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<p class="MsoNormal"><span style="color:#1F497D">I just want to confirm what Moritz wrote.<o:p></o:p></span></p>
<p class="MsoNormal"><span style="color:#1F497D">We also have an EM-GP (not the GP2). The automated blotting works great. But I actually don’t always use it, because even without it, we find the EM-GP to be very reproducible. However indeed it’s the position
of grid with respect to the paper that is critical for the initial contact being reproducible. Every time I have to replace the tweezers and align a new pair in the “head” piece, I need to re-calibrate the height and distance parameters.
<o:p></o:p></span></p>
<p class="MsoNormal"><span style="color:#1F497D">My most hated job is aligning those tweezers, but its critical.<o:p></o:p></span></p>
<p class="MsoNormal"><span style="color:#1F497D">Good luck<o:p></o:p></span></p>
<p class="MsoNormal"><span style="color:#1F497D">Sharon<o:p></o:p></span></p>
<p class="MsoNormal"><span style="color:#1F497D"><o:p> </o:p></span></p>
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<p class="MsoNormal"><b>From:</b> 3dem <3dem-bounces@ncmir.ucsd.edu> <b>On Behalf Of
</b>Hunkeler, Moritz<br>
<b>Sent:</b> Monday, 6 December, 2021 15:41<br>
<b>To:</b> Rebecca Thompson <R.F.Thompson@leeds.ac.uk>; Andrei Moiseenko <postmoiseenko@gmail.com><br>
<b>Cc:</b> 3dem@ncmir.ucsd.edu<br>
<b>Subject:</b> Re: [3dem] Leica EM GP2 best practices<o:p></o:p></p>
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<p class="MsoNormal"><o:p> </o:p></p>
<p class="MsoNormal">Hi all,<o:p></o:p></p>
<p class="MsoNormal"><o:p> </o:p></p>
<p class="MsoNormal">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.
<o:p></o:p></p>
<p class="MsoNormal"><o:p> </o:p></p>
<p class="MsoNormal">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.<o:p></o:p></p>
<p class="MsoNormal"><o:p> </o:p></p>
<p class="MsoNormal">Best,<o:p></o:p></p>
<p class="MsoNormal">Moritz<o:p></o:p></p>
<p class="MsoNormal"><o:p> </o:p></p>
<p class="MsoNormal">btw: I also use exactly the term ‘full, firm and flat’ contact when training :-)<o:p></o:p></p>
<p class="MsoNormal"><o:p> </o:p></p>
<p class="MsoNormal"><o:p> </o:p></p>
<p class="MsoNormal"><o:p> </o:p></p>
<p class="MsoNormal"><o:p> </o:p></p>
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<p class="MsoNormal"><b><span style="font-size:12.0pt;color:black">From: </span></b><span style="font-size:12.0pt;color:black">3dem <<a href="mailto:3dem-bounces@ncmir.ucsd.edu">3dem-bounces@ncmir.ucsd.edu</a>> on behalf of Rebecca Thompson <<a href="mailto:R.F.Thompson@leeds.ac.uk">R.F.Thompson@leeds.ac.uk</a>><br>
<b>Date: </b>Monday, December 6, 2021 at 05:20<br>
<b>To: </b>Andrei Moiseenko <<a href="mailto:postmoiseenko@gmail.com">postmoiseenko@gmail.com</a>><br>
<b>Cc: </b>"<a href="mailto:3dem@ncmir.ucsd.edu">3dem@ncmir.ucsd.edu</a>" <<a href="mailto:3dem@ncmir.ucsd.edu">3dem@ncmir.ucsd.edu</a>><br>
<b>Subject: </b>Re: [3dem] Leica EM GP2 best practices<o:p></o:p></span></p>
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<p class="MsoNormal"><o:p> </o:p></p>
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<p><b><span style="color:white;background:red"> External Email - Use Caution </span></b><o:p></o:p></p>
<p class="MsoNormal">Hi Andrei, <o:p></o:p></p>
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<p class="MsoNormal"><o:p> </o:p></p>
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<p class="MsoNormal">We have the EM GP but I have used the GP2 and hopefully this helps;<o:p></o:p></p>
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<p class="MsoNormal"><o:p> </o:p></p>
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<p class="MsoNormal">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. <o:p></o:p></p>
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<p class="MsoNormal"><o:p> </o:p></p>
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<p class="MsoNormal">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. <o:p></o:p></p>
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<p class="MsoNormal"><o:p> </o:p></p>
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<p class="MsoNormal">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.<o:p></o:p></p>
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<p class="MsoNormal"><o:p> </o:p></p>
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<p class="MsoNormal">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. <o:p></o:p></p>
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<p class="MsoNormal"><o:p> </o:p></p>
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<p class="MsoNormal">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)<o:p></o:p></p>
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<p class="MsoNormal"><o:p> </o:p></p>
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<p class="MsoNormal">Hope this helps- good luck with your optimisation. <o:p></o:p></p>
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<p class="MsoNormal"><o:p> </o:p></p>
