[3dem] mammalian cell freezing question for continuous carbon grids

[Anderson, Erik David]

Hi all,

Thank you for the replies! @Chen I am using a Leica Emgp, but we do have a vitrobot, and so re Dr. Kirchweger’s suggestion will try using that. And thank you for the paper and sharing your experience Dr. Hu, I agree holey carbon seem to work the best for this sort of system (I will likely just need to wait for our order to arrive). I had tried manual plunging when I did side blot, but perhaps I could try it with backblotting and see if that helps improve ice.

Thanks again for the replies and advice!

-Erik
________________________________
From: Peter Kirchweger <peter.kirchweger at weizmann.ac.il>
Sent: Sunday, May 29, 2022 3:33:55 AM
To: Anderson, Erik David <Erik.Anderson at bcm.edu>; 3dem at ncmir.ucsd.edu <3dem at ncmir.ucsd.edu>
Subject: Re: mammalian cell freezing question for continuous carbon grids

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________________________________
Dear Eric,

I can feel your pain ... I have also tried many things as you describe.
The only way I could at least partially successful plunge continuous carbon grids was by using the Vitrobot and by blotting from both sides.
This, however, can create deroofed cells when doing that (which happened to me). Additionally, I usually only got half of the grid with good ice thickness, while the upper half was too thick.
So, I stopped the continuous carbon again and went back to holey carbon.

Good luck and best regards ...

Dr. Peter Kirchweger
​-------------------------------------------------------------
Fass Lab
Postdoctoral fellow
Department of Chemical and Structural Biology
Weizmann Institute of Science
Herzl Street, 760001 Rehovot
ISRAEL
​-------------------------------------------------------------

________________________________
From: 3dem <3dem-bounces at ncmir.ucsd.edu> on behalf of Anderson, Erik David <Erik.Anderson at bcm.edu>
Sent: Saturday, May 28, 2022 7:32 AM
To: 3dem at ncmir.ucsd.edu <3dem at ncmir.ucsd.edu>
Subject: [3dem] mammalian cell freezing question for continuous carbon grids


Dear cryo colleagues,



I am wondering if anyone on this list has successfully frozen/imaged mammalian cells using continuous carbon grids, and if so whether they would be willing to provide tips regarding their freezing conditions?



Brief background, I’ve been trying to grow/image neurons (tried Hela as well) for tomography using continuous carbon grids of varying thickness (tested 3-10nm carbon layer, 150-200 mesh types, all gold), but have not had success during freezing step. I’m using a Leica EMGP, and have attempted back and front blots, respectively (tested 5-20 sec blot times, as well as different aliquots of media from 0-2uL, in 90-99% humidity). I’ve even tried side blotting, but still no success. All attempts have resulted in either broken grid holes or ice that is too thick (back blot generally causes more breakage, while front generally causes ice that’s too thick). I’ve verified the carbon remains (mostly) intact prior to freezing (at least much more intact than what is seen after freezing), and am mindful of how I take the grid out of the liquid prior to freezing. I should also add I’ve successfully grown/imaged the neurons on holey carbon grids, which are my (and it seems the literature’s) preferred grids for this system, but our order is delayed, and so that’s why I’m trying to make continuous carbon work.



If anyone has had success with these grids to image any kind of mammalian cell I’d be very appreciative of any advice/tips!



Thank you,



Erik Anderson

MD-PhD Candidate | Baylor College of Medicine

B.S. Microbiology and Music | University of Wisconsin-Madison | 2015

Ludtke Lab<https://urldefense.proofpoint.com/v2/url?u=https-3A__www.bcm.edu_research_faculty-2Dlabs_steven-2Dludtke-2Dlab&d=DwMF-g&c=-35OiAkTchMrZOngvJPOeA&r=L7-zyQ-04fFCMRqzLIOnx7H0exGZHwIQe_wMPuY600I&m=0pToNIycF_Lo1X5zhTGdAjwpMQnFME8apFLnrmQ7x6eIuOOouehtrBpEXZZVf5FJ&s=fil6vpim5X2wVq7qP-h05TIMTBY6OX5uLrldcv_CcKg&e=>




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