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

Wed Jan 5 08:43:16 PST 2022

One possibility is that the clip ring is not holding the cryo grid well and
tightly in the Gatan holder tip. I would suggest trying a new clip ring
first.

Best regards,
Dong-Hua

On Wed, Jan 5, 2022 at 6:47 AM Grassucci, Robert <rg2502 at cumc.columbia.edu>
wrote:

> 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
>
>
>
>
>
>
>
> 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> <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
>
> 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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>
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>
> https://mail.ncmir.ucsd.edu/mailman/listinfo/3dem <https://urldefense.proofpoint.com/v2/url?u=https-3A__mail.ncmir.ucsd.edu_mailman_listinfo_3dem&d=DwMDaQ&c=G2MiLlal7SXE3PeSnG8W6_JBU6FcdVjSsBSbw6gcR0U&r=DHQ5sxLdEeh7GliHapLyXDQKaXurrg9EcaYzNZr3hks&m=7fbPWhNdvHyIl9XjV53WMWKijxXJDdJiO4k-9bNFAjc&s=J6hrFv817PIg3WZYkht5NKWERoYkWskTByTUCtglkk4&e=>
>
>
>
> --
>
>
>
> ****************************
>
> 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
>
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