[3dem] Insertion of Holder 626

[Patricia Grob]

Hi Ariel,


This sounds like a very long airlock pumping time to me. I am not sure about the TALOS airlock but on our Tecnai microscopes we use a 60-90 s airlock cycle time.

You might have a leak in the airlock if it is not sufficient, either from the holder end or on the stage, which you’ll have to determine (or the pump is not very good!). -154 degrees is indeed pretty high, we typically get -170 or lower during transfer with our  DH 626 Gatan cryo-holders. You could try and monitor the holder  temperature change during the whole transfer time and see when it starts rising. Make sure you start with a holder at -190 degrees C when you first transfer it form the workstation to the airlock. If the rise is immediate, maybe there is an issue with your holder (leak in the dewar at any seal point, sorbent needing to be replaced or envelope needing to be cleaned, or all of the above). You can monitor the temperature of the holder in the pumping station with liquid nitrogen in the dewar (only pumping down on the tip, not the dewar), and make sure that the lowest temperature stays in the -170 to -180 range once cooled, otherwise the dewar is not maintaining vacuum and needs maintenance. 

Good luck.
Cheers,

Patricia

_______________________________
Patricia Grob, Ph.D.
Research Specialist
Nogales Lab
HHMI at UC Berkeley
Molecular & Cell Biology Department / QB3,
Stanley Hall #742
University of California, Berkeley
Berkeley, CA 94720-3220
Phone: (510)666-3335	Fax: (510)666-3336
E.M. suite B307: (510)666-3339
URL: cryoem.berkeley.edu
Email: pgrob at berkeley.edu




> On Feb 24, 2020, at 5:13 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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> 3dem at ncmir.ucsd.edu
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