Unit: Electron Microscopy Platform
Director: Marie-Christine PRÉVOST
The Electron Microscopy Platform is involved in research and development of ultrastructural and morphological analysis in cellular biology, virology and microbiology. These thematics are developed in collaboration with different researchers on the campus. In addition to this activity, the platform utilises a more specialised methodology for immunocytochemical labelling and cryotechnique.
Service activity involves the quality control of different biological samples including cells, primary cells, viruses, bacteria, and fungi. We are also involved 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 (P. Sansonetti) but it is open to all. It's mission is to offer the scientific community of the Institut Pasteur the possibility of working on subjects for which an ultrastructural approach would be essential in the research theme.
The laboratory is split between three sites, and is equipped with 4 transmission electron microscopes ( a Philips CM10 and CM12, a Jeol JEM 1010 and 1200EX) and a high resolution scanning electron microscope (a Jeol JSM-6700F with a Cold FEG emission cathode). This scanning electron microscope was installed in November 2002 and has allowed us to achieve a resolution of 2 to 3 nm at 1 Kv with biological samples.
In November 2003, we equipped our Jeol 1200EX electronic microscope with a high resolution CCD camera (1.4 million pixels), with a large angle (large observation field), and real-time data acquisition which allows the transmission electron microscope to be observed and modified. In addition, the camera is equipped with an analysis programme and image archiving with the "Pro" version (archiving, editing, preparation of reports, addition of annotations, interactive measurements and automatic morpho-mathematic operations, complex filters, and an advanced programming language).
The laboratory possesses a collection of material which allows a large assortment of techniques from different domains 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 the Institut Pasteur in 2003 (two modules in January and November).
Work is carried out under collaboration contracts. The objectives are to define the project(s) with the researchers. Both teams agree to engage themselves concerning the possibilities, risks, and technological orientations, as well as confidentiality, 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 three people (Antony Pugsley, Olivier Schwartz and Jean-Pierre Bourgeois).
The Platform also proposes the development of new technologies and methods in leading domains 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 have been involved in collaborations with different units on the campus involving diverse themes (64 collaboration contracts). These themes required an involvement of electronic microscopy not only using classical techniques, but also with more specialised techniques such as the immunogold labelling with colloidal gold using different methods (cryosections, cryofixations and freeze substitution).
In 2003, we have initiated 24 collaboration contracts.
Here are several examples of these collaborations :
Study of the entry pathways of HIV in primary cells (lymphocytes, macrophages and dendritic cells) and the role in entry by endocytosis, phagocytosis, and macropyncytosis. For this study, we used ultrastructural microscopy, immunomicroscopy, and high resolution scanning electron microscopy.
Collaboration with the group of Olivier Schwartz, 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 baculoviruses which express chimeric structural precursors.
Collaboration with the group of Annette Martin, Molecular Genetics of Respiratory Tract Viruses.
Structural analysis and cellular location of the muted or non-muted pre-integration HIV complex in the DNA flap. Using the electron microscope and immunogold, we have already visualised structures just at the exterior of the nuclear pores. We also visualise 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 the group of Pierre Charneau, Molecular Virology and Vectorology Group.
Subcellular localization of antigenic proteins from Mycobacterium tuberculoisis : Serine-Threonine-Protein-Kinases B, D, E and F, regulator proteins involved in many metabolic process.
Collaboration with Stewart Cole, Bacterial molecular Genetics Unit.
Structure and Dynamic of Genomes
Analysis of the structure of the cell wall of the conidia of Aspergillus fumigatus showed that the outer hydrophobin layer of the conidia played an essential role in the resistance to phagocytosis.
Collaboration with Jean-Paul Latge, Aspergillus Unit
Ecosystems and Epidemiology of infectious diseases
Interaction between Bordetella pertussis and human tracheal cells : Ultrastructural analysis of invasive potential and intracellular survival of Bordetella pertussis, Bordetella parapertusis and Bordetella bronchiseptisca.
Collaboration with Nicole Guiso and Pascale Gueirard
Service Activities The Electron Microscopy Platform verifies different biological samples (viruses, primary cell lines and continuations, bacteria and fungi). These analyses are carried out for units on the campus that require verifications in order to pursue their research investigations.
Teaching Activities The different electron microscopy techniques are taught to researchers, lecturers, and laboratory technicians who wish to use these techniques independently.
It is for this reason that two modules of courses were initiated at the Institut Pasteur in 2003.
1: Dendritic cell in interaction with a T 4 lymphocyte infected with HIV viewed by the scanning electron microscope. Source : N. Sol, S. Guadagnini, O. Schwartz et M.C. Prévost.
2: Scanning Electron micrographs of A.fumigatus conidia of the wild-type G10 strain (a) and of transformants Δ rodB-02(b), Δ rodA-47(c) and Δ rodA Δ rodB-26 (d). Bar 100nm. (Paris and al.)
Keywords: Electron Microscopy, Ultrastructur, Immunogold, Cryo sections, Cryo fixation