The ‘missing wedge’ is a long-standing problem in electron tomography, caused by the use of slab-like flat specimens, which increase in thickness when tilted to high angles. Attempts have been made to reduce the undesirable effects caused by the missing wedge, but the problem remains, particularly for the radiation-sensitive frozen-hydrated specimens used for high resolution imaging. Specimens with cylindrical symmetry offer a way to overcome this problem, since the thickness remains constant at all viewing angles. However, while this has been suggested before, it has never been demonstrated for frozen-hydrated specimens. In this work, I present a way to make cylindrical specimens for electron cryo-tomography, using thin-walled carbon tubes as specimen holders. The tubes are made in a multi-step process, involving carbon deposition on glass micropipette templates and subsequent removal of the glass. Tube diameters are typically a few hundred nanometres, with a wall thickness of 10–20 nm. To make frozen-hydrated specimens, the tubes are filled with an aqueous sample and then plunge-frozen in liquid ethane. Electron images acquired from the tubes have equal quality at all viewing angles, with a tilt range restricted only by the physical limits of the microscope. Tomograms from specimens such as gold particles and liposomes show that the effects of the missing wedge are substantially reduced, with much improved resolution along the electron beam axis. Structural features oriented in all directions are visible in the reconstructions, in marked contrast to tomograms acquired over a more restricted angular range. These results are promising, however some technical challenges remain before this method can be used routinely.

Des colloïdes à base de silice et de polystyrène ont été synthétisés. Les particules d’oxyde ont d’abord été élaborées et modifiées en surface, puis ont servi de germes au cours d’une étape de polymérisation du styrène. Deux procédés de polymérisation en phase hétérogène ont été utilisés (émulsion ou dispersion) menant à des colloïdes aux morphologies originales et contrôlées. Une étude morphologique par tomographie électronique a permis de mieux comprendre les mécanismes de croissance et d’organisation des particules de latex autour des germes de silice. La synthèse de particules Janus pour l’imagerie biomédicale est aussi décrite. Ces particules de silice ont été modifiées en surface par un chromophore biphotonique et un agent de reconnaissance de certaines cellules tumorales. Des études spectroscopiques et des tests de cytotoxicité ont été entrepris. / Hybrid colloids based on silica and polystyrene have been synthesized. Oxide particles were first elaborated, surface modified, and then used as seed in a styrene polymerization step. Two heterogeneous polymerisation proceeds were employed (emulsion or dispersion) leading to colloids with original and controlled morphologies. A morphological study by electronic tomography enabled to better understand growth and organisation mechanisms of latexes around silica seeds. Janus particles synthesis for biomedical imaging is also described. Silica particles were surface modified with a biphotonic chromophore and a tumor cells targeting agent. Spectroscopic studies and cytotoxicity tests were investigated.

Le travail présenté dans ce manuscrit a montré l’importance du développement méthodologique et technique pour identifier et débloquer les verrous empêchant l’analyse de matériaux hybrides et complexes qui se dégradent sous irradiation par un faisceau d’électrons. Nous avons mis en évidence que des dégâts sur l’échantillon produits par les électrons n’apparaissent qu’au-dessus d'un certain seuil de densité de courant électronique qui dépend de la nature du matériau et de ses caractéristiques morphologiques et structurales. Ces développements couplés à la Cryo-EM, nous ont permis de mettre en évidence l’architecture des matériaux hybrides à base de carbone, la variation de la distance lamellaire dans une pérovskite en fonction de la molécule insérée et le positionnement du métal, d’identifier les interactions à l’interface entre deux cristaux moléculaires et la quantification 3D de la fonctionnalisation d’un MOF. Dans la dernière partie, nous avons mis en évidence les processus de nucléation et de croissance d’oxyde de fer par MET in-situ en phase liquide. / The present manuscript shows the importance of methodological and technical development to identify and to unblock locks preventing the analysis of hybrid and complex materials that undergo degradation under electron beam irradiation. We have shown that beam-induced damage to the sample only appears above some specific threshold of current density. Such a threshold depends on the nature of the material and on its morphological and structural characteristics. These developments in synergy with the use of Cryo-EM, allowed us to expose the architecture of carbon-based hybrid materials, measure the variation of the lamellar distance in a perovskite according to the molecular spacer and to the positioning of the metal, identify the interactions at the interface between two molecular crystals, and the 3D quantification of the functionalization within a MOF. Lastly, we brought to light the processes of nucleation and growth of iron oxide by in-situ liquid phase TEM.