[3dem] Weird droplets

Thu Oct 20 06:59:26 PDT 2016

Dear Sergej,

I remember seeing a picture similar to this a few years ago. Some people suggested it was dirt/oil on the grids, other suggested it was due to not glow discharging the grids long enough.

I googled my replay from a few years back and found it. Here's what I suggested a few years ago if you suspect this may be dirt or oil. When I was at the NYSBC we used to called those stains/droplets Mickey Mouse because they used to look like two little ears surrounding the hole. And we also applied the sample to the carbon side, but glow discharged for 10-30s.

If you have access to a scope that does STEM and EDS you could pop one of them in and see what those Micky Mice are made of. It usually just takes a few mins. We do this at our centre when we suspect there's something funky going on with grids or samples.

This is what I wrote back then:

When I was in grad school we used to order those grids. If I recall correctly, we used to do a wash with acetone, a wash (or two with chloroform), a wash with ethyl acetate
and a wash with diethyl ether (though I'm not sure about that one). We'd set the grids on top of a filter paper, in a glass petri dish, add a few drops of each solvent, in that order, one after each round of the
previous solvent had dried. Used to take about a day or so. We also baked them, which should've helped getting rid of any residual plastic. But that plastic was indeed a pain to dissolve. Susan Saban at
Vanderbilt showed me that protocol, perhaps she can offer further insight.

Good luck!

Mariena

PS. Now we purchase C-coated grids directly from QF that come pre-coated
with just carbon, no plastic. They work just as well and eliminated the
hassle of having to dissolve the plastic and waiting. Alternatively,
Protochips has the C-flat grids that, from what I understand, may have a
lighter coat of carbon compared to QF, and also lack the plastic. Hope
this helps.

________________________________
From: 3dem <3dem-bounces at ncmir.ucsd.edu> on behalf of bestdz at gmail.com <bestdz at gmail.com>
Sent: Wednesday, October 19, 2016 9:51:01 PM
To: Sergej Masich; 3dem at ncmir.ucsd.edu
Subject: Re: [3dem] Weird droplets

Dear Sergej,
I believe those are (almost) dried out buffer of your sample. They are black because of high concentration of salt. You observe this because you have carbon film, otherwise they will be just empty holes.
You should check your humidifier (less likely), or drastically reduce your blot time. If you are working with Vitrobot, and already have a very short blot time, say 1s, then you can try manual plunger, or concentrate your sample. Diluted samples are less viscous and thus easier to be blotted away. Also, the blot time should be much shorter when you have a carbon film.
Good luck!

Best,

Xinghong Dai
Postdoctoral Scholar
Department of Molecular and Medical Pharmacology, UCLA
+1 310 447 4330

From: Sergej Masich<mailto:Sergej.Masich at ki.se>
Sent: Wednesday, October 19, 2016 6:30 AM
To: 3dem at ncmir.ucsd.edu<mailto:3dem at ncmir.ucsd.edu>
Subject: [3dem] Weird droplets

Dear List,

I probably missed the discussion since many of you had observed this phenomenon I suppose (see attached images). This is a Quantifoil grid with additional continuous layer of thin (5 nm) carbon. The grid was glow-discharged during 120 seconds, the solution was applied to the “proper” side (facing grid-box centre), blotted and plunge-frozen in liquid ethane. Why do these droplets form? I do not see the droplets after applying solution to the opposite side of the grid. Shouldn’t the carbon surface inside the holes be homogeneously hydrophilic? The surface outside the holes seems to be completely hydrophobic. Is it due to residual plastic?

Thank you in advance.

Sergej
=============================
Sergej Masich, Ph.D.
Dept. of Cell and Molecular Biology
Karolinska Institutet
171 77 Stockholm, Sweden
tel: (+)46 - 8 - 524 873 61
mobile: (+)46 - 736 - 833 693
e-mail: Sergej.Masich at ki.se<mailto:Sergej.Masich at ki.se>
[cid:86227476-7EB8-401D-8ECE-07FA4A306FC4]
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