[3dem] Insertion of Holder 626

Mon Feb 24 13:53:40 PST 2020

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> 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
>>>>> 
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>> 
>> -- 
>> David L. Stokes
>> Skirball Institute, Dept. of Cell Biology
>> NYU School of Medicine
>> http://stokeslab.med.nyu.edu/ <http://stokeslab.med.nyu.edu/>
>> tel: 212-263-1580
>> fax: 212-263-2150
>> 
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