<div dir="ltr"><div dir="ltr">
<div style="font-family:Aptos,Aptos_EmbeddedFont,Aptos_MSFontService,Calibri,Helvetica,sans-serif;font-size:12pt;color:rgb(0,0,0)" class="gmail-x_elementToProof">Hi David,</div><div style="font-family:Aptos,Aptos_EmbeddedFont,Aptos_MSFontService,Calibri,Helvetica,sans-serif;font-size:12pt;color:rgb(0,0,0)" class="gmail-x_elementToProof"><br></div><div style="font-family:Aptos,Aptos_EmbeddedFont,Aptos_MSFontService,Calibri,Helvetica,sans-serif;font-size:12pt;color:rgb(0,0,0)" class="gmail-x_elementToProof">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).</div><div style="font-family:Aptos,Aptos_EmbeddedFont,Aptos_MSFontService,Calibri,Helvetica,sans-serif;font-size:12pt;color:rgb(0,0,0)" class="gmail-x_elementToProof"><br></div><div style="font-family:Aptos,Aptos_EmbeddedFont,Aptos_MSFontService,Calibri,Helvetica,sans-serif;font-size:12pt;color:rgb(0,0,0)" class="gmail-x_elementToProof">As per the <a title="https://www.dartmouth.edu/emlab/docs/quorum_technologies_glocube_glow_discharge_system_manual.pdf" id="gmail-LPlnk689890" rel="noopener noreferrer" target="_blank" href="https://urldefense.com/v3/__https://www.dartmouth.edu/emlab/docs/quorum_technologies_glocube_glow_discharge_system_manual.pdf__;!!Mih3wA!DylVFho-0R-WvQTxUrPSaAdK2igDbjxIhbnMq6bVWxYA0Bmi8HrtkIAczWbASsaa36Xu2EP4sIuWJjIDEYeZ8N-jFm3M$">gloqube manual</a>,
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:</div><div style="font-family:Aptos,Aptos_EmbeddedFont,Aptos_MSFontService,Calibri,Helvetica,sans-serif;font-size:12pt;color:rgb(0,0,0)" class="gmail-x_elementToProof"><br></div><div style="font-family:Aptos,Aptos_EmbeddedFont,Aptos_MSFontService,Calibri,Helvetica,sans-serif;font-size:12pt;color:rgb(0,0,0)" class="gmail-x_elementToProof">-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.</div><div style="font-family:Aptos,Aptos_EmbeddedFont,Aptos_MSFontService,Calibri,Helvetica,sans-serif;font-size:12pt;color:rgb(0,0,0)" class="gmail-x_elementToProof"><br></div><div style="font-family:Aptos,Aptos_EmbeddedFont,Aptos_MSFontService,Calibri,Helvetica,sans-serif;font-size:12pt;color:rgb(0,0,0)" class="gmail-x_elementToProof">-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 <a title="https://www.nature.com/articles/nature10774" id="gmail-LPlnk840095" rel="noopener noreferrer" target="_blank" href="https://urldefense.com/v3/__https://www.nature.com/articles/nature10774__;!!Mih3wA!DylVFho-0R-WvQTxUrPSaAdK2igDbjxIhbnMq6bVWxYA0Bmi8HrtkIAczWbASsaa36Xu2EP4sIuWJjIDEYeZ8M-IMjfD$">here</a>.
