[3dem] Help! Plastic films on my holey grids are KILLING ME

Tue Jul 2 10:49:55 PDT 2019

Dear Cindi,

I, too, had this problem with Quantifoils several years ago.  Another side effect is that even if you do manage to remove the plastic from the holes without destroying them, the carbon layer will be much different in height than particles in holes.   Even after manually correcting for Z height, all defocus was off by 10microns and it could be either + or - .

It turned out that they carbon coated one side, removed the plastic layer, and then flipped them over and carbon coated the back.   The 10 microns was the thickness of the carbon/ plastic/ carbon sandwich. I suppose you could manually focus on particles in an adjacent hole to get your focus correct, but you'd have to be super fast.

And if you are working with small particles, you probably will have trouble getting the ice thin enough with the extra deep holes.

If they were mine, I'd send them back.

Cheers,

Valorie

Valorie Bowman

vdb at purdue.edu
765-494-5643
EM Facility Laboratory Manager/
Senior Research Electron Microscopist
Purdue Cryo EM Facility
Purdue University
________________________________
From: 3dem <3dem-bounces at ncmir.ucsd.edu> on behalf of Vladan Lucic <vladan at biochem.mpg.de>
Sent: Tuesday, July 2, 2019 1:12 PM
To: 3dem at ncmir.ucsd.edu
Subject: Re: [3dem] Help! Plastic films on my holey grids are KILLING ME

Several years ago we had a similar problem, suddenly our usual samples, imaged under the same conditions were bubbling at what used to be the normal dose. After a while I complained and Quantifoil told me they changed something in the cleaning procedure, probably changed or reverted procedure and after that the new grids were fine again. Perhaps that's the same problem that James referred to as being solved quite a while back. I also recommend contacting the manufacturer or the supplier.

Good luck,
Vladan

--

Vladan Lucic, PhD
Max Planck Institute of Biochemistry
Dept of Molecular Structural Biology
Am Klopferspitz 18
82152 Martinsried, Germany

Pnone: +49 89 8578-2647
Fax: +49 89 8578-2643
Web: http://www.biochem.mpg.de/277447/15_ContentSynCompl

On 7/2/19 6:11 PM, Schwartz, Cindi (NIH/NIAID) [E] wrote:

James,

Thanks for that insight. We do end up with some direct from quanitfoil and some from the US supplier (EMS in our case). But, we are having issues with both. Right now, I tried a pretty extensive cleaning (sent to me yesterday) protocol on these quantifoil supplied grids and I have films after negative staining apoferritin. It's been a disappointing morning looking at these grids. I will definitely send these back to quantifoil and at least we know that quantifoil says we can't remove the films once they've been shipped so we should quit trying. The worst part is that each grid is different, so in our hands checking randomly 5 out of 25 isn't enough. The two grids illustrated here are from the same side of the same grid box chosen at random after cleaning. That's why I wanted to bake on colloidal gold fiducials on every grid so I could see if there was gold in the holes, but alas, the colloidal gold doesn't seem to want to stick to this plastic. I will also try this with different kids of gold (maybe FAB-secondary anti-body gold will stick to this plastic).

Here's the protocol I used from Dewight Williams and Mariena Sylvestry-Ramos from when they were in Phoebe Stewart's lab:

The key to dissolving the plastic on quantifoils is to make the plastic flow away from the grid surface. When you dissolve it and it does not migrate away it just reabsorbs back on to the grid support surfaces. To get this to happen you need to set up a thin layer chromatography system, where you have a mobile phase of solvent moving up a filter paper. We have used dichloraethane as the solvent in combination with ethyl acetate. Pre-equilibrate a glass petri dish with a saturated ethyl acetate soaked set of Whatman filter papers with half a box of quantifoid grids, carbon side up (plastic layer down at a slight incline (we used the grid box to prop up one end.  We also added a sharpie or other permanent ink spot to the filter paper before starting to confirm we had a mobile phase which would make the dye migrate (I saw the dye migrate both times). Add to the bottom of the slightly inclined petri dish a few mls of dichloroethane. Place the lid on so the vapor will leave at the top of the inclined petri dish (30 min). We then washed the grids in chloroform (15 min) and acetone (15 min) the same way. In the old days, we did this twice (I switched to a fresh clean glass petri dish and fresh filter paper and repeated). Then baked in a 60 C oven O/N. This morning I glow-discharged and then negative stained apoferritin with Nano-Van.

Attached are the results. Disappointing to say the least.

Looks like I keep plugging away at it. Thanks everyone for your ideas. BTW, other ideas I got were to plasma clean the grids more, but I’m kind of scared to try this as I don’t want to etch away all the carbon too!

Cheers,

Cindi L. Schwartz

Electron Microscopist

Rocky Mountain Labs/NIAID/NIH

903 South 4th Street

Hamilton, MT  59840

406-363-9228

Cindi.Schwartz at NIH.gov<mailto:Cindi.Schwartz at NIH.gov>

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-----Original Message-----
From: Conway, James Frederick <James.Conway at pitt.edu><mailto:James.Conway at pitt.edu>
Sent: Tuesday, July 2, 2019 8:26 AM
To: 3dem at ncmir.ucsd.edu<mailto:3dem at ncmir.ucsd.edu>
Cc: Schwartz, Cindi (NIH/NIAID) [E] <cindi.schwartz at nih.gov><mailto:cindi.schwartz at nih.gov>
Subject: Re: [3dem] Help! Plastic films on my holey grids are KILLING ME

It was my understanding that Quantifoil solved this problem quite a while back, but nonetheless we kept receiving plastic-contaminated grids from one US supplier for some time after. When I contacted Quantifoil, they said that removing the plastic after the factory was near impossible, and when I told them the serial number of the grid boxes, they disavowed knowledge of them. My dark suspicion is that sending them back to the supplier just put them in the pipeline for someone else to receive, but that would be rank speculation. However, buying directly from Quantifoil has resulted in near perfect grids for over 5 years now. I suggest sending the bad batches of grids back for a refund, and if you are not already dealing directly with Quantifoil, try doing so or with a factory-recommended local supplier. If these grids are from Quantifoil, then certainly complain, send them back - it shouldn't be your time and money to fix up the grids. In my experience C-flats behave differently and (in my hands) inconsistently, but I understand there was a period of manufacturing issues that are now resolved and I look forward to trying them again.

James Conway

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James Conway, PhD.,

Professor, Department of Structural Biology Director, Molecular Biophysics and Structural Biology Graduate Program University of Pittsburgh School of Medicine Biomedical Science Tower 3, Room 2047

3501 5th Ave

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U.S.A.

Phone: +1-412-383-9847

Fax:   +1-412-648-8998

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Web:   http://www.structbio.pitt.edu/index.php/12-faculty/18-james-conway

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