[3dem] 3dem Digest, Vol 150, Issue 65

Mon Feb 24 14:38:42 PST 2020

H,

I'll add to Denis protocol

1) Rotate the cryo-stage to -60?
2) Pre-pump the airlock (wait until red light goes off)
3) insert the cryo-holder and wait (again) until red light goes off (time
fixed by yourself, between 30 sec and one min of pumping).
4) stop breathing*
5) Hold the cryo-holder and reset the stage to 0?. Again, wait until red
light goes off!
6) Rotate the cryo-holder and insert it gently (don?t let the vacuum aspire
it abruptly inside the stage!) Look at the vacuum overview panel for the
IGP1 and if you see it start getting up stop rotate the holder for sec. and
then keep going slowly.
7) Check that the vacuum didn?t went to high during insertion
8) Breath*
* optional

On Mon, Feb 24, 2020 at 3:53 PM <3dem-request at ncmir.ucsd.edu> wrote:

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> Today's Topics:
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>    1. Re: Insertion of Holder 626 (Conway, James Frederick)
>    2. Re: Insertion of Holder 626 (Denis Chr?tien)
>
>
> ----------------------------------------------------------------------
>
> Message: 1
> Date: Mon, 24 Feb 2020 21:49:27 +0000
> From: "Conway, James Frederick" <James.Conway at pitt.edu>
> To: "3dem at ncmir.ucsd.edu" <3dem at ncmir.ucsd.edu>
> Subject: Re: [3dem] Insertion of Holder 626
> Message-ID: <E5D60D29-3309-40AA-9B05-1B8695B4459B at pitt.edu>
> Content-Type: text/plain; charset="utf-8"
>
> Ariel, and others,
>
> I have two 626 holders and a 910 (3-grid) cryoholder - none cause the TF20
> vacuum to crash. I do not change o-rings often (once a year if that), don't
> use vacuum grease (it collects dust), and pump for 60-secs in the airlock.
> I follow a common procedure: insert with the stage at -60-degrees tilt to
> maintain liquid N2 in the cryoholder dewar, wait for the 1-minute pump,
> return the stage to 0 tilt holding the dewar steady, then rotate the dewar
> upright - all to keep the relative motion slow between the o-ring and the
> airlock. Some thoughts on what sounds like a real pain:
>
> 1. I use ethane:propane to freeze - its stays liquid and I expect it
> evaporates in the airlock before entry into the column. Ethane ice may
> instead reach the column, as Wim Hagen suggested. Its easy to do - fill the
> cup 2/3 with ethane, top it off with propane, and do the plunge, etc, as
> usual. The ethane/propane mix can become jelly-like, so I bubble a bit of
> propane in to warm it up and it becomes absolutely clear. I drilled holes
> in the styrofoam platform so that liquid nitrogen is surrounding the brass
> cup, but that's not really necessary.
>
> 2. Check the o-ring for cuts. If you replace the o-ring (make sure its the
> correct size), and the vacuum breaks again, check it again for new cuts. If
> you see damage to the surface, there may be something sharp inside the
> cryo-transfer station or inside the airlock that is ripping up the o-ring.
> Try inserting the holder into each (at room temp) in turn, and inspect the
> o-ring afterwards with a magnifying glass.
>
> 3. My turbo pump was replaced a couple of years ago and the spin-up time
> on the new pump was much shorter. We hadn't noticed how the pump had
> degraded over time because it was very gradual. I'm not sure if this
> affected the quality of the vacuum, maybe just caused a long (5-6 minute)
> wait for spin-up that is now 2-3 minutes. However, if its long, maybe the
> turbo pump isn't working so well.
>
> James Conway
> ----------------------------------------------------------------------
> James Conway, PhD.,
> Professor, Department of Structural Biology
> University of Pittsburgh School of Medicine
> Biomedical Science Tower 3, Room 2047
> 3501 5th Ave
> Pittsburgh, PA 15213
> U.S.A.
> Phone: +1-412-383-9847
> Fax:   +1-412-648-8998
> Email: jxc100 at pitt.edu<mailto:jxc100 at pitt.edu>
> Web:
> https://nam04.safelinks.protection.outlook.com/?url=http%3A%2F%2Fwww.structbio.pitt.edu%2Findex.php%2F12-faculty%2F18-james-conway&data=02%7C01%7Celad.binshtein%40vanderbilt.edu%7C492084a4820d4ce22f4108d7b974169e%7Cba5a7f39e3be4ab3b45067fa80faecad%7C0%7C0%7C637181780583744211&sdata=gbOwrKLha4eTt9bLtPA9BIkn81NlUXrjK2FKwX5g%2Bxs%3D&reserved=0
