[3dem] Re: specimen preparation (Kimberley Cheng)

L.E.Franken l.e.franken at rug.nl
Thu Nov 7 05:07:46 PST 2013 

Hi Kimberley,
I had trouble with ice-thickness as well, we found out that our brand of blotting paper had changed. You can try several blot papers, there are papers that are faster at blotting and others that are slower, this also changes the strength of blotting and helps better than simply changing the blotting time (which did not work in our case).

with kind regards,
Linda Franken


On 06-11-13, 3dem-request at ncmir.ucsd.edu wrote:
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> Today's Topics:
> 
>  1. specimen preparation (Kimberley Cheng)
>  2. Re: specimen preparation (Bob Grassucci)
>  3. RE: specimen preparation (Bertin Aurelie)
>  4. Where to find info on REC-files in order to handle them in
>  MATLAB? (Charlotte Hamngren Blomqvist)
> 
> 
> ----------------------------------------------------------------------
> 
> Message: 1
> Date: Wed, 6 Nov 2013 13:34:43 +0000
> From: Kimberley Cheng <Kimberley.Cheng at ki.se>
> Subject: [3dem] specimen preparation
> To: "3dem at ncmir.ucsd.edu" <3dem at ncmir.ucsd.edu>
> Message-ID:
> 	<B491E5EE09A76243BE86D6F4F03B022287A0E5E4 at KIMSX02.user.ki.se>
> Content-Type: text/plain; charset="iso-8859-1"
> 
> Dear all,
> 
> I have difficulties preparing cryoEM specimens of my samples, and I would appreciate any help I can get. Both samples contain lipid nanoparticles, and I use a Vitrobot to prepare the frozen-hydrated specimens. The problems are the following
> 
> Sample 1: Majority of the grid has no ice at all. If I am lucky, a small region of the grid (1/4 of a grid square) is covered with ice that is very thick. What can be the problem? Any advice on how to improve the procedure?
> 
> Sample 2: Ice thickness and particle distribution is good. The concentration is, however, very low. Any suggestions on how to increase the on-grid particle concentration? I have already tried preparing the sample on continuous carbon instead of perforated carbon film.
> 
> I am very interested in hearing your opinions in this.
> 
> Thanks in advance,
> Kimberley Cheng
> 
> 
> 
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> ------------------------------
> 
> Message: 2
> Date: Wed, 06 Nov 2013 09:28:10 -0500
> From: Bob Grassucci <rg2502 at columbia.edu>
> Subject: Re: [3dem] specimen preparation
> To: 3dem at ncmir.ucsd.edu
> Message-ID: <527A51FA.8050707 at columbia.edu>
> Content-Type: text/plain; charset=ISO-8859-1; format=flowed
> 
> Hi Kimberly,
> Which Version of the Vitrobot do you have? With the Mark IV model they 
> changed the mechanism that adjusts the blot force. They now adjust the 
> distance that the blotting pads from each other and unfortunately this 
> can change over time. You want to blot with a more positive force and 
> if that does not work blot longer. This should help with sample 1.
> 
> For sample 2 to get an increase in sample concentration increase the 
> wait time. We use 30 seconds for most of our ribosomes . We use thin 
> carbon over the holes. I hope these suggestions helpRegards,
> Bob
> 
> On 11/6/2013 8:34 AM, Kimberley Cheng wrote:
> > Dear all,
> >
> > I have difficulties preparing cryoEM specimens of my samples, and I 
> > would appreciate any help I can get. Both samples contain lipid 
> > nanoparticles, and I use a Vitrobot to prepare the frozen-hydrated 
> > specimens. The problems are the following
> >
> > Sample 1: Majority of the grid has no ice at all. If I am lucky, a 
> > small region of the grid (1/4 of a grid square) is covered with ice 
> > that is very thick. What can be the problem? Any advice on how to 
> > improve the procedure?
> >
> > Sample 2: Ice thickness and particle distribution is good. The 
> > concentration is, however, very low. Any suggestions on how to 
> > increase the on-grid particle concentration? I have already tried 
> > preparing the sample on continuous carbon instead of perforated carbon 
> > film.
> >
> > I am very interested in hearing your opinions in this.
> >
> > Thanks in advance,
> > Kimberley Cheng
> >
> >
> >
> >
> >
> > _______________________________________________
> > 3dem mailing list
> > 3dem at ncmir.ucsd.edu
> > https://mail.ncmir.ucsd.edu/mailman/listinfo/3dem
> 
> 
> 
> 
> ------------------------------
> 
> Message: 3
> Date: Wed, 6 Nov 2013 14:32:05 +0000
> From: Bertin Aurelie <aurelie.bertin at curie.fr>
> Subject: RE: [3dem] specimen preparation
> To: Bob Grassucci <rg2502 at columbia.edu>,	"3dem at ncmir.ucsd.edu"
