Unit: Electron Microscopy (Platform)
Director: Marie-Christine PREVOST
The Electron Microscopy Platform is implicated in the development and the performance of techniques for ultrastructural and morphological analysis in cellular biology, virology and microbiology. These thematics are developed in collaboration with different researchers within the campus. In addition to these activities, the platform uses more specialized methodologies for immunocytochemical labelling and cryotechnique.
Service activities involve the quality control of different biological samples including cells, primary cells, viruses, bacteria and fungi. The Electron Microscopy Platform also participates in teaching and training within the Institut Pasteur.
The PFME (Electron Microscopy Platform) was created by the Strategic Technologies Department (S. Cole) and is directly attached to the Cell Biology and Infection Department (Ph. Sansonetti) but it is open to departments of Pasteur. Its mission is to offer the scientific community of the Institut Pasteur the possibility to work on research topics which require an ultrastructural approach.
The laboratories of the platform are localized at 3 different sites of the campus,. The platform is equipped with 4 transmission electron microscopes (Philips CM10 and CM12, Jeol JEM 1010 and 1200EX) and a high resolution scanning electron microscope (Jeol JSM-6700F with a cold FEG emission cathode). This scanning electron microscope was installed in November 2002 and has a resolution of 2 to 3nm at 1 Kv with biological samples. In November 2004, we completed the equipment of this microscope by a transfer system "Alto 2500" from Gatan. This enables us to observe directly the sample after freezing or cryofacture. After the cryoimmobilisation of the sample by freezing in slush liquid nitrogen, a substitution of the ice is performed and without the usual chemical fixation different metals or carbon are evaporated on the sample
The laboratory possesses a collection of material which allows a large assortment of techniques from different fields to be used for the preparation of biological samples.
The platform also offers training for technicians, engineers and researchers who wish to use the material independently. A training course was initiated at Institut Pasteur in 2003 (2 modules in January and November).
All work is performed with collaboration contracts. The objectives are to define the projects with the researchers. Both teams agree to engage themselves concerning the possibilities, risks and technological orientations as well as confidentially, results and external acknowledgment.
The collaborations may involve several different laboratories and research groups. The collaboration contracts are validated by a Steering Committee which consists of 3 people (Antony Pugsley, Olivier Schwartz and Jean-Pierre Bourgeois).
The platform also proposes the development of new technologies and methods in leading fields of ultrastructural analysis such as cryogenic techniques linked to cellular biology. This requires the approval of the Director of Strategic Technology and of the Steering Committee.
Since the creation of the Electron Microscopy Platform, we were involved in collaboration with different units on the campus on various topics (90 collaboration contracts). These topics required besides classical electron microscopy techniques also more specialized techniques such as the immuno labelling with colloïdal gold using different methods (cryosections, cryofixations and freeze substitution).
In 2004, the platform has initiated 26 collaboration contracts.
The following are several examples of these collaborations.
Cell Biology and Infection
This study analyses the lateral polyosidic chains of Shigella flexneri LPS in a direct and indirect approach and compares mutant strains which are affected in the composition and/or structure of these chains (Photo 2]
Collaboration with Philippe Sansonetti - Molecular Microbial Pathogenesis.
Study of the entry pathways of HIV in primary cells (lymphocytes, macrophages and dendritic cells) and the role in HIV entry of endocytosis, phatocytosis and macropynocytosis. For this study, we used ultrastructural microscopy, immuno electron microscopy and high resolution scanning electron microscopy.
Collaboration with Olivier Schwartz's group - Virus and Immunity.
Study of the possibility of assembling capsid proteins and heterologous envelope derived from Hepatitis C and GBV-B (a non-classified flavivirus) into pseudo-particle viral chimeras (VLPs): examination by negative coloration and immunolabelling with anti-envelope antibodies from preparations of VLPs purified from insect cells infected by recombinant baculaviruses which express chimeric structural precursors.
Collaboration with Annette Martin's group - Molecular Genetics of Respiratory Tract Viruses.
Structural analysis and cellular location of the muted or non-muted pre-integration HIV complex in DNA flap. Using the electron microscope and immunogold, we have already visualized structures just at the exterior of the nuclear pores. We also visualize the purified surface of the nucleus using the high resolution scanning electron microscope which will allow us to carry out a quantitative immunological analysis.
Collaboration with Pierre Charneau's group - Molecular Virology and Vectorology Group
Subcellular localization of antigenic protein from Mycobactreium tuberculosis : Serine-Thronine-Protein-Kinases B, D, E, and F regulator proteins involved in many metabolic process.
Collaboration with Stewart Cole - Bacterial Molecular Genetics Unit.
Photo 1 : Nuclear pore from Hela cell by cryoscanning electon microscopy Jeol 6700 F and cryotransfert Gantan Alto 2500 : N Arhel, S. Guadagnini, P. Charneau, M-C Prévost
Keywords: Electron microscopy, ultrastructure, immunogold, cryosection, cryofixation