Unit: Electron Microscopy (Platform)
Director: PREVOST Marie-Christine
The Electron Microscopy Platform (PFME) offers service activity, In addition, as response to the request of scientists but also to enlarge their own experience, the PFME is involved in the development of new techniques for ultrastructural morphological study and for immunocytochemical analysis. This involves techniques such as Cryofixation, cryoscanning analysis and pre-embedding immunolabelling.The activity of the PFME concerns all biological domains such as cellular biology, virology, microbiology and parasitology. Moreover, the PFME is implicated in training new users in electron microscopy.
The PFME is integrated in the Imagopole directed by Spencer Shorte. It is directly attached to the Cell Biology and Infection Department (Philippe Sansonetti) but it is open to all. The mission of the PFME is to offer the scientific community of the Institut Pasteur the possibility of working on subjects that require an ultrastructural approach or an immunolocalisation analysis.
The service is divided in three main activities:
To follow the quality of different biological samples (established cell lines, primary cell lines, viruses, bacteria, and fungi, etc) and also perform classic ultrastructural analysis.
Development activities based on new techniques like the visualization of immunocytochemical labelling with " nanogold probe " which is revealed after a silver-enhancement in TEM (Martin Sachse).
Also, it is planed to set up for year 2006, cryofixation by high pressure freezing. This technique allows a rapid, physical fixation, which results in a better morphological preservation. It gives the possibility to observe certain samples which normaly disappear after chemical fixation as biofilms (figure 1).
Every year, an internal training course " Cours de Microscopie Electronique " is performed for 8 to 12 persons. This training course is open to engineers, searchers, technicians of Pasteur but also to employees as OREX.
It gives the opportunity to learn and to be formed to the tools and techniques of the ultrastructural analysis. In the same way, we formed new users who wish to use the materiel independently.
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.
The collaboration contracts, the development of new technologies and methods are validated by a Steering Committee which consists of Antony Pugsley, Olivier Schwartz and Jean-Pierre Bourgeois.
Since the creation of the platform in 2001, we were involved in several collaborative contracts with different units on the campus on various topics (105 collaboration contracts).
In 2005, we had initiated 14 collaboration contracts and we are currently following 31 initiated during 2004 and 2003. This year 2 collaboration contracts were performed with outside people of Pasteur (Necker, Institut Curie).
Currently, we have 17 autonomous users, 7 were trained in the PFME. The formation of new trainees is possible.
Thanks to the financial support of Spenser Shorte, it was possible for the PF's personel but also to outsiders to follow a training for the cryoscanning approach (Gatan Company). This will enable us to form new users to this technique.
Example of projects performed in the PFME.
Cell Biology and infection.
Studies of the surface on Shigella flexneri :
This study analysis of the lateral polyosic 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.
Collaboration with Philippe Sansonetti's group - Molecular Microbial Pathogenesis
Studies on HTLV :
The study analysis of the distribution of the HTLV-1 during the infection of T-Lymphocytes. HTLV-1 may modify the capacity of infected T lymphocytes to polarize upon contact with other T cells in a way that facilitate virus transmission directly from cell to cell. The goal is to characterize morphogicaly the sites of T cell polarisation and virus transmission. By different approach which take place in PFME (ultrastructural analysis, immunogold labeling on thawed cryosections or immunolabeling previous to flat embedding in Epon) we analyse the distribution of the viral proteins.
Collaboration with Andrès Alcover's group - Lymphocyte Cell Biology and Martin Sachse - PFME
Studies on Aspergillus fumigatus :
Study of the biofilm on the surface of conidies from Aspergillus fumigatus after the visualization by cryoscanning In addition, we also localized and characterized proteins of the cell wall of conidies and mycelium of A. fumigatus and their relation to receptors of macrophagic's ( Dectin,etc).
Collaboration with Jean-Paul Latgé' group - Aspergillus Unit.
Keywords: Electron microscopy, ultra structure, immunogold, tweed cryosections, cryofixation