[3dem] temperature depentend cryoTEM

[Svetomir B Tzokov]

Dear Frank,

You could try an experiment where the sample is incubated on the grid for  
no more than 30 seconds. Maybe keeping it in contact with the grid for  
10-15 min interferes with the structures your polymer is forming?

For example, incubate the sample for 10-15 min in the Vitrobot chamber  
before loading it on the grid, then load, wait 30 sec for the sample to  
adsorb, and plunge in the liquid ethane. Our Vitrobot (Mark III) has a  
little holder for a micro tube, yours should probably have it too.

But maybe even before trying the Vitrobot experiment, make sure that your  
sample is OK. Check the particle size by an alternative method - for  
example DLS. Also, have you tried simple negative staining EM? Sometimes  
it can give you a valuable information, as well.

Kind regards,
Svet




On Wed, 29 Jan 2014 08:28:10 -0000, Frank Polzer  
<frank.polzer at physik.hu-berlin.de> wrote:

>
> Dear all,
>
>
> I have recently observed trouble when I was trying to investigate  
> polymer colloids by cryoTEM >that show a temperature-dependence in  
> water. I would like to investigate the particles at >10°C and 40°C  
> solution temperature by cryoTEM. They are expected to be bigger at low  
> >temperatures. Size should decrease about 30% upon heating to 40°C. This  
> has already been >demonstrated by Talmon et al.  
> (http://pubs.acs.org/doi/abs/10.1021/la053198f). I am not able >to  
> reproduce this using the Vitrobot. I end up with particle size at low  
> temperature being >considerably smaller than expected and at high  
> temperature considerably bigger than expected >(the latter I could  
> explain that upon approaching the liquid ethane interface the thin film  
> >cools down).
>
> I am using FEI Vitrobot Mark IV and do the following procedure:
>
>
> -          Equilibrating the dispersion with a thermostat for at least 1  
> h (checked the thermostat >already if it sets the temp. correctly)
>
> -          Equilibrating the pipette tips in the same way
>
> -          Equilibrating the Vitrobot for at least 1 h at 100% humidity  
> and the desired with >tweezers and grids inserted (checked the Vitrobot  
> already if it sets the temp. >correctly)
>
> -          Then I apply 5µl of tempered solution onto the tempered grid  
> in the tempered Vitrobot >chamber
>
> -          I wait for another 10-15 min to avoid thermal fluctuations  
> due to sample transfer
>
> -          I blot and shoot the grid in liquid Ethane using no drain  
> time and transfer it into the >Gatan 914 holder
>
>
> Does anyone have an idea what might change the solution temperature and  
> herein the particle >size during vitrification (filter paper, cold  
> ethane vapor, etc.)?
>
>
> Thank you for your help,
>
>
> Frank
>
>
> Dr. Frank PolzerTEM GroupInstitute of PhysicsHumboldt Universität zu  
> BerlinNewtonstraße 1512489 - Berlin Tel.: +49 30 2093-4995 (office)Tel.:  
> +49 30 2093-7829 (TEM)Fax: +49 30 2093-7886
>  frank.polzer at physik.hu-berlin.de
>
>



-- 
Svetomir B Tzokov
Electron Microscopy Facility Manager
Krebs Institute for Biomolecular Research
Dept Molecular Biology and Biotechnology
The University of Sheffield
Firth Court, Western Bank
Sheffield S10 2TN
United Kingdom
Tel: +44 (0)114 222 4674
Fax: +44 (0)114 222 4243
Mob: +44 (0)7905 832358
Email: s.b.tzokov at sheffield.ac.uk
-------------- next part --------------
An HTML attachment was scrubbed...
URL: <https://mail.ncmir.ucsd.edu/mailman/private/3dem/attachments/20140129/da74299a/attachment-0001.html>