Freezing on carbon coated grid

Hey Guys

I am trying to freeze my virus sample on a Quantifoil R1.2/1.3 grid with 2nm thin Carbon film, as it wasn’t coming in the holes on a holey carbon grid.
While freezing the carbon film broke but I am seeing the particles in the holes.

Can I collect a data set from this grid? Please see the images for reference.


Likely the thin carbon broke during glow discharge - it can happen.

Given there are particles in your holes, and ice looks fine, why not? I would collect!

Quite an unusual particle distribution - in your low mag it looks like in a lot of cases your particles form a distinct ring, I guess it likes a very specific ice thickness!



Thank you @olibclarke for the quick response.


As @olibclarke says, I would collect as well. Concentration is a bit low, but run a Live job to monitor and see how acquisition is going, I would think you’ll end up with something worthwhile. For icosahedral viruses which are structurally rigid it doesn’t take many particles to get to high resolution. :smiley: Then you can play with concentration later if you decide the acquisition isn’t optimal.

GIven particle distribution in the hole, and how few particles there appear to be, I’d be tempted to acquire at lower magnification (to image as much of the hole as possible) and if high resolution is achieved there is a way to exceed that… :smiley:

Good luck and welcome to the forum. :smiley:

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Thanks :blush:
Will start with the collection then.


Hey @rbs_sci and @olibclarke

I Did a glow discharge for 45s, negative polarity, 20mA on this grid on our ‘Quorum 150 ES plus’ but the film broke.
But I have seen papers where groups have glow discharged for 1 minute as well.
Can this be a problem with the grid or shall I decrease the GD time.
Can you suggest some initial condition for GD that I can start with.

Decrease glow discharge time. :slight_smile: Our lab rarely goes over 30 seconds.

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If this is the commercial “2nm CC” type of grid, the film is very easy to damage, as you your micrographs. Everyone is correct in saying to reduce glow discharge time, but in some cases the shorter times may not be enough. Also, if these are auto grids is the carbon being further damaged by clipping and handling?You could get away with home-sourced thicker carbon since it is a virus. If you concentration is so low then yes, it will be hard to get this without continuous support. Some Negative stain could help find a good concentration range. Alternatively, graphene oxide should not need any glow discharge. Some tools in Cryosparc’s curation are helpful for selecting micrographs that have a mix of support vs. non/damaged support - we have had this in a few data sets.

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Hey @Mark-A-Nakasone
I am collecting the data on broken parts as suggested by @olibclarke and @rbs_sci.
But I just want to know the problem associated with the carbon film. The sample concentration is very good. The problem is it’s not coming inside the holes. I am sharing a reference image where I froze the sample on normal R1.2/1.3 Cu grid without any carbon film.


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agreed @Kit1234567890 - seems that your virus behaves well on the carbon and some even appear to be falling apart in the hole. Some protein people would brute force it by increasing the concentration to force some into the hole, however in your case finding a better way to work on carbon would be best. I hope collection goes well and the high symmetry works in your favor.


I definitely think Mark’s suggestion regarding using a thicker film might be the quickest solution, even if it isn’t 100% optimal. If you can even out particle distribution a bit, increasing the acquired particles will likely compensate for any loss from the thicker carbon film. We have a lot of samples which really like carbon too; a lot of viruses do. Also as he suggests, increasing concentration might be a brute force route. Your virus is, what, 25-30nm? Another possibility might be decreasing blotting time and searching for those areas where the ice is a little thicker… I’m less concerned about thick ice than many, as a number of samples we work on guarantee that ice is going to be 300nm+… :sweat_smile:


You can try all sorts of tricks, one thing that has worked well for me in the past was increasing salt concentration in the buffer. You could also try a gold grid. Or maybe 2x sample applications and blots on the grid (apply sample, blot, apply more sample, blot again, then freeze). All of those have worked, depending on the sample.


@rbs_sci the thicker film could be the easier. I just mention it because I used to work with SCMI/MRC-CVR and they always had their own carbon on mica. Yes it was thicker than 2nm, but it never had the issues of commercial 2nm CC grids. If you have your own coater it could be worth it, otherwise you can ask around if someone could mail/“post” you some carbon on mica or even order some. Then using your holey carbon Quantifoil you can float the carbon on. With a large virus and your ice thickness I think you should be safe.

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