[3dem] Enquiry regarding spare parts of CRYO-TEM FEI G2 POLARA

Tue Feb 25 00:06:24 PST 2020

Hello everyone,

We are looking for a old spare 'INSERTION ROD" for CRYO-TEM FEI G2 POLARA.
Since FEI has stopped its production and replacement costs are too high, we
are searching for an old/used/second-hand spare of some POLARA FACILITY.

Thanks and regards,

Anshu Dutta
*CRYO-EM FACILITY MANAGER,*
*CENTRAL INSTRUMENTATION FACILITY, CSIR-IICB, KOLKATA*

On Tue, Feb 25, 2020, 4:34 AM <3dem-request at ncmir.ucsd.edu> wrote:

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> Today's Topics:
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>    1. Re: Insertion of Holder 626 (Patricia Grob)
>    2. Re: Insertion of Holder 626 (Shi, Dan (NIH/NCI) [E])
>
>
> ----------------------------------------------------------------------
>
> Message: 1
> Date: Mon, 24 Feb 2020 14:57:49 -0800
> From: Patricia Grob <pgrob at berkeley.edu>
> To: TALAVERA PEREZ Ariel <Ariel.Talavera at ulb.be>
> Cc: "3dem at ncmir.ucsd.edu" <3dem at ncmir.ucsd.edu>
> Subject: Re: [3dem] Insertion of Holder 626
> Message-ID: <21B285DD-8F19-4F3A-B201-EEDE5B579B3D at berkeley.edu>
> Content-Type: text/plain; charset="utf-8"
>
> Hi Ariel,
>
>
> This sounds like a very long airlock pumping time to me. I am not sure
> about the TALOS airlock but on our Tecnai microscopes we use a 60-90 s
> airlock cycle time.
>
> You might have a leak in the airlock if it is not sufficient, either from
> the holder end or on the stage, which you?ll have to determine (or the pump
> is not very good!). -154 degrees is indeed pretty high, we typically get
> -170 or lower during transfer with our  DH 626 Gatan cryo-holders. You
> could try and monitor the holder  temperature change during the whole
> transfer time and see when it starts rising. Make sure you start with a
> holder at -190 degrees C when you first transfer it form the workstation to
> the airlock. If the rise is immediate, maybe there is an issue with your
> holder (leak in the dewar at any seal point, sorbent needing to be replaced
> or envelope needing to be cleaned, or all of the above). You can monitor
> the temperature of the holder in the pumping station with liquid nitrogen
> in the dewar (only pumping down on the tip, not the dewar), and make sure
> that the lowest temperature stays in the -170 to -180 range once cooled,
> otherwise the dewar is not maintaining vacu
>  um and needs maintenance.
>
> Good luck.
> Cheers,
>
> Patricia
>
> _______________________________
> Patricia Grob, Ph.D.
> Research Specialist
> Nogales Lab
> HHMI at UC Berkeley
> Molecular & Cell Biology Department / QB3,
> Stanley Hall #742
> University of California, Berkeley
> Berkeley, CA 94720-3220
> Phone: (510)666-3335    Fax: (510)666-3336
> E.M. suite B307: (510)666-3339
> URL: cryoem.berkeley.edu
> Email: pgrob at berkeley.edu
>
>
>
>
> > On Feb 24, 2020, at 5:13 AM, TALAVERA PEREZ Ariel <Ariel.Talavera at ulb.be>
> wrote:
> >
> > Dear all,
> >
> > I am using a Gatan 626 holder on a side entry TALOS microscope. I am
> > getting problems with very frequent crystalline ice formation on the
> > grids. Some times I get a mix of vitreous and crystalline water, but
> > must of the time I only get crystalline water. I have been extra careful
> > during sample preparation, and transferring the grind on to the holder,
> > and I have done 48 hours holder bake out before insertion, but I still
> > get ice on the grid.
> >
> > I monitored the temperature of the holder during the insertion process,
> > including 3 minutes for the Airlock pump. During those 3 minutes the
> > temperature rises up to -152.2. Can this be the reason of the unwanted
> > ice formation? How high can be the temperature of the holder to ensure a
> > nice vitreous water?
> >
> > I am also getting vacuum problems while inserting the holder. This
> > process has resulted to be extremely, extremely sensitive. After the 3
> > minutes Airlock pumping (I cannot give longer time because of the
> > aforementioned temperature issue) as soon as I open the column port the
> > vacuum either rises to 99 Log or completely crashes the column vacuum.
> > This has happened with two different holders either cooled with liquid
> > nitrogen or at room temperature. So, my question: Is this insertion
> > process always that  picky or it can be that there is something wrong
> > with the holders/Airlock pump?
> >
> > Thanks a lot in advanced.
> >
> > Best regards.
> >
> > Ariel
> >
> > --
> > Ariel TALAVERA PEREZ, PhD
> > Center for Microscopy and Molecular Imaging (CMMI)
> > Laboratory of Microscopy
