[3dem] Insertion of Holder 626

Tue Feb 25 00:17:48 PST 2020

Dear All,

The vacuum system of the Talos is significantly different than that of the old Tecnai. Since a turbo pump has been removed it is required to remove most of the water that one introduces to the microscope with the stage pumping. The Talos L120 has a split flow pumping to the stage, this means that a turbo pump is now pumping the airlock. This should be used and therefor the pumping of the airlock should be set to maximum.

If you have a correct functioning Gatan 626 holder, it should keep its temperature. By tilting to -55 or -60 (does not really matter) you will have the holder at an angle (and not up side down) when inserting, thus some liquid nitrogen remains in the holder. This should be enough to keep the holder temp low for 3 min pumping.

A holder of -150 degree is probably already devitrifying the sample, since the tip is going to be warmer that what is measured (the measurement is not at the tip). In the past we had on purpose warmed up holders to see the effect on the sample. Around -150 we could see the sample starting to change with the so called “leopard skin” formation first and then dark black spots. Upon insertion and during pumping the holder should not warm up much. Most well behaving 626 holders should be colder than -170.

Then the insertion. The “old” of holding the holder with one hand while tilting back to zero should NOT be used. The problem with this method is that people tend to pull or push the holder to one side and while rotating the stage simultaneously, the chances of leakage is higher. It is better to:

While at -55 degrees after the airlock pump time of 3 minutes and the red light is off (don’t forget to select and confirm the holder in the UI), you slowly turn the holder to the column insertion position without turning the stage! You do this very very gentility. At about 1/3 of the way the airlock wil open. You need to watch the vacuum and the moment you see it go up, you wait and stop turning. You keep the holder with your hand at that position and wait for the vacuum to go down again.  Then you continue and it will spike again. You wait again. Continue doing this until you can go in. When in, you rotate the stage back to 0 degree.

The cryo box needs to be inserted and cooled since it is the most effective pump for water!

For the Talos 200KV side entry there is no split flow. It will almost certain overload the igp. Therefore this system has a check option for inserting cryo-holders that switches off the igp temporary during insertion and will wait for the vacuum to recover and the cryo-box to “pump away” the inserted water, before switching back on. The procedure as described above for insertion is the same though.

I always checked the o-ring fir any particles and removed them. I never used grease.

I will see if I can still get a hold of an instruction video for side entry insertion and otherwise i’ll make one during the upcoming cold spring harbor cryo-EM course in march.

I hope this helps: a Talos is not a Tecnai, the vacuum system is different.

Cheers

Matthijn Vos

On Feb 25, 2020, at 00:04, Shi, Dan (NIH/NCI) [E] <dan.shi at nih.gov> wrote:

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 Chrétien <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 févr. 2020 à 22:01, Denis Chrétien <denis.chretien at univ-rennes1.fr<mailto:denis.chretien at univ-rennes1.fr>> a écrit :

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

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
Université Libre de Bruxelles
Rue Adrienne Bolland, 8
B-6041 Gosselies, Belgium
Phone +32 (0)26509866

_______________________________________________
3dem mailing list
3dem at ncmir.ucsd.edu<mailto:3dem at ncmir.ucsd.edu>
https://urldefense.proofpoint.com/v2/url?u=https-3A__mail.ncmir.ucsd.edu_mailman_listinfo_3dem&d=DwIGaQ&c=slrrB7dE8n7gBJbeO0g-IQ&r=hgbDG_ZdQaorZsucl01zHg&m=Yn2a7P10qylNPjdZ499rPbNzetpn8RyAV870SJbr0hg&s=wwZt0ruHRcbRcKBOUphvqWKC9QI8lDUPt_L8YiYwln4&e=
_______________________________________________
3dem mailing list
3dem at ncmir.ucsd.edu<mailto:3dem at ncmir.ucsd.edu>
https://urldefense.proofpoint.com/v2/url?u=https-3A__mail.ncmir.ucsd.edu_mailman_listinfo_3dem&d=DwIGaQ&c=slrrB7dE8n7gBJbeO0g-IQ&r=hgbDG_ZdQaorZsucl01zHg&m=Yn2a7P10qylNPjdZ499rPbNzetpn8RyAV870SJbr0hg&s=wwZt0ruHRcbRcKBOUphvqWKC9QI8lDUPt_L8YiYwln4&e=
_______________________________________________
3dem mailing list
3dem at ncmir.ucsd.edu<mailto:3dem at ncmir.ucsd.edu>
https://urldefense.proofpoint.com/v2/url?u=https-3A__mail.ncmir.ucsd.edu_mailman_listinfo_3dem&d=DwIGaQ&c=slrrB7dE8n7gBJbeO0g-IQ&r=hgbDG_ZdQaorZsucl01zHg&m=Yn2a7P10qylNPjdZ499rPbNzetpn8RyAV870SJbr0hg&s=wwZt0ruHRcbRcKBOUphvqWKC9QI8lDUPt_L8YiYwln4&e=

--
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

_______________________________________________
3dem mailing list
3dem at ncmir.ucsd.edu<mailto:3dem at ncmir.ucsd.edu>
https://mail.ncmir.ucsd.edu/mailman/listinfo/3dem

_______________________________________________
3dem mailing list
3dem at ncmir.ucsd.edu<mailto:3dem at ncmir.ucsd.edu>
https://mail.ncmir.ucsd.edu/mailman/listinfo/3dem

_______________________________________________
3dem mailing list
3dem at ncmir.ucsd.edu
https://mail.ncmir.ucsd.edu/mailman/listinfo/3dem
-------------- next part --------------
An HTML attachment was scrubbed...
URL: <http://mail.ncmir.ucsd.edu/pipermail/3dem/attachments/20200225/04838457/attachment-0001.html>