[3dem] contamination...leopard?

[jb]

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> 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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-- 

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