> > Universit? Libre de Bruxelles
> > Rue Adrienne Bolland, 8
> > B-6041 Gosselies, Belgium
> > Phone +32 (0)26509866
> >
> > _______________________________________________
> > 3dem mailing list
> > 3dem at ncmir.ucsd.edu
> > https://mail.ncmir.ucsd.edu/mailman/listinfo/3dem
>
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> Message: 2
> Date: Mon, 24 Feb 2020 23:03:42 +0000
> From: "Shi, Dan (NIH/NCI) [E]" <dan.shi at nih.gov>
> To: "3dem at ncmir.ucsd.edu" <3dem at ncmir.ucsd.edu>
> Subject: Re: [3dem] Insertion of Holder 626
> Message-ID:
>         <
> BL0PR0901MB454665C3D181129EF1325B8DF2EC0 at BL0PR0901MB4546.namprd09.prod.outlook.com
> >
>
> Content-Type: text/plain; charset="utf-8"
>
> The modified procedure from the traditional one for our L120C is
>
>
>   1.  Start the turbo pump and wait it reaching 100% speed and set
> ?airlock pump time? to 30 ? 60 seconds
>   2.  Set the alpha at -55 to -60 degree and click on ?set alpha? to tilt
> the compustage
>   3.  Insert the 626 holder to the engaging position and select the
> cryo-ST holder from the screen
>   4.  Hold the cryo-holder and click on ?set alpha? to rotate the
> compustage back to 0 degree at least 10 seconds before the airlock pump
> time expired
>   5.  Wait until the red light on the compustage off, rotate the
> cryo-holder to the insertion position and insert the holder in smoothly.
>   6.  Feel free to breath
>
> Best,
>
> Dan
>
> From: Denis Chr?tien <denis.chretien at univ-rennes1.fr>
> Sent: Monday, February 24, 2020 4:54 PM
> To: 3dem at ncmir.ucsd.edu
> Subject: Re: [3dem] Insertion of Holder 626
>
> Sorry, I mentioned Thermo (for the O-rings), but for the 626 it would be
> Gatan (still cost about one arm and a leg ...)
>
> I forgot to mention also the -60? insertion procedure that Wim also
> quoted. It really improved our ability to insert cryo-holders without leaks
> (and risks of warming-up the specimen by spilling all the LN2 outside the
> cryo-holder):
>
> 1) Rotate the cryo-stage to -60?
> 2) Pre-pump the airlock (wait until red light goes off)
> 3) insert the cryo-holder and wait (again) until red light goes off (time
> fixed by yourself, between 30 sec and one min of pumping).
> 4) stop breathing*
> 5) Hold the cryo-holder and reset the stage to 0?. Again, wait until red
> light goes off!
> 6) Rotate the cryo-holder and insert it gently (don?t let the vacuum
> aspire it abruptly inside the stage!)
> 7) Check that the vacuum didn?t went to high during insertion
> 8) Breath*
> * optional
>
> Denis
>
>
> Le 24 f?vr. 2020 ? 22:01, Denis Chr?tien <denis.chretien at univ-rennes1.fr
> <mailto:denis.chretien at univ-rennes1.fr>> a ?crit :
>
> If we order O-rings from Thermo, it cost one arm? Does anyone have
> references for ? standard ? O-rings that we can buy elsewhere for a much
> cheaper price?
>
> To come back to the original questions, we still use 626 and other
> side-entry holders. Sometime we face vacuum problems. In our experience
> these are mainly due to (as already said):
>
> - Dusts on the O-ring (or inside the airlock system). Inspect the O-ring
> and also try to blow dry-air inside the airlock. Maybe the airlock tube is
> damage, then ask Thermo for repair ? :-(
>
> - Dry O-rings: regularly put some (not a lot !) vacuum grease on it. Be
> careful not to scratch the metal when removing it or damaging the O-ring.
> Change it if dry or obviously damaged.
>
> - One frequent issue with cryo-holders: do not fill the cryo-holder
> station with to much LN2, you may freeze the O-ring and it will inevitably
> lead to a vacuum leak (very reproducible!). To limit this issue, we
> slightly lift the cryo-holder station with a rectangular grid box to avoid
> LN2 going into contact with the O-ring. We (not every one of us) cool the
> cryo-stage inside the microscope. Then the work-station is cooled just
> before inserting the specimen grid on the holder. This also limits the risk
> of cooling the O-ring.
>
> - We ? pre-pump ? the airlock, and pump for 1 min before inserting (on a
> Tecnai G2 Sphera). 3 min of pumping seams a lot!
>
> - Change of the zeolithe, grease on the valve O-ring of the cryo-holder,
> periodic zeolithe cycle (we do it every session): it improves stability on
> our side.
>
> Best
>
> Denis
>
>
> Le 24 f?vr. 2020 ? 21:26, David Stokes <stokes at nyu.edu<mailto:
> stokes at nyu.edu>> a ?crit :
>
> I used to do the following prior to every session:
> Remove o-ring from holder.
> Clean o-ring off with kimwipes and fingers (I didn't bother to use any
> solvent).
> Clean o-ring groove with kimwipes.
> Blow away fibers with compressed air.