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.</div><div style="font-family:Aptos,Aptos_EmbeddedFont,Aptos_MSFontService,Calibri,Helvetica,sans-serif;font-size:12pt;color:rgb(0,0,0)" class="gmail-x_elementToProof"><br></div><div style="font-family:Aptos,Aptos_EmbeddedFont,Aptos_MSFontService,Calibri,Helvetica,sans-serif;font-size:12pt;color:rgb(0,0,0)" class="gmail-x_elementToProof">-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). </div><div style="font-family:Aptos,Aptos_EmbeddedFont,Aptos_MSFontService,Calibri,Helvetica,sans-serif;font-size:12pt;color:rgb(0,0,0)" class="gmail-x_elementToProof"><br></div><div style="font-family:Aptos,Aptos_EmbeddedFont,Aptos_MSFontService,Calibri,Helvetica,sans-serif;font-size:12pt;color:rgb(0,0,0)" class="gmail-x_elementToProof">-We ended up using 6 uL drops because the grids were hydrophobic, but it probably didn't make much of a difference.</div><div style="font-family:Aptos,Aptos_EmbeddedFont,Aptos_MSFontService,Calibri,Helvetica,sans-serif;font-size:12pt;color:rgb(0,0,0)" class="gmail-x_elementToProof"><br></div><div style="font-family:Aptos,Aptos_EmbeddedFont,Aptos_MSFontService,Calibri,Helvetica,sans-serif;font-size:12pt;color:rgb(0,0,0)" class="gmail-x_elementToProof">-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.</div><div style="font-family:Aptos,Aptos_EmbeddedFont,Aptos_MSFontService,Calibri,Helvetica,sans-serif;font-size:12pt;color:rgb(0,0,0)" class="gmail-x_elementToProof"><br></div><div style="font-family:Aptos,Aptos_EmbeddedFont,Aptos_MSFontService,Calibri,Helvetica,sans-serif;font-size:12pt;color:rgb(0,0,0)" class="gmail-x_elementToProof">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.</div>
<div id="gmail-Signature" class="elementToProof"><div style="font-family:Aptos,Aptos_EmbeddedFont,Aptos_MSFontService,Calibri,Helvetica,sans-serif;font-size:12pt;color:rgb(0,0,0)"><br></div><div style="font-family:Aptos,Aptos_EmbeddedFont,Aptos_MSFontService,Calibri,Helvetica,sans-serif;font-size:12pt;color:rgb(0,0,0)">Talya Levitz, PhD</div><div style="font-family:Aptos,Aptos_EmbeddedFont,Aptos_MSFontService,Calibri,Helvetica,sans-serif;font-size:12pt;color:rgb(0,0,0)">Scientist I, Dana Farber Cancer Institute</div><div style="font-family:Aptos,Aptos_EmbeddedFont,Aptos_MSFontService,Calibri,Helvetica,sans-serif;font-size:12pt;color:rgb(0,0,0)"><i>she/they</i></div></div>
<br></div><br><div class="gmail_quote gmail_quote_container"><div dir="ltr" class="gmail_attr">On Mon, May 19, 2025 at 3:00 PM <<a href="mailto:3dem-request@ncmir.ucsd.edu">3dem-request@ncmir.ucsd.edu</a>> wrote:<br></div><blockquote class="gmail_quote" style="margin:0px 0px 0px 0.8ex;border-left:1px solid rgb(204,204,204);padding-left:1ex">Send 3dem mailing list submissions to<br>
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Today's Topics:<br>
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1. Re: Positively charged carbon grids (Benjamin Apker)<br>
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----------------------------------------------------------------------<br>
<br>
Message: 1<br>
Date: Mon, 19 May 2025 14:16:28 -0400<br>
From: Benjamin Apker <<a href="mailto:benjamin.apker@mitegen.com" target="_blank">benjamin.apker@mitegen.com</a>><br>
To: PEREZ-MORGA David <<a href="mailto:David.Perez-Morga@ulb.be" target="_blank">David.Perez-Morga@ulb.be</a>><br>
Cc: "<a href="mailto:3dem@ncmir.ucsd.edu" target="_blank">3dem@ncmir.ucsd.edu</a>" <<a href="mailto:3dem@ncmir.ucsd.edu" target="_blank">3dem@ncmir.ucsd.edu</a>><br>
