[3dem] Insertion of Holder 626

Mon Feb 24 13:11:20 PST 2020

To order O-rings, you may contact Tom. He repairs Gatan stuff. I once bot
some O-rings from him. He is also our goto if anything wrong with out of
warranty Gatan thing. Do check with Gatan first.

Tom Schmelzer

*TGS Technologies LLC*

*USA*

724-453-3865

email: tom at tgstechnologies.net

On Mon, Feb 24, 2020 at 4:01 PM Denis Chrétien <
denis.chretien at univ-rennes1.fr> wrote:

> 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 févr. 2020 à 21:26, David Stokes <stokes at nyu.edu> a écrit :
>
> 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> 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>
> 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
> Université 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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