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<p style="margin-top:0;margin-bottom:0">Hi Cindi, <br>
</p>
<p style="margin-top:0;margin-bottom:0"><br>
</p>
<p style="margin-top:0;margin-bottom:0">I'm so sorry. Years ago, in Phoebe's lab, we used to get the grids with just plastic, then C-coat them ourselves, then dissolve the plastic. And indeed, that plastic was a nightmare to remove.
<br>
</p>
<p style="margin-top:0;margin-bottom:0"><br>
</p>
<p style="margin-top:0;margin-bottom:0">A few yrs ago I shared this email with the list:
<a href="https://mail.ncmir.ucsd.edu/pipermail/3dem/2012-December/002760.html" class="OWAAutoLink" id="LPlnk435924" previewremoved="true">
https://mail.ncmir.ucsd.edu/pipermail/3dem/2012-December/002760.html</a></p>
<p style="margin-top:0;margin-bottom:0"><br>
</p>
<p style="margin-top:0;margin-bottom:0">From your email, it looks like everything you're doing is pretty much what we did too, but in our experience (if I remember correctly), the plastic did come off.
<br>
</p>
<p style="margin-top:0;margin-bottom:0"><br>
</p>
<p style="margin-top:0;margin-bottom:0">The only thing I didn't see in your email was baking the grids. In Phoebe's lab at Vanderbilt, we put our grids on top of an aluminum disk, took them to a CM12, cranked up the power and left them overnight inside the
microscope (our conditions were, a beam spread slightly inside the 4cm mark, spot size 1, all apertures out). I don't think this is done anymore, but in our case, it seemed to work. Dewight used to also mention how obnoxious and stubborn that plastic was,
but I don't remember if he had an extra step to completely get rid of the plastic. We did 2x wash with each of the 4 solvents I mention in that email, and waited until the filter paper was dry before adding the next solvent down the list.
<br>
</p>
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</p>
<p style="margin-top:0;margin-bottom:0">Finally, IDK if it would be advisable, but for hydrocarbons left behind (this is on holders), we've used an orange peel cleaner (we use Zep, but any other orange-based compound should work similarly). If all else fails,
that stuff should eat through anything nasty. <br>
</p>
<p style="margin-top:0;margin-bottom:0"><br>
</p>
<p style="margin-top:0;margin-bottom:0">Good luck, <br>
</p>
<p style="margin-top:0;margin-bottom:0"><br>
</p>
<p style="margin-top:0;margin-bottom:0">Mariena<br>
</p>
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<div id="divRplyFwdMsg" dir="ltr"><font face="Calibri, sans-serif" style="font-size:11pt" color="#000000"><b>From:</b> 3dem <3dem-bounces@ncmir.ucsd.edu> on behalf of Lars-Anders Carlson <lars-anders.carlson@umu.se><br>
<b>Sent:</b> Monday, July 1, 2019 1:23:10 PM<br>
<b>To:</b> 3dem@ncmir.ucsd.edu<br>
<b>Subject:</b> Re: [3dem] Help! Plastic films on my holey grids are KILLING ME</font>
<div> </div>
</div>
<div class="BodyFragment"><font size="2"><span style="font-size:11pt;">
<div class="PlainText">Hi Cindi,<br>
Can you remove it by just plasma cleaning longer? That could be the <br>
reason why some of us typically don't see this film in the first place <br>
(another reason could of course be that we don't look as closely as you).<br>
<br>
QF grids used to have plastic deposits around the edge of the holes, <br>
which is where they would start bubbling at higher exposures (80-100 <br>
e/Å2), but I haven't seen much of this lately.<br>
<br>
I hope you find a solution since you seem really frustrated about it.<br>
<br>
Cheers,<br>
Lars<br>
<br>
On 2019-07-01 18:38, Schwartz, Cindi (NIH/NIAID) [E] wrote:<br>
> Hi all,<br>
> <br>
> We’ve been suffering with trying to get the plastic films off our <br>
> Quantifoil grids with very inconsistent results. We’ve also had film <br>
> issues on the c-flats, but my understanding is that the Quantifoils use <br>
> a plastic background and the c-flats use a carbon background. Please <br>
> correct me if I’m wrong on that one. We use both copper and gold grids <br>
> with the carbon substrate of various different patterns from R1/4 to <br>
> Multi-A with grids that we purchased 2 years ago to ones we’ve received <br>
> last month.<br>
> <br>
> The list of solvents we’ve tried that should dissolve plastic films:<br>
> <br>
> Ethyl acetate<br>
> <br>
> Chloroform<br>
> <br>
> Ethylene dichloride<br>
> <br>
> Trichlorobenzene<br>
> <br>
> The list of solvents to clean them up a bit:<br>
> <br>
> Acetone<br>
> <br>
> Ethanol<br>
> <br>
> 5% acetic acid<br>
> <br>
> We’ve tried sonication (a disaster)<br>
> <br>
> Submerging in drops<br>
> <br>
> Submerging in vast quantities of liquid<br>
> <br>
> With/without little stir bars while the grids are on a metal platform in <br>
> the solvent<br>
> <br>
> Time ranges from 15 min to overnight<br>
> <br>
> With/without a light glow discharge before cleaning<br>
> <br>
> We’ve put 10 nm colloidal gold fiducials on both sides of the grids <br>
> (like you’d use for plastic section tomography—where they stick to <br>
> Formvar just fine) after cleaning with various permutations from the <br>
> above lists and we see that the carbon has gold, and the ‘holes’ are <br>
> clean with no gold, only they aren’t clean! The colloidal gold does not <br>
> seem to stick to the plastic film because we see the film if we negative <br>
> stain apoferritin on these same grids or we see the films in the big <br>
> scope based on the ctf or the way the ice has blotted or how the films <br>
> have broken away and flipped onto the carbon. It’s rather obvious when <br>
> you also see true holes next to ‘filmed’ holes.<br>
> <br>
> Lastly, something will work for a subset of grids in a box, but later <br>
> on, grids from the same box (even the same SIDE of the box) don’t get <br>
> cleaned using the same protocol.<br>
> <br>
> All I can conclude is that whatever this plastic is, we should get <br>
> Quantifoil to stop using it because nothing seems to dissolve it and <br>
> it’s probably killing the planet somehow. If not, it’s certainly <br>
> detrimental to my mental health. Or better yet, Quantifoil should do the <br>
> research and either clean them better themselves or at least give us a <br>
> protocol to do so in the lab.<br>
> <br>
> Any tips, tricks, or surefire ways to clean grids would be great. And <br>
> please publish it because nothing pops up on Pubmed when I search for it.<br>
> <br>
> Thanks,<br>
> <br>
> Cindi L. Schwartz<br>
> <br>
> Electron Microscopist<br>
> <br>
> Rocky Mountain Labs/NIAID/NIH<br>
> <br>
> 903 South 4th Street<br>
> <br>
> Hamilton, MT 59840<br>
> <br>
> 406-363-9228<br>
> <br>
> Cindi.Schwartz@NIH.gov <<a href="mailto:Cindi.Schwartz@NIH.gov">mailto:Cindi.Schwartz@NIH.gov</a>><br>
> <br>
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> <br>
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<br>
-- <br>
Lars-Anders Carlson<br>
Assistant Professor<br>
Dept of Medical Biochemistry and Biophysics<br>
Wallenberg Centre for Molecular Medicine<br>
Umeå University<br>
901 87 Umeå, Sweden<br>
<a href="http://www.carlsonlab.se/">http://www.carlsonlab.se/</a><br>
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