[3dem] Insertion of Holder 626

Tue Mar 10 02:12:27 PDT 2020

Hi All 

Imperials solution to the vacuum crashing problem during cryo-holder insertion was to replace the Viton orings with non-spec size, 8.4 x 2.1mm.

We ordered various oring sizes from Polymax and eventually found 8.4 x 2.1mm to be the best suiter in terms of no FFT cut-off and successful insertions/retractions.  We have now fitted this size to all 626's including the Elsa and vacuum crashes are a thing of the past!

Good luck, be safe.

Paul

-----Original Message-----
From: 3dem <3dem-bounces at ncmir.ucsd.edu> On Behalf Of Matthijn VOS
Sent: 25 February 2020 21:54
To: Grassucci, Robert <rg2502 at cumc.columbia.edu>
Cc: 3dem at ncmir.ucsd.edu
Subject: Re: [3dem] Insertion of Holder 626

*******************
This email originates from outside Imperial. Do not click on links and attachments unless you recognise the sender. 
If you trust the sender, add them to your safe senders list https://spam.ic.ac.uk/SpamConsole/Senders.aspx to disable email stamping for this address.
*******************
Dear Bob,

In principle one would not need to worry about the tip since your column valves are closed during sample insertion and thus the FEG is protected. I never open the column valves if my column value us not back to normal (say log10 or so). In addition their should be a cryo-box around the specimen area which collects the water. With a vacuum “crash” with a cryo-holder this is caused by water molecules. The IGP senses a current that is too high and to protect it the valves are closed and the igp is switched off. These protections are there so I do not worry to much if I hear the valves closing if the vacuum spikes. All of the water is collected by the cryo-box and is pumped away at a later stage when the cryo/cycle is run. Here the cryo-box is warmed up while the igp is switched off and the remaining turbo pump, pumps away the water. This basically is the state the microscopes goes into during a vacuum “crash”. The water that goes into the igp with a vacuum spike, is collected on the plates in the igp like all the rest that is pumped away during normal operation. This affects the lifetime of the igp. How much we can debate about, but the safety that is activated on a reading above the threshold is there to protect it. Thus a vacuum “crash” is maybe the wrong word people are using. If the cryo-box collects too much water it will saturate at some point, which turns your sample into the coldest point in the microscope. Thus your sample will start to contaminate fast and your working day is over (time for cryo-cycle).  

For the heavier holder types, this might be a design problem by itself if it requires fingers. The holder sits on the V-grove and this can be adjusted by service to be a strong forse or weaker. To strong force will cause it to pick up all vibrations, too loose is a problem for tomography when going to high tilt and the holder slips out. One might need to tighten this V-groove adjustment more to avoid the use of fingers on insertion, but maybe ar a consequence of higher sensitivity to vibrations. 

Hope this helps

Cheer

Matthijn

> On Feb 25, 2020, at 21:18, Grassucci, Robert <rg2502 at cumc.columbia.edu> wrote:
> 
> Hi All,
> I am a bit concerned that anybody considers it acceptable that there are vacuum dumps when inserting the holder.  This leads to junk getting into the column and causes further issues with the vacuum level and tip stability.  One trick that we have used for the heavier versions of cryo dewars like the Elsa is to place your finger beneath the holder rod while doing the final rotation.  The idea is that the heavy dewar leads to uneven pressure on the O-ring which can cause a leak.  Placing your finger in this position gives it the extra support needed so this does not happen.  I totally agree with others who check and clean the O-ring frequently.  These routine checks often get diluted over the generations of training and still need to be remembered.
> Good luck,
> Bob
> 
> 
> -----Original Message-----
> From: 3dem [mailto:3dem-bounces at ncmir.ucsd.edu] On Behalf Of Shi, Dan 
> (NIH/NCI) [E]
> Sent: Tuesday, February 25, 2020 8:50 AM
> To: TALAVERA PEREZ Ariel <Ariel.Talavera at ulb.be>
> Cc: 3dem at ncmir.ucsd.edu
> Subject: Re: [3dem] Insertion of Holder 626
> 
> Dear Ariel,
> 
> If there is any minor leak from the rod O-ring, long time airlock pump would raise your grid temperature because the 'fresh' air constantly flow through the grid to warm it up and the temperature sensor is not on the grid.
> 
> The leak could be caused by multiple reasons as other people pointed out, if you have tried all the suggestions to clean the O-ring and replace different O-ring, the vacuum crash is still happening. Another hidden problem could be the OD of the cryo-holder's rod/O-ring slot is few tens micrometer too small to seal compustage tube well. 
> 
> A simple test will identify where is the problem to cause the leak. You can swap the O-ring of this 626 holder with the one from a good RT holder, insert both holders into the Talos, the vacuum pressure spike results would tell whether the O-ring or the rod is the leak source. If the O-ring is not a problem for the RT holder. You may replace the original O-ring with few tens micrometer thicker O-ring - i.e. the same ID and slightly bigger OD for the cryo-holder.
> 
> An alternative method to minimize the problems you have could be to shorten the airlock pump time to less than 60 seconds and to decrease the final holder rotation angle as we did to our Talos L120C.
> 
> Good luck,
> 
> Dan     
> 
> -----Original Message-----
> From: TALAVERA PEREZ Ariel <Ariel.Talavera at ulb.be>
> Sent: Monday, February 24, 2020 8:13 AM
> 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
> 
> _______________________________________________
> 3dem mailing list
> 3dem at ncmir.ucsd.edu
> https://urldefense.proofpoint.com/v2/url?u=https-3A__mail.ncmir.ucsd.e
> du_mailman_listinfo_3dem&d=DwIGaQ&c=G2MiLlal7SXE3PeSnG8W6_JBU6FcdVjSsB
> Sbw6gcR0U&r=DHQ5sxLdEeh7GliHapLyXDQKaXurrg9EcaYzNZr3hks&m=duZK5NEW2t-O
> voE8gsAGwAh6a5z7Nif0YYzgjpGvhlA&s=R2ftIG9BookDnnL38KPcHGZMDO1o60ZGCeTn
> R89hibI&e= _______________________________________________
> 3dem mailing list
> 3dem at ncmir.ucsd.edu
> https://urldefense.proofpoint.com/v2/url?u=https-3A__mail.ncmir.ucsd.e
> du_mailman_listinfo_3dem&d=DwIGaQ&c=G2MiLlal7SXE3PeSnG8W6_JBU6FcdVjSsB
> Sbw6gcR0U&r=DHQ5sxLdEeh7GliHapLyXDQKaXurrg9EcaYzNZr3hks&m=duZK5NEW2t-O
> voE8gsAGwAh6a5z7Nif0YYzgjpGvhlA&s=R2ftIG9BookDnnL38KPcHGZMDO1o60ZGCeTn
> R89hibI&e= _______________________________________________
> 3dem mailing list
> 3dem at ncmir.ucsd.edu
> https://mail.ncmir.ucsd.edu/mailman/listinfo/3dem
_______________________________________________
3dem mailing list
3dem at ncmir.ucsd.edu
https://mail.ncmir.ucsd.edu/mailman/listinfo/3dem