MRic - TEM presentation

MRic-TEM is the transmission electron microscopy part of the MRic facility. It provides, to academic research teams and private companies, the technology and the expertise to image analyse the ultrastructure. The expertise of the facility is mainly, in biological and chemical field. MRic-TEM offers a large choice of technics to image at different levels : organs, cells, organites, macromolecular complexes...

A large choice of devices are proposed on MRic-TEM to offer you a large amount of technics.

2 microscopes are available on the facility. Jeol 1400 - 120 kV wield field camera for organ and cell imaging and Tecnai G2 Sphera - 200 kV for high resolution imaging.

Fixation is an essential step in EM. 3 technics are proposed on the facility thanks to dedicated devices :

• Chemical fixation, with aldehyde, is the most common technic. It is an easy and fast technic but causes artifacts due to the low fixation speed.
• High Pressure Freezing (HPF) is used for thick samples (up to 200 µm). A high pressure is put on the sample followed by a rapid freezing with liquid nitrogen. A good freezing will vitrified the sample and provides a good preservation of the structures. This technic, combined with methacrylic resins, is used for immunolabeling.
• Plunge Freezing is used for thin layer samples (up to 200 nm). The sample is deposited to a glow discharged EM grids followed by blotting and vitrification by rapid freezing into liquid ethane. This technic is used to vitrified thin layer samples.

Advanced technics, such as immunolabeling, electron tomography, cryo-electron tomography, sub tomogram averaging, modelisation are also available.

The facility members can help you to analyse images, do the 3D reconstructions of the tomograms and do the modelisations.

Different technics are available on the facility depending on the samples :

Thick samples (Tissues, cells) :

Resin preparation

• Chemical fixation or cryo fixation (HPF)
• Embedding with epoxy resins or cryo substitution and cryo inclusion with methacrylic resins
• Semithin (200-500 nm) and ultrathin (60-150 nm) sections
• Positive staining (pre and post embedding)

Immunolabeling technics

• Tokuyasu technic
• Lowicryl

Imaging

• Classical
• Electron tomograms

Isolated particles (extracellular vesicles, liposomes, viruses, macromolecular complexes) :

• Negative contrast - is a fast technics to image isolated particles. The sample is deposited on a grid and contrasted with heavy metal salts. Due to sample dehydration, deformations may appear on some like vesicles.
• Rotary shadowing - is used to image particular samples such as DNA strands.
• Cryo electron microscopy - the samples are vitrified in a thin layer ice after plunge freezing. The samples are close to their native states, no dehydration or chemical fixatives.
• Cryo electron tomography - the samples are vitrified in a thin layer ice after plunge freezing, with nanogolds particles (fiducials for reconstruction). The samples are close to their native states, no dehydration or chemical fixatives. The samples are imaged at different angles (every 2° from -60 to +60°). The images are backprojected to reconstruct the volume.

3D modelisation

Room temperature technics

• Sections - Electron tomography
• Homogeneous samples (negative staining) - Single particles approaches

Cryo-methods

• Heterogeneous samples - Cryo electron tomography
• Homogeneous samples - Single particle approaches

3D modelisation

Softwares

• ImageJ/Fiji
• Etomo
• 3Dmod
• PEET
• Chimera
• Eman2

The facility is accessible to the research units linked to the SFR Biosit. The equipments are also available to the research units external to Biosit and to private companies. The facility offers the possibility to take in charge the projects from sample preparation to image interpretation. Tarification (cost/hour) is based on maintenance contracts and consumable purchase.

Equipment booking is available, after a training by MRic-TEM members, at the following link :

MRic booking

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La plateforme est ouverte à toutes les équipes appartenant aux unités constituantes de la SFR Biosit. Les équipements sont également disponibles aux unités de recherche extérieures à Biosit et aux entreprises privées. La plateforme offre la possibilité de prendre en charge des projets de l'adaption de protocoles de préparation à l'interprétation des images. Les heures d'utilisation des appareils sont facturées aux utilisateurs selon un tarif horaire calculé de façon à payer les contrats d'entretien, les fluides et l'achat des matériels nécessaires à leur bon fonctionnement.