[3dem] Insertion of Holder 626

[Dokland, Terje]

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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