[3dem] CP3 versus EM-GP for SPA

Rebecca Thompson R.F.Thompson at leeds.ac.uk
Wed Sep 20 03:12:44 PDT 2017

Hi Laura,

We have the Vitrobot mk 4 and Leica EM GP, I dont have any experience with the Gatan Cp3. Our facility has a mix of users and we prep everything from macromolecular complexes/viruses/filaments/liposomes/organelles/cells, but the majority of users are working on SPA projects. In our experience;

- Users are split 50/50 between the vitrobot and Leica. The only type of projects where users have a preference for one machine over the other is preparing cells (they go for back side blotting on Leica).
- I’ve prepared all sorts of single particle grids on the Leica and never had any problems getting good ice within a couple of iterations, and most samples I can guess conditions well enough first time (although the sample doesn’t always look good ;) )
- Some thoughts on the Leica

  *   I really like the little ethane bucket, which is quick to fill and has temperature control.
  *   As others have said, the Leica is definitely less reproducible than the Vitrobot, both within the same batch of grids and between different sessions. As the filter paper is unsupported, very slight ‘ruffles’ in the filter paper add another variable to blot time and the distance the arm moves/ position of the grid relative to filter paper (if you leave the filter paper in the high humidity environment it ‘ruffles’ more so we tend to replace filter paper every few grids). You have to get to know the system a bit to adjust for these variables.
  *   (Annoyingly…) It seems every machine is different, but I find with ours, in terms of a range of blot times that gives good results, there is a substantially bigger range on the Leica (Blot times of 0.5-6 seconds may be be useful depending on sample/buffer), compared with the vitrobot (Majority of things look good with a 5.5-6.5 s blot). You’re much more likely to get conditions completely wrong and get grids which are entirely dry/too thick on the Leica, compared with the vitrobot.
  *   Having said that, I personally, I slightly prefer the Leica over the vitrobot because when you do get the conditions right you can get lovely ice over almost the whole grid, whereas on the Vitrobot we tend to get more of a gradient of ice (but this can be handy if you are just starting and have no idea where to start!). I also find I can set it up a little faster.
  *   Long bake out time is annoying- we use a minimum of 1 hour bake (after the nitrogen has evaporated) so you have to leave at least 2 h between sessions. Having said that, we have a lovely 20 % RH freezing room, and it is not uncommon for 2 or even 3 users to use the Leica back to back (over a 2-3-4 h period) and experience no noticeable problems with increased surface contamination, so we just have to be a bit organised when people make bookings.
  *   Both the Leica and the Vitrobot humidifiers have significant room for improvement, perhaps the Gatan model is better..

Hope that helps :) Good luck with your purchase!
Rebecca Thompson
Senior cryo-Electron Microscopy Support Scientist/Facility manager
Astbury Biostructure Laboratory
University of Leeds

Email fbsrft at leeds.ac.uk<mailto:fbsrft at leeds.ac.uk>
Phone 0113 3438957/3438959 (Office/Titan Krios control room)
Location Astbury 8.105
Linkedin   https://uk.linkedin.com/in/1rebeccathompson
Twitter  Bex_16

On 19 Sep 2017, at 21:24, Mariena Silvestry Ramos <ms3289 at cornell.edu<mailto:ms3289 at cornell.edu>> wrote:

Hi all,

Kim: I haven't used the Leica set up but have used a couple of iterations of the Vitrobot (mk 2, 3 and now 4) and the Cp3. I like them all and honestly wish I could have a mix of all the cool features they offer.

Like Wim said, the newer Vitrobot does away with the gas hookup and external PC. The names of a few features (grid offset in older systems) changed to something else (can't remember exactly). But I like that it's simple yet robust. It's literally a take out of the crate, hook up some things kinda system. I still prefer the old humidifier as I liked to see my water bubbling ... but that's possibly just me. I'm having some issues with moving our Vitrobot here at Cornell from a newer building to an older one because (and I kid you not) the city of Ithaca and the fire code need certain features in our (really old) room to be installed if we want to avoid fires (seriously, 5-7mL of liquid ethane and no flames present in our lab and still they want fancy ventilation stuff).

I have a couple of users that have bought the EMS plunging device. They generally like it except for the placement of the button to dunk the sample in ethane which honestly it's a really bad place to put it, but hey, I wasn't asked to help design the thing 😉. It doesn't have external hookups.

