[3dem] Insertion of Holder 626

Mon Feb 24 13:25:50 PST 2020

This company https://www.applerubber.com/products/o-rings/
stocks 300 million o-rings. We should be able to find what we need there!

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On Feb 24, 2020, at 3:01 PM, Denis Chrétien <denis.chretien at univ-rennes1.fr<mailto: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<mailto: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

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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

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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
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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