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Sample preparation

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Plunge-freezing machine, working under humidity and temperature controlled conditions (home made)

Cryovacublock device Reichert-Jung (Escaig et al, 1982) for slam-freezing of bulk samples

During the vitrification step,the sample is cooled down very rapidly at very low temperature and water is transformed into amorphous ice. The vitrification method must be adapted to the nature of the sample:
- small objets in solution are plunged into liquid ethane (plunge-freeze device)
- entire cells or bulk samples are vitrified by slam freezing to liquid Helium temperature (or by using high pressure freezing machines). Thin sections of the material are prepared with a cryomicrotome.

The material (thin film or thin section) is maintained at liquid nitrogen temperature and observed in the microscope, under low doses of electrons (low dose system).


Cryo-ultra microtome (LEICA VC6/FC6)

Cryosectionning of frozen hydrated samples

The method of preparation of thin sections (50-100nm) of the amorphous frozen hydrated material (without any chemical fixation, or embedding in a resin), was initially developped in the group of J. Dubochet (Lausanne) (Al-Amoudi et al, (2004), voir également le site CEMOVIS ). This method is now perfectly handled in our group.


Cryo-sections of the lamello-columnar phase of nucleosomes. Nucleosomes (10 nm in diameter) are seen in top view (left and in side view (right) and can be compared to similar orientations of the cristallographic structure (Leforestier et al, 2001)

Freeze-fracturing

Freeze-fracture method is commonly used in biology to study the ultrastructure of the cell. It is also widely used to analyze membrane systems. This method has proved itself extremely powerful in the understanding of the organisation of the ordered phases of DNA and nucleosomes (Livolant, 1991, Livolant et Leforestier, 2000).


Freeze-fracture device (BALZERS BAF 400T)

Observation of DNA fragments in one layer of the columnar hexagonal phase, after virrification and freeze-fracture of the sample.

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