[3dem] Insertion of Holder 626

Mon Feb 24 12:26:56 PST 2020

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