Subject: Re: [3dem] Positively charged carbon grids<br>
Message-ID:<br>
<<a href="mailto:CALdUJgtror0TV7pqL84cuaYwYq4F_PSn_uimDT-GOYcr1yLKGw@mail.gmail.com" target="_blank">CALdUJgtror0TV7pqL84cuaYwYq4F_PSn_uimDT-GOYcr1yLKGw@mail.gmail.com</a>><br>
Content-Type: text/plain; charset="utf-8"<br>
<br>
Dear David,<br>
<br>
As mentioned by the others, a positive charge can be achieved either by<br>
discharging in a plasma with an amylamine atmosphere or post-discharge<br>
treatment of the grid.<br>
<br>
A quick note that the amylamine atmosphere will contaminate/coat the<br>
surfaces of your glow discharge system for future discharges. The GloQube<br>
Plus Dual-chamber System (system page here<br>
<<a href="https://urldefense.com/v3/__https://www.mitegen.com/product/glow-discharge-system-for-tem-grids/__;!!Mih3wA!FBrH2ZBiX5cmFjBrqijbiMRH8ZSVHf1bFeipwQ4ml1GDsLNy0aqyA4gNX9CWGQqU-l4H7Fxsi75_80hS-z0lV8YwmfT-$" rel="noreferrer" target="_blank">https://urldefense.com/v3/__https://www.mitegen.com/product/glow-discharge-system-for-tem-grids/__;!!Mih3wA!FBrH2ZBiX5cmFjBrqijbiMRH8ZSVHf1bFeipwQ4ml1GDsLNy0aqyA4gNX9CWGQqU-l4H7Fxsi75_80hS-z0lV8YwmfT-$</a> >) has<br>
separate chambers to accommodate alternative discharge atmospheres and<br>
avoid cross-contamination.<br>
<br>
There was an informative discussion on this topic (and whether it results<br>
in a hydrophilic or hydrophobic surface) on this forum back in 2009 (thread<br>
here <<a href="https://mail.ncmir.ucsd.edu/pipermail/3dem/2009-February/001505.html" rel="noreferrer" target="_blank">https://mail.ncmir.ucsd.edu/pipermail/3dem/2009-February/001505.html</a> ><br>
).<br>
<br>
Cheers,<br>
Ben<br>
<br>
On Mon, May 19, 2025 at 7:45?AM PEREZ-MORGA David via 3dem <<br>
<a href="mailto:3dem@ncmir.ucsd.edu" target="_blank">3dem@ncmir.ucsd.edu</a>> wrote:<br>
<br>
> Dear 3dem users,<br>
> Any advice on how to posively charge TEM carbon-film grids?<br>
> Thanks<br>
> David<br>
><br>
> David P?rez-Morga<br>
><br>
><br>
><br>
> *Molecular Parasitology Lab.<br>
> <<a href="https://urldefense.com/v3/__http://parasmol.ulb.be/__;!!Mih3wA!GCidJgEO8IR5DBtmsHlZ1xMn5tStEEgMF3x2YYQr6hsFcnpO4LeTFOMnSCWWcR_etu3hIvvHCr2PjsVOmOUFMk5JlLkK6g$" rel="noreferrer" target="_blank">https://urldefense.com/v3/__http://parasmol.ulb.be/__;!!Mih3wA!GCidJgEO8IR5DBtmsHlZ1xMn5tStEEgMF3x2YYQr6hsFcnpO4LeTFOMnSCWWcR_etu3hIvvHCr2PjsVOmOUFMk5JlLkK6g$</a>>**<br>
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><br>
> *Electron Microscopy Lab.<br>
> <<a href="https://urldefense.com/v3/__https://www.cmmi.be/microscopy__;!!Mih3wA!GCidJgEO8IR5DBtmsHlZ1xMn5tStEEgMF3x2YYQr6hsFcnpO4LeTFOMnSCWWcR_etu3hIvvHCr2PjsVOmOUFMk5KQGcYng$" rel="noreferrer" target="_blank">https://urldefense.com/v3/__https://www.cmmi.be/microscopy__;!!Mih3wA!GCidJgEO8IR5DBtmsHlZ1xMn5tStEEgMF3x2YYQr6hsFcnpO4LeTFOMnSCWWcR_etu3hIvvHCr2PjsVOmOUFMk5KQGcYng$</a>>*<br>
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<br>
-- <br>
Benjamin Apker<br>
Chief Executive Officer<br>
MiTeGen<br>
Expanding Tools for Cryo-EM:<br>
<a href="https://urldefense.com/v3/__https://www.mitegen.com/product-category/cryo-em/__;!!Mih3wA!FBrH2ZBiX5cmFjBrqijbiMRH8ZSVHf1bFeipwQ4ml1GDsLNy0aqyA4gNX9CWGQqU-l4H7Fxsi75_80hS-z0lVyLn4MYX$" rel="noreferrer" target="_blank">https://urldefense.com/v3/__https://www.mitegen.com/product-category/cryo-em/__;!!Mih3wA!FBrH2ZBiX5cmFjBrqijbiMRH8ZSVHf1bFeipwQ4ml1GDsLNy0aqyA4gNX9CWGQqU-l4H7Fxsi75_80hS-z0lVyLn4MYX$</a> <br>
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