[3dem] Carbon peeling from C flat gold grids

[Julika Radecke]

Dear Janet,

You may have already received a similar message several times by now, so I'll keep it short.
Do you have a specific reason for using C flats?
If so, then it is going to be very tricky, they are super fragile. You wrote you used Quantifoils before, if you can, I would change back to those or any other grid that is suitable and less fragile than a C flat.
Just like you, for growing primary neurons, in my case, I use 200 mesh R2/1 or R2/2 Quantifoil, no coating, just fire sterilization and later freezing with the Vitrobot.

Best,
Julika


-----Original Message-----
From: 3dem <3dem-bounces at ncmir.ucsd.edu> On Behalf Of Reinhard Rachel via 3dem
Sent: dimanche, 1 mars 2026 19:19
To: Janet Meehl <janet.meehl at colorado.edu>; 3dem at ncmir.ucsd.edu
Subject: [3dem] Carbon peeling from C flat gold grids

Hi Janet, 

> I’m having issues with C flat grids that I recently purchased.  The 
> grids
are 
> holey carbon 2/2 and 1.2/1.3 gold 200 mesh grids purchased from EM Sciences.

> Prior to seeding cells on grids, they are glow discharged, UV 
> sterilized in
a 
> tissue culture hood, dipped briefly in 70% ethanol followed by water 
> and placed in a culture dish. Poly lysine is added to the dish and 
> grids sit overnight at 4C before being rinsed several times with 
> water. Media and
cells 
> are added. Grids are plunge frozen using a Leica GP2 and are clipped 
> to be imaged in a Krios microscope.  In the light microscope, carbon 
> films appear

> intact prior to freezing but I think the carbon is very fragile and 
> will
tear 
> upon the slightest perturbation.  In the last experiment, every grid 
> (8) had

> large areas of carbon missing.  I have used Quantifoils for this same 
> protocol with no issues at all.

I am just trying hard to understand this protocol - not only as an electron microscopist (yes, I do have quite some experience), but as a microbiologist. 
If you do the glow discharging: did you test those grids after glow discharging, to which extent you are likely to contain any further microbiological contamination, in the follow-up procedures? means: live, viable cells? Glow discharging is likely to result in quite some reduction of viable microorganisms on these grids - if there were any viable microbes (consider:  -
a) vacuum, b) UV). Whether this is sufficient? (you may test this). 
The urgent question for me arises whether you really need further steps like
(b) UV sterilization, and (c) ethanol treatment. My gut feeling: at least one of these steps is not needed, in terms of sterile surfaces on grids. Probably both?
I am just curious: how long (minutes) is the exposure to the Poly-Lys (which is sterile, I suppose)?
Which media?
which kind of cells are added, and then how long is the 'incubation'? 

My argument: any type of grids for cryo (or: for bio-TEM) are highly labile structure, 10-20 nm thin carbon films, with holes. Whatever you do, they are likely to break, and the more steps you "do" with treatments, the more are they likely to break. - second argument: sterile is sterile. One treatment to reach sterility is fine.
- and: the number of viable (!) cells on grids is low anyway. 
just my 2 cents. 
Kind regards,
Reinhard

--
Prof. Dr. Reinhard Rachel
University of Regensburg
TEM+SEM / Anatomy
Faculty of Biology & Preclin. Med.
Universitaetsstrasse 31
D-93053 Regensburg - Germany
tel +49 941 943 -2837, -1666
mail reinhard.rachel at biologie.uni-regensburg.de
office: VKL 3.1.29
member of the IFSM board

Next microscopy conference:
- IMC21, The 21st Intl. Microscopy Congr 2026 in Liverpool, UK: Aug 31-Sept 4,
2026
- next Microbiol. conference:  VAAM 22.3.-25.3.2026 - Berlin


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