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

[John Rubinstein]

Hi Cindy,

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.

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.

Best,
John

--
John Rubinstein
Molecular Medicine Program
The Hospital for Sick Children Research Institute
686 Bay Street, Rm. 20-9705
Toronto, ON
Canada
M5G 0A4
Tel: (+001) 416-813-7255
Fax: (+001) 416-813-5022
www.rubinsteinlab.org<http://www.rubinsteinlab.org>



On Jul 1, 2019, at 12:38 PM, Schwartz, Cindi (NIH/NIAID) [E] <cindi.schwartz at nih.gov<mailto:cindi.schwartz at nih.gov>> wrote:

Hi all,

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.

The list of solvents we’ve tried that should dissolve plastic films:
Ethyl acetate
Chloroform
Ethylene dichloride
Trichlorobenzene

The list of solvents to clean them up a bit:
Acetone
Ethanol
5% acetic acid

We’ve tried sonication (a disaster)
Submerging in drops
Submerging in vast quantities of liquid
With/without little stir bars while the grids are on a metal platform in the solvent
Time ranges from 15 min to overnight
With/without a light glow discharge before cleaning

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.

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.

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.

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.

Thanks,
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>
<image001.png>  Please remember our Earth before printing this email <image001.png>
*************************************************************************
The information in this e-mail and any of its attachments is confidential and may contain sensitive information. It should not be used by anyone who is not the original intended recipient. If you have received this e-mail in error please inform the sender and delete it from your mailbox or any other storage devices. National Institute of Allergy and Infectious Diseases shall not accept liability for any statements made that are sender's own and not expressly made on behalf of the NIAID by one of its representatives.
*************************************************************************

_______________________________________________
3dem mailing list
3dem at ncmir.ucsd.edu<mailto:3dem at ncmir.ucsd.edu>
https://mail.ncmir.ucsd.edu/mailman/listinfo/3dem

-------------- next part --------------
An HTML attachment was scrubbed...
URL: <http://mail.ncmir.ucsd.edu/pipermail/3dem/attachments/20190702/891aa40b/attachment-0001.html>