> ----------------------------------------------------------------------
>
> -----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<mailto: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 Universit? Libre de Bruxelles Rue Adrienne Bolland, 8
> B-6041 Gosselies, Belgium
> Phone +32 (0)26509866
>
>
>
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>
> ------------------------------
>
> Message: 2
> Date: Mon, 24 Feb 2020 22:53:40 +0100
> From: Denis Chr?tien <denis.chretien at univ-rennes1.fr>
> To: "3dem at ncmir.ucsd.edu" <3dem at ncmir.ucsd.edu>
> Subject: Re: [3dem] Insertion of Holder 626
> Message-ID: <A582622E-C993-451F-8D23-5E6573E8B849 at univ-rennes1.fr>
> Content-Type: text/plain; charset="utf-8"
>
> Sorry, I mentioned Thermo (for the O-rings), but for the 626 it would be
> Gatan (still cost about one arm and a leg ...)
>
> I forgot to mention also the -60? insertion procedure that Wim also
> quoted. It really improved our ability to insert cryo-holders without leaks
> (and risks of warming-up the specimen by spilling all the LN2 outside the
> cryo-holder):
>
> 1) Rotate the cryo-stage to -60?
> 2) Pre-pump the airlock (wait until red light goes off)
> 3) insert the cryo-holder and wait (again) until red light goes off (time
> fixed by yourself, between 30 sec and one min of pumping).
> 4) stop breathing*
> 5) Hold the cryo-holder and reset the stage to 0?. Again, wait until red
> light goes off!
> 6) Rotate the cryo-holder and insert it gently (don?t let the vacuum
> aspire it abruptly inside the stage!)
> 7) Check that the vacuum didn?t went to high during insertion
> 8) Breath*
> * optional
>
> Denis
>
> > Le 24 f?vr. 2020 ? 22:01, Denis Chr?tien <denis.chretien at univ-rennes1.fr>
> a ?crit :
> >
> > If we order O-rings from Thermo, it cost one arm? Does anyone have
> references for ? standard ? O-rings that we can buy elsewhere for a much
> cheaper price?
> >
> > To come back to the original questions, we still use 626 and other
> side-entry holders. Sometime we face vacuum problems. In our experience
> these are mainly due to (as already said):
> >
> > - Dusts on the O-ring (or inside the airlock system). Inspect the O-ring
> and also try to blow dry-air inside the airlock. Maybe the airlock tube is
> damage, then ask Thermo for repair ? :-(
> >
> > - Dry O-rings: regularly put some (not a lot !) vacuum grease on it. Be
> careful not to scratch the metal when removing it or damaging the O-ring.
> Change it if dry or obviously damaged.
> >
> > - One frequent issue with cryo-holders: do not fill the cryo-holder
> station with to much LN2, you may freeze the O-ring and it will inevitably
> lead to a vacuum leak (very reproducible!). To limit this issue, we
> slightly lift the cryo-holder station with a rectangular grid box to avoid
> LN2 going into contact with the O-ring. We (not every one of us) cool the
> cryo-stage inside the microscope. Then the work-station is cooled just
> before inserting the specimen grid on the holder. This also limits the risk
> of cooling the O-ring.
> >
> > - We ? pre-pump ? the airlock, and pump for 1 min before inserting (on a
> Tecnai G2 Sphera). 3 min of pumping seams a lot!
> >
> > - Change of the zeolithe, grease on the valve O-ring of the cryo-holder,
> periodic zeolithe cycle (we do it every session): it improves stability on
> our side.
> >
> > Best
> >
> > Denis
> >
> >> Le 24 f?vr. 2020 ? 21:26, David Stokes <stokes at nyu.edu <mailto:
> stokes at nyu.edu>> a ?crit :
> >>
> >> I used to do the following prior to every session:
> >> Remove o-ring from holder.
> >> Clean o-ring off with kimwipes and fingers (I didn't bother to use any
> solvent).
> >> Clean o-ring groove with kimwipes.
> >> Blow away fibers with compressed air.
> >> Lightly grease o-ring.
> >> Inspect o-ring and o-ring groove under dissecting scope and carefully
> remove any fibers prior to re-installation onto the holder.
> >> Don't scratch the o-ring groove. In fact, don't touch it with forceps.
> Use compressed air.
> >>