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<p class="MsoNormal">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 <a href="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=DwMGaQ&c=-35OiAkTchMrZOngvJPOeA&r=L7-zyQ-04fFCMRqzLIOnx7H0exGZHwIQe_wMPuY600I&m=OtggXvQDbjHEoUQWNgchtCIICf4_v9B4ZYkL2ZX3uAHph3Z-1Hny-jO8YuLdFFyp&s=LIZQNqN55HqVy9mFynPD9kt5NUYY27xNelzdAFlB2aY&e=">here</a>. <o:p></o:p></p>
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<p class="MsoNormal"><o:p> </o:p></p>
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<p class="MsoNormal">Best wishes,<o:p></o:p></p>
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<p class="MsoNormal">Becky<o:p></o:p></p>
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<p class="MsoNormal"><o:p> </o:p></p>
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<p class="MsoNormal"><span style="color:black">______________________________________________________________<br>
<br>
Rebecca Thompson (She/her)<br>
Head of Faculty Biological Sciences Research Facilities<br>
Deputy Director, Astbury Biostructure Laboratory Electron Microscopy <br>
<br>
University of Leeds<br>
Phone 0113 3433384/3438957 (office/Titan Krios control room)<br>
Mobile 07816179813<br>
Location Roger Stevens 5.40<br>
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Sometimes I may send emails out of 'normal’ core working hours. I do not expect you to read, or respond outside of your own working hours!<o:p></o:p></span></p>
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<p class="MsoNormal"><span style="color:black"><o:p> </o:p></span></p>
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<p class="MsoNormal"><span style="color:black"><o:p> </o:p></span></p>
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<p class="MsoNormal"><o:p> </o:p></p>
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<p class="MsoNormal" style="margin-bottom:12.0pt"><o:p> </o:p></p>
<blockquote style="margin-top:5.0pt;margin-bottom:5.0pt">
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<p class="MsoNormal">On 4 Dec 2021, at 08:55, Andrei Moiseenko <<a href="mailto:postmoiseenko@gmail.com">postmoiseenko@gmail.com</a>> wrote:<o:p></o:p></p>
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<p class="MsoNormal"><o:p> </o:p></p>
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<p class="MsoNormal">Dear all,<o:p></o:p></p>
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<p class="MsoNormal">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.<span class="apple-converted-space"> </span><o:p></o:p></p>
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<p class="MsoNormal">Specifically:<o:p></o:p></p>
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<p class="MsoNormal" style="text-indent:-.25in">1.<span style="font-size:7.0pt;font-family:"Times New Roman",serif"> <span class="apple-converted-space"> </span></span>Do you use the blotting sensor? In my case it fails very often and it seems that the
results are better without it.<span class="apple-converted-space"> </span><o:p></o:p></p>
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<p class="MsoNormal" style="text-indent:-.25in">2.<span style="font-size:7.0pt;font-family:"Times New Roman",serif"> <span class="apple-converted-space"> </span></span>In case you use the blotting sensor, what range of “additional movement” do you use?<o:p></o:p></p>
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<p class="MsoNormal" style="text-indent:-.25in">3.<span style="font-size:7.0pt;font-family:"Times New Roman",serif"> <span class="apple-converted-space"> </span></span>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?<o:p></o:p></p>
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<p class="MsoNormal" style="text-indent:-.25in">4.<span style="font-size:7.0pt;font-family:"Times New Roman",serif"> <span class="apple-converted-space"> </span></span>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.<o:p></o:p></p>
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<p class="MsoNormal" style="text-indent:-.25in">5.<span style="font-size:7.0pt;font-family:"Times New Roman",serif"> <span class="apple-converted-space"> </span></span>What range of blotting time and specimen volume do you typically use for SPA samples
at conditions specified?<o:p></o:p></p>
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<p class="MsoNormal">Any advice on using the device is very welcome.<o:p></o:p></p>
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<p class="MsoNormal"> <o:p></o:p></p>
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<p class="MsoNormal" style="margin-bottom:10.0pt;line-height:12.65pt">Best regards,<br>
Andrey Moiseenko<o:p></o:p></p>
<p class="MsoNormal" style="margin-bottom:10.0pt;line-height:12.65pt">Electron Microscopy Lab @ Biology Department<br>
Moscow State University<br>
<a href="mailto:postmoiseenko@gmail.com">postmoiseenko@gmail.com</a><o:p></o:p></p>
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<p class="MsoNormal"> <o:p></o:p></p>
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<p class="MsoNormal"><span style="font-size:9.0pt;font-family:"Helvetica",sans-serif">_______________________________________________<br>
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