[3dem] Positively charged carbon grids

Tue May 20 11:10:10 PDT 2025

 Hi David,

We have done a bit of experimenting with this recently, so I am happy to
share all I have learned (much of which involved combing through 3DEM
archives).

As per the gloqube manual
<https://urldefense.com/v3/__https://www.dartmouth.edu/emlab/docs/quorum_technologies_glocube_glow_discharge_system_manual.pdf__;!!Mih3wA!DylVFho-0R-WvQTxUrPSaAdK2igDbjxIhbnMq6bVWxYA0Bmi8HrtkIAczWbASsaa36Xu2EP4sIuWJjIDEYeZ8N-jFm3M$ >,
posttreatment of the grid with 5 mM magnesium acetate or 0.01% polylysine
should make the grid positively-charged but still hydrophilic. As others
have mentioned, glow discharging with amylamine present in the chamber can
render the grids both positively charged and hydrophobic. Some notes:

-As Ben noted, amylamine is both toxic and destroys pumps by generating
crystallized enol in the pumping system. If you try using amylamine, do it
in a dedicated glow discharger, and be potentially ready to sacrifice a
vacuum pump. If you run a long (~15 minute) IPA flush of the pump after
using (if you have a gloqube or similar) and change the pump oil every ~6
months they say it will prolong the pump life, but we ran the flush and
could still smell amylamine coming out the other end for a while
afterwards. We glow discharged the grids for the same amount of time as we
would normally, but with opposite polarity (we actually tried both negative
and positive and it didn't seem to make a difference). We were also advised
to use a shorter blotting time (~1 second), although this ended up being
slightly too short for us. Since the grid is hydrophobic, ice quality is
more difficult than on hydrophilic grids.

-The only actual protocol we could find online treating with polylysine or
magnesium acetate (we ordered Sigma P2636 and M5661) was Gabe Lander's
(negative stain) protocol here <https://urldefense.com/v3/__https://www.nature.com/articles/nature10774__;!!Mih3wA!DylVFho-0R-WvQTxUrPSaAdK2igDbjxIhbnMq6bVWxYA0Bmi8HrtkIAczWbASsaa36Xu2EP4sIuWJjIDEYeZ8M-IMjfD$ >.
We tried following it for cryo grids by treating them like negative stain
grids (manual wicking and washing) before sample application: treated with
5 uL 0.1% polylysine or 5 mM MgAcetate immediately after GD, waited 90
seconds, wicked excess away with Whatman #1, immediately added 5 uL water,
wicked, added another 5 uL water, wicked again, and let sit ~5 mins to
dryness. We also tried no washes, but results were basically the same.

-We found that the polylysine and MgAcetate also made our grids somewhat
hydrophobic (polylysine similarly to amylamine, magnesium acetate slightly
less so but sample size was small). For our sample the polylysine
completely denatured the sample, but that might be protein-dependent. We
did need a lower blot time for these grids as well (1-2 seconds).

-We ended up using 6 uL drops because the grids were hydrophobic, but it
probably didn't make much of a difference.

-Our ultimate conclusion was that there is a good reason that more people
don't do this and that there are very few instances of this working in the
literature — the whole thing increases grid handling and therefore
breakage, good ice is much harder to get, and results are questionable (in
our case at least). The added step of needing a continuous carbon layer (I
have heard anecdotally people using holey carbon without continuous carbon
and getting results, but haven't seen it myself) is not ideal as well.

Good luck — hope you have more success than we did. We also used a Leica
GP1; it's possible double-sided blotting would produce better ice in the
case of hydrophobic grids.

Talya Levitz, PhD
Scientist I, Dana Farber Cancer Institute
*she/they*

On Mon, May 19, 2025 at 3:00 PM <3dem-request at ncmir.ucsd.edu> wrote:

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>    1. Re: Positively charged carbon grids (Benjamin Apker)
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> ----------------------------------------------------------------------
>
> Message: 1
> Date: Mon, 19 May 2025 14:16:28 -0400
> From: Benjamin Apker <benjamin.apker at mitegen.com>
> To: PEREZ-MORGA David <David.Perez-Morga at ulb.be>
> Cc: "3dem at ncmir.ucsd.edu" <3dem at ncmir.ucsd.edu>
> Subject: Re: [3dem] Positively charged carbon grids
> Message-ID:
>         <
> CALdUJgtror0TV7pqL84cuaYwYq4F_PSn_uimDT-GOYcr1yLKGw at mail.gmail.com>
> Content-Type: text/plain; charset="utf-8"
>
> Dear David,
>
> As mentioned by the others, a positive charge can be achieved either by
> discharging in a plasma with an amylamine atmosphere or post-discharge
> treatment of the grid.
>
> A quick note that the amylamine atmosphere will contaminate/coat the
> surfaces of your glow discharge system for future discharges. The GloQube
> Plus Dual-chamber System (system page here
> <
> https://urldefense.com/v3/__https://www.mitegen.com/product/glow-discharge-system-for-tem-grids/__;!!Mih3wA!FBrH2ZBiX5cmFjBrqijbiMRH8ZSVHf1bFeipwQ4ml1GDsLNy0aqyA4gNX9CWGQqU-l4H7Fxsi75_80hS-z0lV8YwmfT-$
> >) has
> separate chambers to accommodate alternative discharge atmospheres and
> avoid cross-contamination.
>
> There was an informative discussion on this topic (and whether it results
> in a hydrophilic or hydrophobic surface) on this forum back in 2009 (thread
> here <https://mail.ncmir.ucsd.edu/pipermail/3dem/2009-February/001505.html 
> >
> ).
>
> Cheers,
> Ben
>
> On Mon, May 19, 2025 at 7:45?AM PEREZ-MORGA David via 3dem <
> 3dem at ncmir.ucsd.edu> wrote:
>
> > Dear 3dem users,
> > Any advice on how to posively charge  TEM carbon-film grids?
> > Thanks
> > David
> >
> > David P?rez-Morga
> >
> >
> >
> > *Molecular Parasitology Lab.
> > <
> https://urldefense.com/v3/__http://parasmol.ulb.be/__;!!Mih3wA!GCidJgEO8IR5DBtmsHlZ1xMn5tStEEgMF3x2YYQr6hsFcnpO4LeTFOMnSCWWcR_etu3hIvvHCr2PjsVOmOUFMk5JlLkK6g$
> >**
> > <
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> >*
> >     IBMM-Universit? Libre de Bruxelles
> >
> > *Electron Microscopy Lab.
> > <
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> >*
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>
> --
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> <
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> >
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