[3dem] carbon detaching from grids

[Vinod Nair]

Hi all,
 We have been lucky not to have the problem of carbon detaching from the
grids. However of late, we are seeing residual substrate spanning the holes
of both Quantifoil and C flat grids.  This film is  not visible under
routineTEM analysis unless you add some kind of fiducial on them.We have
started inspecting grids at random from a new box by adsorbing 3
microliters of colloidal gold on the grid. In most cases we are observing
10nm gold in the holes which shouldn't be the case if they are true hole.
To our misfortune, we have over half dozen boxes with grids without true
hole.
We have tried various solvent wash protocols, ethylene dichloride soak, (
Passmore  & Russo, Methods  Enzymol. 2016 technique) including the ones
suggest by Quantifoil. The results have been highly variable depending on
the batch of grids. Some clean reasonably well where as other batches of
grids are quite resilient to even an over night soak in the solvents.
We have noticed the same kind of substrate even on the Aufoil grids.

The question I have for the cryo EM community is whether any of you are
facing this problem. More importantly, if you have a  grid cleaning
protocol that works well.

Thanks in advance for your valuable insight and suggestions

Vinod Nair,
Staff Scientist
NIAID/NIH



On Fri, Dec 7, 2018 at 10:55 AM Briegel, A. <a.briegel at biology.leidenuniv.nl>
wrote:

> Dear Cheri and Anna,
> We also had this problem recently with our standard samples (with same
> glow discharger, plunge freezer and same buffers), so it might  indeed be
> related to the new manufacturing process.
> I hope the problem can be solved.
> Best wishes,
> Ariane
>
>
> Sent from my iPhone
>
> On 7 Dec 2018, at 18:26, Anna Sartori Rupp <anna.sartori.rupp at gmail.com>
> wrote:
>
>
> Hi Cheri,
> We recently experienced exactly the same problem with the two latest
> purchased boxes of quantifoil gold R2/2 grids. We used the same glow
> discharge machine (ELMO) and the same settings that we normally use and we
> experienced that when we put the grids in a buffer like PBS or HEPES + 2%
> PFA the carbon was detaching on almost all the grids.
> To see where the problem could come from we used two very old batches of
> quantifoil grids and repeated the same procedure , but in this case the
> carbon remained nicely attached to the grids.
>
> So it could be that your problem might not be due to your plasma cleaner
> but rather to the new way quantifoil is manifacturing their grids.
>
>  I contacted quantifoil and sent them the corresponding pictures, they
> told me that in fact they recently changed their carbon film deposition
> parameters (he talked about different plasma cleaner conditions...) and
> they told me that they would look into it , but I haven t heard back from
> them yet.
> It would be great if you could also contact them to report your problem.
> I hope this helps,
> Ciao
> Anna
>
> -------------------------------------------------
>
> Anna Sartori-Rupp , PhD
> Ultrastructural Bioimaging Unit
> Institut Pasteur,
> 28 rue du Dr Roux
> 75015 Paris, France
>
>
> Il giorno Gio 6 Dic 2018, 9:12 PM HAMPTON, CHERI M CTR USAF AFMC AFRL/RXAS
> <cheri.hampton.ctr at us.af.mil> ha scritto:
>
>> Hi all,
>>
>>
>> I have suddenly begun experiencing the carbon detaching from my QF R2/1
>> grids when submersed in buffer and when applying proteins for blotting and
>> cryo-plunging. I have tried two different lots of grids, and now I am
>> suspicious of the facility's plasma cleaner that I have been using to glow
>> discharge. All settings and timings have remained constant on my part. I
>> plan to seek out another plasma cleaner to do a comparison test, but
>> meanwhile, what kinds of solvents or materials placed in the plasma
>> cleaner/glow discharge unit might linger and cause problems? I'd like to
>> justify having a "clean" one just for TEM :)
>>
>>
>> Thanks for your input,
>>
>> Cheri
>>
>>  ~'~'~'~'~'~'~'~'~'~'~
>> Cheri M. Hampton, PhD
>> Research Microscopist
>> UES, Inc.
>> AFRL/RXAS
>> 2179 12th St, B652/R122
>> Wright-Patterson AFB, OH 45433-7750
>> (937) 255-9081
>> cheri.hampton.ctr at us.af.mil
>>
>>
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