> >> During the inspection process, you will see whether there are any nicks
> or flat spots in the o-ring. If so, then consider a replacement. If there
> is a scratch in the o-ring groove, then you might need a new holder (or
> maybe not).
> >> Also make sure you really have the right sized o-ring. At some point in
> ancient history, they changed the size slightly, such that CM200 o-ring was
> a bit fatter than the one for the CM12 stage.
> >>
> >> David
> >>
> >>
> >> On 2/24/2020 3:10 PM, Sharon G Wolf wrote:
> >>> Terje
> >>> We also have suffered for years from vacuum breaks. We tried
> EVERYTHING. The most effective thing was to (believe it or  not) change the
> o-ring every single session.
> >>> Recently we understood this was due to the fibers and other "dirt"
> that we observed on the o-rings every time we looked with the binocular
> after inserting and removing the holder from the microscopes, transfer
> stations or pumping station. We embarked on a ?cleaning program? where we
> gently swab all the above mentioned devises. It?s now slowly getting
> better. We are still changing the o rings but we hope it will soon no
> longer be necessary, once all the "stuff" that accumulated over the years
> is cleaned out from those places.
> >>> Sharon
> >>>
> >>>
> >>> Sent from my iPhone
> >>>
> >>>> On 24 Feb 2020, at 19:28, Dokland, Terje <dokland at uab.edu <mailto:
> dokland at uab.edu>> wrote:
> >>>>
> >>>> ?We have also had persistent vacuum break problems with our 626
> holder in our Tecnai F20 over the past two years. About 50% of the time
> inserting the holder breaks the vacuum. I though it was unique to this
> holder, but with the Elsa holder that we recently acquired it is even
> worse: vacuum break about 90% if the time. We?ve changed O rings, had the
> airlock disassembled and cleaned, but the problem persists.
> >>>>
> >>>> That being said, it never affected ice quality, and the temperature
> usually doesn?t go above -165 C. When the vacuum recovers it?s usually at
> 25-30 log or so and back to <10 within a half hour. It?s really annoying
> though.
> >>>>
> >>>> In my experience as long as the temperature doesn?t rise above -150C
> you should be ok.
> >>>>
> >>>> It helps to make sure the tip is very cold by submerging it
> completely in lN2 before inserting.
> >>>>
> >>>> Terje
> >>>>
> >>>> Sent from my iPhone
> >>>>
> >>>>> On Feb 24, 2020, at 7:13 AM, TALAVERA PEREZ Ariel <
> Ariel.Talavera at ulb.be <mailto: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
> >>>>> Universit? Libre de Bruxelles
> >>>>> Rue Adrienne Bolland, 8
> >>>>> B-6041 Gosselies, Belgium
> >>>>> Phone +32 (0)26509866
> >>>>>
> >>>>> _______________________________________________
> >>>>> 3dem mailing list
> >>>>> 3dem at ncmir.ucsd.edu <mailto:3dem at ncmir.ucsd.edu>
> >>>>>
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> >
> >>>> _______________________________________________
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> >
> >>> _______________________________________________
> >>> 3dem mailing list
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> >>>
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> >
> >>
> >> --
> >> David L. Stokes
> >> Skirball Institute, Dept. of Cell Biology
> >> NYU School of Medicine
> >>
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> >> tel: 212-263-1580
> >> fax: 212-263-2150
> >>
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> Subject: Digest Footer
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> ------------------------------
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> End of 3dem Digest, Vol 150, Issue 65
> *************************************
>

-- 
________________________________
Elad Binshtein, Ph.D.
Cryo-EM specialist - Senior Scientist
Crowe lab - Vanderbilt Vaccine Center (vvc)
MRB4 Room 11475
Vanderbilt University Medical Center
Nashville, TN
Mobile: +1-615-481-4408
https://www.linkedin.com/in/elad-binshtein-6bb89428/
E-Mail: elad.binshtein at vanderbilt.edu <eladbi at gmail.com>
twitter @EladBinshtein
________________________________
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