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Cryoelectron Microscopy

par douarche - publié le , mis à jour le

 

Cryo-TEM at LPS. (A) Cubosome (phytantriol/polymer/water). (B) Supra-crystal of gold nanoparticles. (C) Bacteriophage capsid containing a DNA molecule condensed into a toroid. (C) Liquid crystalline phase of nucleosomes. Scale bar 20 nm.

 
CryoEM let us visualize biological and soft matter objects while preserving their hydrated state and native ionic environment, to access their conformations and interactions. One major interest of the method is to be able to explore conformational changes of the macromolecular complexes under physicological conditions, in the aim to relate these changes to their functional activities.
We use both thin film cryoEM, to image macromolecular complexes and nano-objects in solution, and CEMOVIS (Cryo-Electron Microscopy Of VItreous Sections) to image biologiocal tissue and cells, or bulk liquid crystals.

The SOBIO team hosts the CRYO-LPS platform of the National facility METSA

 
THE CRYO-TEM


JEOL 2010F-CRYO, 200kV FEG, equipped with a Gatan 626 cryo-holder and a Gatan Utrascan 4K

 
 

SAMPLE VITRIFICATION


Two methodes are used to vitrify the samples at low temperature :
- The plunging method is used for small objects suspended in solution. The lab is equipped with 2 home made plunge-freezing devices and 1 Vitrobot Mark IV vitrification robot (Thermofischer).
- The « slam-freezing » method (an alternative to high pressure freezing) is used for entire cells and bulk samples. We use the Liquid Helium Cryovacublock device (Reichert).
 

  • Thin film vitrification  
    Freeze-plunging device (home-made) Vitrobot Mark IV (Thermofischer)
  • Bulk sample vitrification  
    Slam-freezing Cryovacublock

 

CYO-ELECTRON MICROSCOPY OF VITREOUS SECTIONS (CEMOVIS)


The CEMOVIS method, initially developped in the group of J. Dubochet (Lausanne), relies on the obtention of ultra-thin sections (40-100nm) of vitrified biological and soft matter samples.
Our cryo-microtome (Leica UC6/FC6) is installed in a temperature and humidity controlled room (RH <20%), and coupled to a fluorescence microscope to target Regions Of Interest.

 
We use CEMOVIS to explore a variety of systems : cultured eukaryotic cells and bacteria, unicellular eukaryotes (Paramecium, Euglena, yeast), tissues (Caenorhabditis elegans), polymers and colloids in concentrated solutions, lyotropic liquid crystals, ...

 

 

FREEZE FRACTURE


Freeze fracture is used to analyse the liquid crystalline phases of DNA and nucleosomes.


Freeze-fracture device (BALZERS BAF 400T)


Visualisation of DNA fragments in a layer of the columnar hexagonal phase, after vitrification of the sample and freeze-fracture.

 

REFERENCES


LIVOLANT F. (1991) Supramolecular organization of double-stranded DNA molecules in the columnar hexagonal liquid crystalline phase. An electron microscopic analysis using freeze-fracture methods. J Mol Biol. 218(1):165-81.

LEFORESTIER A. , F. LIVOLANT (1993) Supramolecular ordering of DNA in concentrated solution. An ultrastructural analysis of the cholesteric liquid crystalline structure. Biophysical Journal 65, 56-72.

LEFORESTIER A. , J. DUBOCHET, F. LIVOLANT (2001) Bilayers of Nucleosome Core Particles. Biophysical Journal, 81, 2414-2421.

LEFORESTIER A, LEMERCIER N., LIVOLANT F. (2012) Contribution of cryo-electron microscopy of vitreous sections to the understanding of biological membrane structure. Proc. Natl. Acad. Sci. (USA) 109 (23) 8959-8964.

MULLER F, DEGROUARD J, SALONEN A. (2013) Stabilization of Lipid-Based Lyotropic Liquid Crystalline Phases by Nanoparticles. Chapter in book : Advances in Planar Lipid Bilayers and Liposomes. Volume 18 Chapter 8 Elsevier 209-236.

LEFORESTIER A., P. LEVITZ, T. PREAT, P. GUTTMANN, L.J. MICHOT, TCHENIO P. (2014) Imaging Drosophila brain by combining cryo-soft X-ray microscopy of thick vitreous sections and cryo-electron microscopy of ultrathin vitreous sections. J Struct Biol. 188 (2), 177-82.

MULLER F, DEGOUSEE T, DEGROUARD J, BRULET A, SALONEN A. (2015) Probing structure in submicronic aqueous assemblies of emulsified microemulsions and charged spherical colloids using SANS and cryo-TEM. J Colloid Interface Sci. 446:114-121.

BEAUDOIN E, ABECASSIS B, CONSTANTIN D, DEGROUARD J, DAVIDSON P. (2015) Strain-controlled fluorescence polarization in a CdSe nanoplatelet–block copolymer composite. Chem. Commun. 51 (19) 4051-4054.

SCHMITT J, HAJIW S, LECCHI A, DEGROUARD J, SALONEN A, IMPEROR-CLERC M, PANSU B. (2016) Formation of Superlattices of Gold Nanoparticles Using Ostwald Ripening in Emulsions : Transition from fcc to bcc Structure. J. Phys. Chem. B. 120 (25) 5759-66.

THIBAULT T, DEGROUARD J, BARIL P, PICHON C, MIDOUX P, MALINGE JM. (2017) Production of DNA minicircles less than 250 base pairs through a novel concentrated DNA circularization assay enabling minicircle design with NF-κB inhibition activity. Nucleic Acids Research. 45 (5) e26.

FERREIRA J, MIKHAILOVSKAYA A, CHENNEVIERE A, RESTAGNO F, COUSIN F, MULLER F, DEGROUARD J, SALONEN A, MARQUES EF. (2017) Interplay between bulk self-assembly, interfacial and foaming properties in a catanionic surfactant mixture of varying composition. Soft Matter. 13 (39) 7197-7206.

ELTSOV M., GREWE D., LEMERCIER N., FRANGAKIS A., LIVOLANT F., LEFORESTIER A. (2018) Nucleosome conformational variability in solution and in interphase nuclei evidenced by cryo-electron microscopy of vitreous sections. Nucleic Acid Research, 46, 9189-9200. doi : 10.1093/nar/gky670.

M. CHEVREUIL, D. LAW-HINE, J. CHEN, S. BRESSANELLI, S. COMBET, D. CONSTANTIN, J. DEGROUARD, J. MÖLLER, M. ZEGHAL, G. TRESSET (2018) Nonequilibrium self-assembly dynamics of icosahedral viral capsids packaging genome or polyelectrolyte. Nat. Commun. 9 3071.