[3dem] Insertion of Holder 626

[Conway, James Frederick]

Ariel, and others,

I have two 626 holders and a 910 (3-grid) cryoholder - none cause the TF20 vacuum to crash. I do not change o-rings often (once a year if that), don't use vacuum grease (it collects dust), and pump for 60-secs in the airlock. I follow a common procedure: insert with the stage at -60-degrees tilt to maintain liquid N2 in the cryoholder dewar, wait for the 1-minute pump, return the stage to 0 tilt holding the dewar steady, then rotate the dewar upright - all to keep the relative motion slow between the o-ring and the airlock. Some thoughts on what sounds like a real pain:

1. I use ethane:propane to freeze - its stays liquid and I expect it evaporates in the airlock before entry into the column. Ethane ice may instead reach the column, as Wim Hagen suggested. Its easy to do - fill the cup 2/3 with ethane, top it off with propane, and do the plunge, etc, as usual. The ethane/propane mix can become jelly-like, so I bubble a bit of propane in to warm it up and it becomes absolutely clear. I drilled holes in the styrofoam platform so that liquid nitrogen is surrounding the brass cup, but that's not really necessary.

2. Check the o-ring for cuts. If you replace the o-ring (make sure its the correct size), and the vacuum breaks again, check it again for new cuts. If you see damage to the surface, there may be something sharp inside the cryo-transfer station or inside the airlock that is ripping up the o-ring. Try inserting the holder into each (at room temp) in turn, and inspect the o-ring afterwards with a magnifying glass.

3. My turbo pump was replaced a couple of years ago and the spin-up time on the new pump was much shorter. We hadn't noticed how the pump had degraded over time because it was very gradual. I'm not sure if this affected the quality of the vacuum, maybe just caused a long (5-6 minute) wait for spin-up that is now 2-3 minutes. However, if its long, maybe the turbo pump isn't working so well.

James Conway
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James Conway, PhD.,
Professor, Department of Structural Biology
University of Pittsburgh School of Medicine
Biomedical Science Tower 3, Room 2047
3501 5th Ave
Pittsburgh, PA 15213
U.S.A.
Phone: +1-412-383-9847
Fax:   +1-412-648-8998
Email: jxc100 at pitt.edu<mailto:jxc100 at pitt.edu>
Web:   http://www.structbio.pitt.edu/index.php/12-faculty/18-james-conway
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-----Original Message-----
From: 3dem [mailto:3dem-bounces at ncmir.ucsd.edu] On Behalf Of TALAVERA PEREZ Ariel
Sent: 24 February 2020 14:13
To: 3dem at ncmir.ucsd.edu<mailto: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

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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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