[3dem] Insertion of Holder 626

[Shi, Dan (NIH/NCI) [E]]

Dear Ariel,

If there is any minor leak from the rod O-ring, long time airlock pump would raise your grid temperature because the 'fresh' air constantly flow through the grid to warm it up and the temperature sensor is not on the grid.

The leak could be caused by multiple reasons as other people pointed out, if you have tried all the suggestions to clean the O-ring and replace different O-ring, the vacuum crash is still happening. Another hidden problem could be the OD of the cryo-holder's rod/O-ring slot is few tens micrometer too small to seal compustage tube well. 

A simple test will identify where is the problem to cause the leak. You can swap the O-ring of this 626 holder with the one from a good RT holder, insert both holders into the Talos, the vacuum pressure spike results would tell whether the O-ring or the rod is the leak source. If the O-ring is not a problem for the RT holder. You may replace the original O-ring with few tens micrometer thicker O-ring - i.e. the same ID and slightly bigger OD for the cryo-holder.

An alternative method to minimize the problems you have could be to shorten the airlock pump time to less than 60 seconds and to decrease the final holder rotation angle as we did to our Talos L120C.

Good luck,

Dan     

-----Original Message-----
From: TALAVERA PEREZ Ariel <Ariel.Talavera at ulb.be> 
Sent: Monday, February 24, 2020 8:13 AM
To: 3dem at ncmir.ucsd.edu
Subject: [3dem] Insertion of Holder 626

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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