[3dem] Insertion of Holder 626

[Sharon G Wolf]

It very well could be an o-ring issue. That could explain the vacuum breaks. Make sure the o-ring is completely clean - no hairs etc, and is VERY lightly greased. Try replacing it with a new one.  We have had horrible problems with vacuum breaks due to garbage on the o-rings. Don’t let people into the room with sweaters, for instance.
The crystalline ice could also mean that you need to replace the clip ring. If it is not clamping the grid tightly, you will have ice. Also there is a spring that lines the depression where the grid sits. That also occasionally needs replacing. 
Finally, its worth finding out that your controller is actually functioning properly during bake-out. Is your holder actually heating up? Is the vacuum in the pumping station sufficient?
Good luck
Sharon


Sent from my iPad

> On 24 Feb 2020, at 15:35, Lars-Anders Carlson <lars-anders.carlson at umu.se> wrote:
> 
> Hi Ariel,
> Is that a Talos 120? Our Talos 120 has the worst airlock mechanism of any scope I've used. Just in terms of the tactile aspects it feels more "flimsy" than e.g. Tecnais did. New users find it difficult to insert even the RT holder, and although I'm fine with the RT holder I've almost never managed to insert the 626 without a vacuum break. So we rarely use this scope for cryo, for this reason.
> 
> As for how you do it, I would consider trying a shorter pumping time (try 60 or 90 s) and just accepting that the vacuum will crash (since it will anyway). Once the holder is in you can refill it and then it won't warm up that much.
> 
> Another thing that may help is to pretilt the stage when you're inserting the holder, in case you aren't already doing this.
> 
> If none of this helps, you may have a problem with the shutter of the 626.
> 
> Hope this helps,
> Lars
> 
> 
> 
>> On 2020-02-24 14:13, TALAVERA PEREZ Ariel 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
> 
> -- 
> Lars-Anders Carlson
> Assistant Professor
> Dept of Medical Biochemistry and Biophysics
> Wallenberg Centre for Molecular Medicine
> Umeå University
> 901 87 Umeå, Sweden
> http://www.carlsonlab.se/
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