[3dem] Insertion of Holder 626

[Sharon G Wolf]

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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