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Hi Cindy,
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<div class="">Quantifoil grids are manufactured using the method in Ultramicroscopy 74 (1998) 75—81, although it may have evolved since it was first described. Briefly, a silicon wafer is coated with a sacrificial layer of glutaraldehyde cross-linked gelatin,
which is subsequently coated with photoresist. The desired pattern of holes is produced in the photoresist by photolithography. The cross-linked gelatin layer is then digested with proteinase K, releasing the holey photoresist film so that it can be floated
onto EM grids. The holey film should be soluble in organic solvent (they mention ethanol in the manuscript). I wonder if some of the cross-linked gelatin could remain on your grids.</div>
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<div class="">C-flat grids are manufactured using the method described in Microsc Microanal 13 (2007) 365-71. With this method, a water-soluble liftoff agent (similar to what Ken Downing used in a method described in 2003), is applied to a silicon wafer that
has the desired pattern of holes etched into it. Carbon is then evaporated onto the wafer and floated onto EM grids without the use of any plastics.</div>
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<div class="">Best,</div>
<div class="">John</div>
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-- <br class="">
John Rubinstein<br class="">
Molecular Medicine Program<br class="">
The Hospital for Sick Children Research Institute<br class="">
686 Bay Street, Rm. 20-9705<br class="">
Toronto, ON<br class="">
Canada<br class="">
M5G 0A4<br class="">
Tel: (+001) 416-813-7255<br class="">
Fax: (+001) 416-813-5022<br class="">
<a href="http://www.rubinsteinlab.org" class="">www.rubinsteinlab.org</a></div>
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<div class="">On Jul 1, 2019, at 12:38 PM, Schwartz, Cindi (NIH/NIAID) [E] <<a href="mailto:cindi.schwartz@nih.gov" class="">cindi.schwartz@nih.gov</a>> wrote:</div>
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Hi all,<o:p class=""></o:p></div>
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<o:p class=""> </o:p></div>
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We’ve been suffering with trying to get the plastic films off our Quantifoil grids with very inconsistent results. We’ve also had film issues on the c-flats, but my understanding is that the Quantifoils use a plastic background and the c-flats use a carbon
background. Please correct me if I’m wrong on that one. We use both copper and gold grids with the carbon substrate of various different patterns from R1/4 to Multi-A with grids that we purchased 2 years ago to ones we’ve received last month.<o:p class=""></o:p></div>
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<o:p class=""> </o:p></div>
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The list of solvents we’ve tried that should dissolve plastic films:<o:p class=""></o:p></div>
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Ethyl acetate<o:p class=""></o:p></div>
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Chloroform<o:p class=""></o:p></div>
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Ethylene dichloride<o:p class=""></o:p></div>
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Trichlorobenzene<o:p class=""></o:p></div>
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<o:p class=""> </o:p></div>
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The list of solvents to clean them up a bit:<o:p class=""></o:p></div>
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Acetone<o:p class=""></o:p></div>
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Ethanol<o:p class=""></o:p></div>
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5% acetic acid<o:p class=""></o:p></div>
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<o:p class=""> </o:p></div>
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We’ve tried sonication (a disaster)<o:p class=""></o:p></div>
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Submerging in drops<o:p class=""></o:p></div>
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Submerging in vast quantities of liquid<o:p class=""></o:p></div>
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With/without little stir bars while the grids are on a metal platform in the solvent<o:p class=""></o:p></div>
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Time ranges from 15 min to overnight<o:p class=""></o:p></div>
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With/without a light glow discharge before cleaning<o:p class=""></o:p></div>
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<o:p class=""> </o:p></div>
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We’ve put 10 nm colloidal gold fiducials on both sides of the grids (like you’d use for plastic section tomography—where they stick to Formvar just fine) after cleaning with various permutations from the above lists and we see that the carbon has gold, and
the ‘holes’ are clean with no gold, only they aren’t clean! The colloidal gold does not seem to stick to the plastic film because we see the film if we negative stain apoferritin on these same grids or we see the films in the big scope based on the ctf or
the way the ice has blotted or how the films have broken away and flipped onto the carbon. It’s rather obvious when you also see true holes next to ‘filmed’ holes.<o:p class=""></o:p></div>
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<o:p class=""> </o:p></div>
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Lastly, something will work for a subset of grids in a box, but later on, grids from the same box (even the same SIDE of the box) don’t get cleaned using the same protocol.<o:p class=""></o:p></div>
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<o:p class=""> </o:p></div>
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All I can conclude is that whatever this plastic is, we should get Quantifoil to stop using it because nothing seems to dissolve it and it’s probably killing the planet somehow. If not, it’s certainly detrimental to my mental health. Or better yet, Quantifoil
should do the research and either clean them better themselves or at least give us a protocol to do so in the lab.<o:p class=""></o:p></div>
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<o:p class=""> </o:p></div>
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Any tips, tricks, or surefire ways to clean grids would be great. And please publish it because nothing pops up on Pubmed when I search for it.<o:p class=""></o:p></div>
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<o:p class=""> </o:p></div>
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Thanks,<o:p class=""></o:p></div>
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<span style="font-size: 12pt; font-family: "Lucida Handwriting";" class="">Cindi L. Schwartz</span><span style="font-size: 12pt; font-family: Arial, sans-serif;" class=""> <o:p class=""></o:p></span></div>
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<span style="" class=""><o:p class=""> </o:p></span></div>
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<span style="" class=""><o:p class=""> </o:p></span></div>
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<span style="font-size: 12pt; font-family: Arial, sans-serif;" class="">Electron Microscopist</span><span style="" class=""><o:p class=""></o:p></span></div>
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<span style="font-size: 12pt; font-family: Arial, sans-serif;" class="">Rocky Mountain Labs/NIAID/NIH</span><span style="" class=""><o:p class=""></o:p></span></div>
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<span style="font-size: 12pt; font-family: Arial, sans-serif;" class="">903 South 4th Street</span><span style="" class=""><o:p class=""></o:p></span></div>
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<span style="font-size: 12pt; font-family: Arial, sans-serif;" class="">Hamilton, MT 59840</span><span style="" class=""><o:p class=""></o:p></span></div>
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<span style="font-size: 12pt; font-family: Arial, sans-serif;" class="">406-363-9228</span><span style="" class=""><o:p class=""></o:p></span></div>
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<span style="font-size: 12pt; font-family: Arial, sans-serif;" class=""><a href="mailto:Cindi.Schwartz@NIH.gov" target="_blank" style="color: rgb(149, 79, 114); text-decoration: underline;" class=""><span style="color: blue;" class="">Cindi.Schwartz@NIH.gov</span></a></span><span style="" class=""><o:p class=""></o:p></span></div>
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