[3dem] Leica EM GP2 best practices

Mon Dec 6 05:41:24 PST 2021

Hi all,

my experience is only on the EM-GP, not GP2, but I would like to echo Guenter’s reply. I would recommend using the blot sensor. The reason (in our case, when we encountered these problems initially) for the sensor not detecting the wetting of the paper properly (resulting in ‘overbending’ of the grid – sometimes 90º bends) was that we had set the grid too low in respect to the paper. The sensor only works properly when the top rim of the grid is aligned with the top of the filter paper. This does indeed lead to a problem that sometimes (depending on how wrinkly the paper is) every second blot is a bit weaker then every other (since the paper wrinkles in a frequency of about 1 grid/blot). We either use that as an additional screening step (:-)), discard too weak blots (they are easily spotted through the microscope), changing the paper more often, or skipping every position where the paper wrinkles back (by looking at the paper before the blot you can anticipate whether it will be a weak or a strong blot). Leica might argue that the blotting sensor should exactly overcome the problem of the wrinkly paper, but it clearly doesn’t, since the ‘position’ setting (210-220) basically sets the arrival speed of the paper on the grid. The arm slows down before it’s predetermined stopping point, and so depending on whether the paper already made contact (which also leads to slowing down) or not, the speed and will be different, and thus the blotting force will effectively be different. However, in our/my hands that’s still much more consistent than manual blotting. Alternatively, the position can be set to a pretty high level, but some people don’t like the very strong resulting blotting force.

Again, this might all be very different on the EM-GP2, though. But I am always interested to hear other people’s experiences with the EM-GP, since they are very divergent. In our hands, the EM-GP is very reproducible, and I just love the handling (filling the ethane cup in seconds, ethane not freezing over in long sessions, blot sensor). We had some problems with our unit not being able to hold the set humidity, but this got fixed. My go-to starting condition is 10s pre-blot, 3s blot, 1-3s post-blot, front side, 4 µl sample, 90%, 10 ºC.

Best,
Moritz

btw: I also use exactly the term ‘full, firm and flat’ contact when training :-)

From: 3dem <3dem-bounces at ncmir.ucsd.edu> on behalf of Rebecca Thompson <R.F.Thompson at leeds.ac.uk>
Date: Monday, December 6, 2021 at 05:20
To: Andrei Moiseenko <postmoiseenko at gmail.com>
Cc: "3dem at ncmir.ucsd.edu" <3dem at ncmir.ucsd.edu>
Subject: Re: [3dem] Leica EM GP2 best practices

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Hi Andrei,

We have the EM GP but I have used the GP2 and hopefully this helps;

1) My personal experience with the blotting sensor on the GP is that it fails- I didnt have much better experience with the GP2- however we held a course last week where a participant reported very good consistent results- but personally I cannot recommend.

3) This is subtly different if you plan front blot or back blot of the grids. The phrase I use when training users is ‘full, firm and flat’ contact- if doing back blotting this typically needs to be a shade ‘firmer’ in contact and benefits from 0.5 ul being on the back of the grid to ensure the liquid draws through the grid.

The tweezers should not cause the grid to bend. Be aware- With the GP, the movement of the arm between the settings menu and main workflow is annoyingly slightly different-so optimise. You can check by running an actual cycle on the main menu and you may have to adjust again- ours the actual motion tends to be 1-3 mm short, so it looks like the contact is too firm on the settings menu but just right when you actually run the cycle. May not be a problem with the GP2 but one to watch.

4) My recommendation is always to position the grid further down towards the centre of the filter paper for precisely the reason you state, this helps to over come the wrinkling. I do also recommend replacing the filter paper fairly often (before it becomes too wrinkly) this also improves reproducibility.

5) I typically use 2.5-4s, ‘front blotting’ for SPA projects with 3ul. Front blotting tends to be a bit more reproducible in my hands, and I only use back blotting if I have a need (eg cells on grids, trying to concentrate filaments onto a grid)

Hope this helps- good luck with your optimisation.

For anyone interested in learning more on sample preparation and also data acquisition, you can check out the (presently remote) courses we are running alongside other partner sites in the UK here<https://urldefense.proofpoint.com/v2/url?u=https-3A__secure-2Dweb.cisco.com_1qLgk-2DkuCXnxRGcYss1gHQUXEiobjbK0z-5FkignjINLwPjStTOT68xLVQUA-2DDg7-5FjA53I2xGNEIs5RfuCLB5QL9TZMefT-2DQ19wiyxgfgnq4rfNXAn-5FM06QCaxo42rXAyRq5XXiONUUnDhKoRy9jmXX-5FQhaVpgceWmrHw-2D2BHQ0Ig7a329QDI2YL-2DsMizkUy28eM2q2rbSkhc8vlMYlDQXDXOw2v54gCgAWFLqWy-2De4PeRiOa6w7dVz5OgiFMlw7K8rG9OwqOt-2DjShhH4LMUrGoxfW5bbf1q3Ywaz-5FcG-2Da4puJZ-2DjRPSChI1zjzi9NrblvYnG76CYoecUKe1OjLfBmzlg_https-253A-252F-252Furldefense.proofpoint.com-252Fv2-252Furl-253Fu-253Dhttps-2D3A-5F-5Fastbury.leeds.ac.uk-5Ffacilities-5Fwellcome-2D2Dmrc-2D2Dtraining-2D2Din-2D2Dcryoem-5F-2526d-253DDwMGaQ-2526c-253D-2D35OiAkTchMrZOngvJPOeA-2526r-253DL7-2DzyQ-2D04fFCMRqzLIOnx7H0exGZHwIQe-5FwMPuY600I-2526m-253DajUw8T5Tk31Hnw2msYTMk39Y6cJZZoWrDhQTMgR46utGxcjT6ZaJQ9WATpjMlmdJ-2526s-253DOpyqv5UQUZPBhnuTtoJF8sxddwgQiQssApfxS7aUqgU-2526e-253D&d=DwIGaQ&c=-35OiAkTchMrZOngvJPOeA&r=L7-zyQ-04fFCMRqzLIOnx7H0exGZHwIQe_wMPuY600I&m=OtggXvQDbjHEoUQWNgchtCIICf4_v9B4ZYkL2ZX3uAHph3Z-1Hny-jO8YuLdFFyp&s=LIZQNqN55HqVy9mFynPD9kt5NUYY27xNelzdAFlB2aY&e= >.

Best wishes,
Becky

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On 4 Dec 2021, at 08:55, Andrei Moiseenko <postmoiseenko at gmail.com<mailto:postmoiseenko at gmail.com>> wrote:

Dear all,
I am trying to get reproducible results with Leica EM GP2. I would be very grateful if you could share any successful best practices on using this plunge freezer.
Specifically:
1.       Do you use the blotting sensor? In my case it fails very often and it seems that the results are better without it.
2.       In case you use the blotting sensor, what range of “additional movement” do you use?
3.       In case you use manual blotting, how do you set the horizontal blot position: just by touching the grid with the filter paper, or slightly bending it?
4.       How do you set the filter paper vertical position: covering the grid only, just touching the forceps tips or higher (how much higher)? It seems that setting the higher position helps to reduce the effects of the filter paper crinkling.
5.       What range of blotting time and specimen volume do you typically use for SPA samples at conditions specified?
Any advice on using the device is very welcome.

Best regards,
Andrey Moiseenko
Electron Microscopy Lab @ Biology Department
Moscow State University
postmoiseenko at gmail.com<mailto:postmoiseenko at gmail.com>

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