[3dem] Leica plunger users

[Rebecca Thompson]

Hi Olga,

Should point out there are two versions of this currently out there- the EM GP and EM GP 2. We have the ‘original’ EM GP, ours had no problems reaching 4 °C (would get there in 15-20 mins from RT), but as Denis has rightly pointed this then causes lots of problems with icing up and the arm not moving properly after a short period of time. We run ours at 8 °C as standard and its fine like this. The EMGP 2 (we have had on trial and I’ve taught on course using it) doesn’t seem to suffer from this so much and has a nice drainage port at the back which helps a little to drain away any water that does accumulate (although isn’t perfect, still seems to accumulate).

With the humidity, we have also seen this, Denis’s trick of unscrewing the water tank has worked well on occasions for us, but once the humidity sensor needed replacing so if it continues worth a call to Leica! Can be worth sticking a probe in the side to independently measure the humidity, this will give you an indication of what is really going on.

We have both the Leica EM GP and Vitrobot Mv IV. For single particles in our hands both are capable of performing equivalently, with some tweaking, so I don’t say one is much better than the other for single particles (and users are quite evenly spread in our facility), but the Leica does have some useful features that can help with some projects;
1) You can choose front blotting (filter paper contacts meniscus directly) or back blotting (sample drawn through the grid), which can give you some useful options if you want to concentrate large samples such as filaments of bacterial cells onto the grid. But for most single particle projects we do front blotting.  If you are growing eukaryotic cells onto grids the back blotting option is perfect.
2) I’ve found the sensor totally useless on the EM GP, it seems a little better on the GP2 but we still seemed to get more reproducible results without using it.
3) Whatever blotting modality you are using, I recommend setting the arm so it makes full, firm and flat contact with the grid, but without bending/flexing the grid at all. We typically use blotting times of 3-5 seconds for single particles using Whatman 1 paper. I also like to position the tweezer arm more towards the centre of the filter paper circle, this helps to iron out some of the irreproducibility caused when the filter paper ‘ruffles’ after a short period in the humid atmosphere.

Hope this helps, and happy to provide some more info if it would be useful :) Good luck!

Best wishes,
Becky


________________________________________________
Rebecca Thompson
Senior cryo-Electron Microscopy Support Scientist/Facility manager
Astbury Biostructure Laboratory
University of Leeds

Email r.f.thompson at leeds.ac.uk<mailto:r.f.thompson at leeds.ac.uk>
Phone 0113 3438957/3438959 (office/Titan Krios control room)
Mobile 07816179813
Location Roger Stevens 5.40
Linkedin   https://uk.linkedin.com/in/1rebeccathompson
Twitter  Bex_16

On 19 Jun 2019, at 20:55, Denis Chrétien <denis.chretien at univ-rennes1.fr<mailto:denis.chretien at univ-rennes1.fr>> wrote:


The one I have in my possession now did not cool down to 4C for nearly 3 hrs,

We have now been using the EM GP for several years. At 4 °C, we faced problems of ice cristallisation blocking the trap and arm systems. It was « solved » by Leica engineers by increasing the lower temperature that the device can reach (I don’t remember, between 5 and 7 °C). This setting can be modified in the service menu if you have the code. One way to overcome this problem if it happens is to increase the temperature for a short time so that the ice melts and then come back to 4 °C (after removing water…). Don’t let the water accumulate and remove it periodically.

and the humidity was going up and down.

Same problem from time to time … One way we overcome this problem is to unscrew the water tank so that air can circulate more freely inside the device.

Is it the normal behaviour?

I won’t say « normal », but yes it can behaves like that :-(

Also, what is the good regime for protein solutions blotting? Could it be compared to Vitrobot?

No comparison, I never used a Vitrobot. Nevertheless, what’s very cool with the Leica is the temperature control of the liquid ethane. We set it to -184 °C and it can last hours at this temperature without refilling.

Hope that helps, waiting also for the feedback of other users,

Denis

Thank you!
Olga


=======
Yours sincerely,
Olga Sokolova, PhD, D.Sc.
Professor RAS, Faculty of biology Moscow State University
phone +7-495-938-0005
cell +7-916-4841251
http://proteom-msu.org/en/index.html
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