[3dem] Insertion of Holder 626

[Tobias Furstenhaupt]

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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