> 	<3dem at ncmir.ucsd.edu>
> Message-ID:
> 	<25C7D31FB8DE01458930977F9C6063B861E23AFB at mbxparis02.recherche.curie.fr>
> 	
> Content-Type: text/plain; charset="iso-8859-1"
> 
> Dear Kimberly,
> Maybe you have an issue with the glow discharge conditions. The inhomogeneity (point 1) might result from that.
> For point 2, you can try to incubate your sample in a humid athmosphere (wet closed box) for several minutes up to an hour before freezing it.
> Best
> Aurelie
> 
> -----Message d'origine-----
> De�: 3dem-bounces at ncmir.ucsd.edu [mailto:3dem-bounces at ncmir.ucsd.edu] <3dem-bounces at ncmir.ucsd.edu]> De la part de Bob Grassucci
> Envoy��: mercredi 6 novembre 2013 15:28
> ��: 3dem at ncmir.ucsd.edu
> Objet�: Re: [3dem] specimen preparation
> 
> Hi Kimberly,
> Which Version of the Vitrobot do you have? With the Mark IV model they changed the mechanism that adjusts the blot force. They now adjust the distance that the blotting pads from each other and unfortunately this can change over time. You want to blot with a more positive force and if that does not work blot longer. This should help with sample 1.
> 
> For sample 2 to get an increase in sample concentration increase the wait time. We use 30 seconds for most of our ribosomes . We use thin carbon over the holes. I hope these suggestions helpRegards, Bob
> 
> On 11/6/2013 8:34 AM, Kimberley Cheng wrote:
> > Dear all,
> >
> > I have difficulties preparing cryoEM specimens of my samples, and I 
> > would appreciate any help I can get. Both samples contain lipid 
> > nanoparticles, and I use a Vitrobot to prepare the frozen-hydrated 
> > specimens. The problems are the following
> >
> > Sample 1: Majority of the grid has no ice at all. If I am lucky, a 
> > small region of the grid (1/4 of a grid square) is covered with ice 
> > that is very thick. What can be the problem? Any advice on how to 
> > improve the procedure?
> >
> > Sample 2: Ice thickness and particle distribution is good. The 
> > concentration is, however, very low. Any suggestions on how to 
> > increase the on-grid particle concentration? I have already tried 
> > preparing the sample on continuous carbon instead of perforated carbon 
> > film.
> >
> > I am very interested in hearing your opinions in this.
> >
> > Thanks in advance,
> > Kimberley Cheng
> >
> >
> >
> >
> >
> > _______________________________________________
> > 3dem mailing list
> > 3dem at ncmir.ucsd.edu
> > https://mail.ncmir.ucsd.edu/mailman/listinfo/3dem
> 
> 
> _______________________________________________
> 3dem mailing list
> 3dem at ncmir.ucsd.edu
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> 
> 
> ------------------------------
> 
> Message: 4
> Date: Wed, 6 Nov 2013 11:12:47 +0000
> From: Charlotte Hamngren Blomqvist
> 	<charlotte.hamngren.blomqvist at chalmers.se>
> Subject: [3dem] Where to find info on REC-files in order to handle
> 	them in	MATLAB?
> To: "3dem at ncmir.ucsd.edu" <3dem at ncmir.ucsd.edu>
> Message-ID:
> 	<431ED9CDFB840545A7B887CBD3411AEFBCE497B2 at wrex.ita.chalmers.se>
> Content-Type: text/plain; charset="iso-8859-1"
> 
> Dear all,
> 
> I have a question regarding the *.rec file format (reconstructed files created using the FEI software Inspect3D):
> 
> Where can I find information about the file specifications for the REC files?
> I try opening them in MATLAB similarly to what I do with the MRC-files as described here: http://www.2dx.unibas.ch/documentation/mrc-software/mrc-file-format-specifications , with the modifications for FEI-created files suggested here: http://www.2dx.unibas.ch/documentation/mrc-software/fei-extended-mrc-format-not-used-by-2dx (larger header to cut away). When doing this for the REC file, there are shifts in the different quadrants of the image, and I suspect that is due to cutting of the header incorrectly.
> 
> I am very grateful if someone has some information about this!
> 
> Best regards
> Charlotte
> 
> _______________________
> Charlotte Hamngren Blomqvist, PhD student
> Filosofie magister i fysik / Msc in Physics
> 
> Teknisk fysik / Applied Physics
> Eva Olsson group
> Chalmers tekniska h�gskola / Chalmers University of Technology
> SE-41296 Gothenburg, Sweden
> Tel +46-(0)31 772 51 80
> www.chalmers.se<http://www.chalmers.se/>
> E-mail: charlotte.hamngren.blomqvist at chalmers.se<mailto:charlotte.hamngren at chalmers.se <charlotte.hamngren at chalmers.se>>
> 
> Visiting address: Fysikgr�nd 3, room F4417b
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