> Lightly grease o-ring.
> Inspect o-ring and o-ring groove under dissecting scope and carefully
> remove any fibers prior to re-installation onto the holder.
> Don't scratch the o-ring groove. In fact, don't touch it with forceps. Use
> compressed air.
>
> During the inspection process, you will see whether there are any nicks or
> flat spots in the o-ring. If so, then consider a replacement. If there is a
> scratch in the o-ring groove, then you might need a new holder (or maybe
> not).
> Also make sure you really have the right sized o-ring. At some point in
> ancient history, they changed the size slightly, such that CM200 o-ring was
> a bit fatter than the one for the CM12 stage.
>
> David
>
>
> On 2/24/2020 3:10 PM, Sharon G Wolf wrote:
>
> Terje
> We also have suffered for years from vacuum breaks. We tried EVERYTHING.
> The most effective thing was to (believe it or  not) change the o-ring
> every single session.
> Recently we understood this was due to the fibers and other "dirt"  that
> we observed on the o-rings every time we looked with the binocular after
> inserting and removing the holder from the microscopes, transfer stations
> or pumping station. We embarked on a ?cleaning program? where we gently
> swab all the above mentioned devises. It?s now slowly getting better. We
> are still changing the o rings but we hope it will soon no longer be
> necessary, once all the "stuff" that accumulated over the years is cleaned
> out from those places.
> Sharon
>
>
> Sent from my iPhone
>
>
> On 24 Feb 2020, at 19:28, Dokland, Terje <dokland at uab.edu<mailto:
> dokland at uab.edu>> wrote:
>
> ?We have also had persistent vacuum break problems with our 626 holder in
> our Tecnai F20 over the past two years. About 50% of the time inserting the
> holder breaks the vacuum. I though it was unique to this holder, but with
> the Elsa holder that we recently acquired it is even worse: vacuum break
> about 90% if the time. We?ve changed O rings, had the airlock disassembled
> and cleaned, but the problem persists.
>
> That being said, it never affected ice quality, and the temperature
> usually doesn?t go above -165 C. When the vacuum recovers it?s usually at
> 25-30 log or so and back to <10 within a half hour. It?s really annoying
> though.
>
> In my experience as long as the temperature doesn?t rise above -150C you
> should be ok.
>
> It helps to make sure the tip is very cold by submerging it completely in
> lN2 before inserting.
>
> Terje
>
> Sent from my iPhone
>
>
> On Feb 24, 2020, at 7:13 AM, TALAVERA PEREZ Ariel <Ariel.Talavera at ulb.be
> <mailto:Ariel.Talavera at ulb.be>> wrote:
>
> Dear all,
>
> I am using a Gatan 626 holder on a side entry TALOS microscope. I am
> getting problems with very frequent crystalline ice formation on the
> grids. Some times I get a mix of vitreous and crystalline water, but
> must of the time I only get crystalline water. I have been extra careful
> during sample preparation, and transferring the grind on to the holder,
> and I have done 48 hours holder bake out before insertion, but I still
> get ice on the grid.
>
> I monitored the temperature of the holder during the insertion process,
> including 3 minutes for the Airlock pump. During those 3 minutes the
> temperature rises up to -152.2. Can this be the reason of the unwanted
> ice formation? How high can be the temperature of the holder to ensure a
> nice vitreous water?
>
> I am also getting vacuum problems while inserting the holder. This
> process has resulted to be extremely, extremely sensitive. After the 3
> minutes Airlock pumping (I cannot give longer time because of the
> aforementioned temperature issue) as soon as I open the column port the
> vacuum either rises to 99 Log or completely crashes the column vacuum.
> This has happened with two different holders either cooled with liquid
> nitrogen or at room temperature. So, my question: Is this insertion
> process always that  picky or it can be that there is something wrong
> with the holders/Airlock pump?
>
> Thanks a lot in advanced.
>
> Best regards.
>
> Ariel
>
> --
> Ariel TALAVERA PEREZ, PhD
> Center for Microscopy and Molecular Imaging (CMMI)
> Laboratory of Microscopy
> Universit? Libre de Bruxelles
> Rue Adrienne Bolland, 8
> B-6041 Gosselies, Belgium
> Phone +32 (0)26509866
>
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>
> --
> David L. Stokes
> Skirball Institute, Dept. of Cell Biology
> NYU School of Medicine
> http://stokeslab.med.nyu.edu/
> tel: 212-263-1580
> fax: 212-263-2150
>
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