The Cp3 (probably a similar model to yours) I used to use when I was at NY Structural worked very well and gave consistent results, IMHO. We did have the misfortune that the heating element go bad, but Bill Rice, Ed and I worked on having an external one installed. That was way back in 2012 or so, so maybe Gatan has made a better design. Alternatively, any good machine shop or even a good engineering department could help you design a heater if you want. It also depends on the cryogen you use. I love liquid ethane because it was what I used in grad school and know, but other people use the ethane/propane mix (and avoid the freezing of the liquid), and yet others use propane. It's ultimately your choice. I have no problem with dunking a pair of tweezers in the ethane cup and liquefying things when needed, but I know some other folks don't like doing that. I've frozen viruses, cell organelles, single particles and other materials in all of these devices and they work with a few adjustments here and there.

Overall I recommend the Vitrobot and the Cp3, so either one would be a good option, IMO.

Best wishes on your purchase,


From: 3dem <3dem-bounces at ncmir.ucsd.edu<mailto:3dem-bounces at ncmir.ucsd.edu>> on behalf of Wim Hagen <hagen at embl.de<mailto:hagen at embl.de>>
Sent: Tuesday, September 19, 2017 3:28:06 PM
To: Laura Kim
Cc: 3dem
Subject: Re: [3dem] CP3 versus EM-GP for SPA

Hi Laura,

We use Leica, Vitrobot Mark 2 and Mark 4. I can confirm Mike's experience:
Leica is flexible but not very reproducible from session to session, things need to be set and checked each time one uses it. We also have problems with the humidifier.

The Vitrobot Mark 2 humidifier died and unfortunately I can't find the original €25  terrarium humidifier model anymore on the Interwebs... (the official FEI quote was several hundreds €).

The Vitrobot Mark 4 behaves fine and people seem to like it for its reproducibility.

Other than the backside blotting I can't name one thing that makes the Leica favorite for certain things here, but I see people experiment on the Leica more, the we-used-to-be-crystallographers crew just want grids and mainly use the Vitrobot.

My personal views:

Very flexible to play with the simplest to the craziest ideas, not so good session-to-session reproducibility, warm-up takes too long for multi-user facilities, no gas hookup, cheap tweezers and a tweezer alignment tool so you can replace tweezers yourself.
I really like its design and features... if they would work well.

Vitrobot Mark 4:
Reproducible, only double side blotting, no gas hookups (they ditched that after the Mark 3), VERY expensive tweezers (for the reproducibility reason).
Once adjusted properly it does one job but it does it well.

One other thing:
Ethane volume on Leica is smaller and our German safety officer says it then does not have to be in a fume-hood, the Vitrobot however can't be outside the fume-hood, could be a German thing, and I could ask our workshop to make a Vitrobot ethane cup with smaller ethane volume.
Just saying this because I think freezing outside the fume-hood is much cleaner, but the true veterans here however also freeze very clean in the (da) hood.

No experience with Gatan but I would love to get my hands on one because I like its features.
What's the main reason you debate/consider changing?


Wim Hagen
EMBL Heidelberg

On Sep 19, 2017, at 20:07, Mike Strauss <mikestrauss13 at crystal.harvard.edu<mailto:mikestrauss13 at crystal.harvard.edu>> wrote:

Hi Laura,

we have a Leica system for plunging. Most of our projects are SPA, and the results are fine.  It takes some getting used to, and the results seem to be considerably less consistent than the Vitrobot. Once you get to know your system, and learn to adjust the various positions and offsets, it behaves well enough.

One thing I would warn you of is the long bake-out time on the Leica plunger.  The cryogen area is inside the system, and cannot be accessed by the user in any practical way, so if you want to have multiple users in the day, they need to be right after one another, or well-separated.  Our system has also had troubles with the humidifier unit, where the (distilled) water level has to be perfect, otherwise it will not work properly.  But this too is a matter of getting to know your system.

I have very little practical experience with the CP3, so I can't comment.


On Mon, Sep 11, 2017 at 5:10 PM, Laura Kim <kimyaunhee at gmail.com<mailto:kimyaunhee at gmail.com>> wrote:
Hi 3DEM,

We are replacing our Gatan CP3, which is quite old and on its way out. We are debating between the purchase of a replacement Gatan CP3 or a Leica EM-GP for our cryo-EM facility, where the majority of projects are single particle. We like that the EM-GP has temperature control and does not require gas hookups, but have heard that most labs using the EM-GP are doing filament-type projects and not SPA. Can any labs using the EM-GP for SPA comment on this? Do people have a preference for one over the other for SPA? Please let me know, thanks.

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