[3dem] Insertion of Holder 626

Mon Feb 24 13:14:00 PST 2020

Our L120C had similar vacuum crash problem when an user inserted the Gatan 626 cryo-holder traditional procedure and we have changed one step from the previous  insertion procedure which most people may use. It seems to drop the column vacuum crash rate quite bit with 30 second - 60 second airlock pumping time and we did not see any association of crystallized ice with the 626 insertion. I would like to share our experience with you.  

We rotated or reset the compustage back to 0 degree while maintaining the 626 holder at the same tilted level at least 10 seconds ahead of the airlock pumping time ended, then rotated the 626 holder for the rest ~90 degree to the insertion position to insert it after pumping time out. When the O-ring seal is not perfect, rotating the compustage back to zero degree and maintaining the holder position might generate a minor leak to the airlock. This modified procedure would keep the airlock being pumped until rotating the holder for the last 90 degree.

All the best,

Dan

Dan Shi
Staff Scientist
SBL, NCI at Frederick

-----Original Message-----
From: Tobias Furstenhaupt <furstenh at mpi-cbg.de> 
Sent: Monday, February 24, 2020 8:55 AM
To: 3dem at ncmir.ucsd.edu
Subject: Re: [3dem] Insertion of Holder 626

Dear Ariel

you are pumping your cold 626 for 3 mins before inserting? Most people (including us) only use 20-30 seconds(!) to pump and then insert it. This way the sample does not warm up so much. Its more risky to crash the column vacuum but in reality this happens not too often.
Also cleaning/exchanging the O-Ring of the 626 helps to maintain a good vacuum. Actually the O-Rings are reasonably cheap and probably we will exchange them every two months as a preventive measure. Of course the O-Ring has to be greased properly (not too much, not too little) and has to be without damage, dust, fibres etc. And make sure that it is not cold when inserting the 626 or the Ring does not seal properly.
Do you also do the 'trick' of rotating the stage to -60 degrees so that you can insert the 626 with its dewar pointing to the right (or left) instead of straight down? This way you keep a bit liquid nitrogen in the dewar which also stabilises the temperature.
If your cryo _and_ roomtemperature holder do crash the vacuum too often then it might be a good time for a service. Especially the roomtemperature holder should be 'safe' for the vacuum. Generally you get more crashes with the cryoholder (its heavier, less balanced and normally used cold and with short pumping times)  but again if you crash your vacuum more than 50% of the insertions then something is wrong.
Hope this helps. Feel free to ask if you have more questions

cheers
Tobias

-----------------------------------
Tobias Fürstenhaupt, PhD
head of Electron Microscopy
Max Planck Institute of Molecular Cell Biology and Genetics (MPI-CBG) Pfotenhauerstrasse 108
01307 Dresden, Germany

phone: (+49) (0)351 210-2690

-----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
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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