Nesta edição: Jean Luc Putaux fala com o QMCWEB

Em Grenoble, na França, reside e trabalha um dos expoentes mundiais no uso da Transmission Electronic Microscopy: Jean Luc Putaux. Pesquisador no CERMAV, um laboratório do CNRS (órgão governamental francês de apoio à pesquisa), Jean Luc - além de uma notoriedade - é também bastante humilde e simpático. Acostumado com o mundo nanoscópico, Jean Luc teve uma paciência gigante e nos concedeu esta entrevista. Saiba mais sobre as técnicas de microscopia e, quem sabe, venha para Grenoble trabalhar no CERMAV. Como vocês, Jean Luc:

QMCWEB:// Who is Jean Luc? Can you talk about your formation and your current position?
Jean Luc :// I graduated from the Ecole Nationale Supérieure d'Electrochimie et d'Electrométallurgie de Grenoble (ENSEEG) in 1987. So I am an engineer. However, I realized quite quickly that I would not like to work for the industry. In 1986, our professor of material physics brought us to the Centre d'Etudes Nucléaires of Grenoble (nowadays, CEA Genoble) to visit the electron microscopy group. It was nearly in love at first sight. When I saw the very big 400 kV microscope that was being installed, I knew I wanted to learn how to use en electron microscope. I did a 3 months training course with Dr. J.M. Penisson in this group. Then I got my DEA (french equivalent to Master Degree) in metallurgy during my third year at ENSEEG and after graduation, I was lucky to get a 3 year grant to do my PhD with Dr. J. Thibault at CEA Grenoble.

I studied the atomic structure of silicon grain boudaries, using high resolution electron microscopy. Then I did a post-doc near Paris (CECM Vitry), studying shape memory metallic alloys. When time came to find a job, I received a phone call from Dr. H. Chanzy from the Centre de Recherches sur les Macromolecules Végétales in Grenoble. He convinced me that applying my expertise of TEM of "hard" materials to the new world (for me) of biopolymers was worth trying. I was lucky to get a permanent position as Research Associate in CNRS the same year. I arrived at CERMAV in 93, at the same time as a brand new microscope. So, as I had no background in polymer science or biology, this may appear as an uncommon path, coming from semiconductors and metallic alloys to presently study the morphology and structure of biopolymers in CERMAV !

QMCWEB://What is TEM and Cryo-TEM? What these techniques have been used for?
Jean Luc :// As opposed to scanning electron microscopy (SEM) where one observes the surface of macroscopic samples using a focused electron beam of medium voltage (5-15 kV), transmission electron microscopy (TEM) looks through micro or nanoscopic specimens which have to be very thin ("transparent", a few hundreds nanometers), using highly accelerated electrons (100-300 kV). We get an image of the volume of the specimen. All transmission electron microscopes also offer the possibility to visualize an electron diffraction pattern from a selected area of the sample. Studying these patterns, one can get information on the atomic or molecular structure of the object. It's important to remember that the sample is submitted to a huge electron irradiation so if it's made from light atoms (like polymers or biological samples), the crystal structure is destroyed within seconds. Moreover, the sample placed in the vacuum of the microscope's column rapidly dries. The use of low temperature (generally -180°) helps minimizing both detrimental effects. A frozen sample "resists" a little longer during irradiation and any solvant will stay inside the object. "Frozen hydrated" biopolymer crystals were first observed by Dr. Chanzy in CERMAV. When the morphology or structure of the sample depends on the liquid environment, one will use a more specific "cryo-TEM" technique developped by Dr. J. Dubochet (Lausanne, Switzerland). The objects are observed at low temperature, embedded in vitreous ice obtained by quench freezing a thin film of the original suspension.

QMCWEB://What are the advantages of Cryo-TEM if compared with other techniques such as AFM or SEM?
Jean Luc ://I think the techniques nicely complement. AFM and SEM only look at the surface of the specimens while TEM will also provide a view of the internal structure, with the possibility to do crystallography. The main problem is to maintain a hydrated environment around the specimen. With cryo-TEM, we use fast-freezing. With AFM, one have to do the observation in a drop of liquid. With SEM, you must use a sophisticated microscope, called an ESEM (Environmental Scanning Electron Microscope). The principle is that, thanks to a clever design of differential pumping, a humid atmosphere is maintained around the sample, even though the rest of the microscope column is at a high vacuum. That is a great (and new) technique providing striking images, for instance, from living dust mites or delicate vegetal structures that would be completely distorted by a regular drying. However, to get closer to the very high resolution provided by TEM and AFM, one would have to use an ESEM with a field emission gun, i.e. a better illumination system with a very sharp and coherent electron beam. In that case, the microscope would be a lot more expensive but the quality of the pictures would be greatly improved.

QMCWEB:// What is the CERMAV? Which are the most important topics studied in that lab?
Jean Luc :// CERMAV (director: Dr. Serge Perez) is a pluridiciplinary laboratory from CNRS (French national research center), created in 1966 and focused on the study of polysaccharides (sugar-based natural polymers). Due to this pluridisciplinarity, it would be very long to give a detailed presentation. I encourage French reading people to connect to our website (www.cermav.cnrs.fr) to get more information. I'm afraid english reading people will have to wait for our updated english presentation. There are 6 groups in the laboratory, studying (in no particular order) biochemistry of plant cell walls, polysaccharide chemistry and physico-chemistry, glycochemistry and oligosaccharides biotechnology, structure, interaction and dynamics of biomolecules and molecular glycobiology. I am part of the "Structure and properties of glycomaterials" group in which some researchers extract from the biomass and chemically modify polysaccharides (cellulose, chitin,...) while others are using these to enhance the mechanical properties of natural or synthtetic polymer-based materials. Using TEM as well as X-ray diffraction and solid-state NMR, we also characterize the morphology and ultrastructure of the polysaccharides either as suspended individual objects or once they have been included in a polymer matrix.

QMCWEB:// In your opinion, how will be the scientific electron microscopy by the end of this decade? Will it change too much? What are the possibilities for the microscopy in the very near future?
Jean Luc :// TEM is developing along different directions. The first one is what I would call the "sub-angstroem race". People studying beam-resistant materials want to achieve a point-to-point resolution less than 0.1 nm. Some technological developments are done on the microscope to reduce the aberrations and improve the monochromaticity of the electron beam. Other people prefer to improve the analysis of the experimental micrographs using image processing. However, these improvements will only have a limited impact on the imaging of beam-sensitive materials. The stability of those limits the image resolution, not the microscope performance. In that case, new developments may come from an easier access to microscopes equipped with helium-cooled stages where the specimen temperature is around 4 K. Those used to be found only in Japan but Philips are now selling their own system. I agree that this is not for routine microscopy. My own expectations are the implementation of automatized tomography procedure in the new microscopes. It will be easier to get a 3D view of our samples, especially in the case of ice-embedded objects observed by cryo-TEM.

QMCWEB:// If a QMCWEB reader is interested in getting a Ph.D. or a postdoc position at CERMAV, to work with cryo-TEM , what should he/she do? Is it possible?
Jean Luc :// By filling application forms to get positions, I learned very early that technique should come in second. The research topic is more important. So although cryo-TEM is very interesting to develop as a technique (which we don't have so much time to do in our laboratory, unfortunately), the subject, the substrate, the object... whatever, will be important and in CERMAV, it has to be related to polysaccharides. Financial aspects are also always critical. I am open to discussions !

QMCWEB:// Would you come to Brazil if you were invited to be a speaker in a conference/meeting?
Jean Luc :// Yes, I would be glad to. I never came to this country.