.rachel at biologie.uni-regensburg.de>

office: VKL 3.1.29

member of the IFSM board



Next microscopy conferences:

- MC2023, DGE: 26.2.-2.3. 2023

- next European EM conf.: EMC2024 in Kopenhagen, August 2024

- IMC20 in Busan, South Korea: Sept, 2023

- next Microbiol. conferences:  VAAM 20-23 Febr 2022 Düsseldorf







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

Jörg Bürger



Charité - Universitätsmedizin Berlin

Institut für Medizinische Physik und Biophysik

Charitéplatz 1, CCO - Virchowweg 6

10117 Berlin



Max Planck Institut für molekulare Genetik

Microscopy and Cryo-EM microscopy group

Ihnestr. 63-73

14195 Berlin



tel. 030-8413-1644/1278

fax. 030-8413-1385



email. buerger at molgen.mpg.de<mailto:buerger at molgen.mpg.de>
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