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• Responsable de formations
• Responsable de diplôme (M2)

• Physique/Matière Condensée/Science des matériaux
  

The aim of this study was to perform a structural characterization of dental pulp tissue using multiphoton microscopy and to compare this to classical histological coloration. Materiel & Method:Sample preparation: Freshly extracted teeth with healthy pulp were used in this study. All teeth were fixed in PFA 4% for 24 hours, rinsed in PBS and decalcified. The samples were then embedded in paraffin. The paraffin blocks were cut with a Microm HM340E microtome with Niagara system to obtain 3 μm thick sections. The samples were then dewaxed with xylene, rehydrated and stained with Hematoxilin/eosin and Masson’s trichrome. Multiphoton Microscopy (MPM) images were recorded using a custom-built multiphoton microscope based on a SliceScope microscope (MPSS-1000P, Scientifica) upright microscope. MPM images were obtained recording the non- linear emission spectra after laser scanning of samples. Two-photon fluorescence (2-PEF) signal was used to image autofluorescent structures. Second harmonic generation signal (SHG) was performed to image collagen. The recorded images were processed by ImageJ software.Results: At the dentin/pulp junction, we observed the dentin, which emitted an important SHG signal due to the abundance of type 1 collagen fibers. The palisade of odontoblasts and odontoblast extensions (2PEF) were imaged towards the dentinal tubules. We imaged blood vessels due to the 2-PEF signal of blood cells. A fibrous structure representing the extracellular matrix emitted a SHG signal, showing the presence of type I collagen fiber. At the apical 1/3 of the root fibers were observed at the periodontal ligament emitting both in SHG and 2PEF, coming in contact to the pulp. The MPM images were then compared to the histological stained samples. Conclusion: Multiphoton microscopy is a convenient label-free minimally invasive technique to image live, vascularized and dynamic tissue such as the pulp. It provides valuable additional information with respect to histological sections observed under an optical microscope.

Nanoindentation based on atomic force microscopy (AFM) can measure the elasticity of biomaterials and cells with high spatial resolution and sensitivity, but relating the data to quantitative mechanical properties depends on information on the local contact, which is unclear in most cases. Here, we demonstrate nonlocal deformation sensing on biorelevant soft matters upon AFM indentation by using nitrogen-vacancy centers in nano-diamonds, providing data for studying both the elasticity and capillarity without requiring detailed knowledge about the local contact. Using fixed HeLa cells for demonstration, we show that the apparent elastic moduli of the cells would have been overestimated if the capillarity was not considered. In addition, we observe that both the elastic moduli and the surface tensions are reduced after depolymerization of the actin cytoskeleton in cells. This work demonstrates that the nanodiamond sensing of nonlocal deformation with nanometer precision is particularly suitable for studying mechanics of soft biorelevant materials.

We report on two selective functionalization strategies to create a chemical contrast between the active rib waveguides and the passive surrounding areas of a chalcogenide-based optical sensing system. In such configuration, the analyte could be concentrated on the waveguides and interact with the evanescent field, producing a stronger optical signature. The rib waveguides are obtained by photolithography and subsequent ion beam etching of amorphous Ge-Se-Te thin films that allow residual resist to remain above the waveguides. The first functionalization strategy consists in the reuse of the resist as a mask during the following surface modification process. It allows the functionalization of all areas around the waveguides. The second strategy consists of depositing a new sacrificial metal layer, leading to a perfect negative functionalization contrast, modifying only the waveguides. For the two strategies developed, three precursors were used. The use of a silylated fluorescein derivative allowed the validation of the protocols, with the observation of a fluorescence contrast between the functionalized and non-functionalized areas. The use of tetraethoxysilane as a hydrophilic precursor and the mixture of tetraethoxysilane and octyl-trimethoxysilane as a hydrophobic precursor created a clear contrast in wettability between the rib waveguides and the surrounding areas. A spore deposition was performed on functionalized components according to the two proposed strategies, with the two hydrophilic/hydrophobic precursors. The spore immobilization rate was increased by making the waveguides more hydrophobic, as well as by making the areas surrounding the guides more hydrophilic, demonstrating the effectiveness of our two strategies.

Spinal cord injuries (SCI) affect between 2.5 and 4 million patients worldwide, with no current curative treatment. To understand the mechanisms underlying the absence of spontaneous regeneration following injury, we are combining the non-linear multiphoton microscopy (MPM) technique with force measurements via atomic force microscopy (AFM), in a mouse model, to monitor the glial scar, a scar that inhibits the axonal regeneration by forming a physical and chemical barrier composed mainly of astrocytes and microglia. We recorded 2-photon excited fluorescence (2PEF) and second harmonic generation (SHG) signals of excised mice SC injured tissues in MPM at 72h, 1week and 6 weeks post-lesion, and further performed polarization dependent measurements of the SHG signal to assess the preferential orientation of the collagen bundles. Our MPM images revealed a strong SHG signal at 1 week post injury, due to the formation of fibrillary collagen fibers (collagen type I) by the injury site. The SHG signal was increased at 6 weeks after injury, and associated with (1) a higher fiber density (2) a shorter fiber length and less fibers oriented in the same direction. AFM based force spectroscopy measurements, performed at the same post-lesion time-points to map the elastic properties of the spared grey and white matters and injured (lesion) parts of the tissue, suggested an increase of the lesion area stiffness over time. These results together indicate the presence of a fibrotic process seven days after injury, that is further increased at later time points. We similarly started to investigate the effect of a treatment (pharmacological transient depletion of microglia/macrophage proliferation) in mice that underwent SCI. Our preliminary results suggested an increase in fibers length in treated tissues, as well as a reduction of the collagen extension around the injury site.

The molecular and cellular mechanisms associated with tissue degradation orregeneration in an infectious context are poorly defined. Herein, we explored the role ofmacrophages in orchestrating either tissue regeneration or degradation in zebrafishembryos pre-infected with the fish pathogen Mycobacterium marinum. Zebrafish wereinoculated with different infectious doses of M. marinum prior to fin resection. While mildinfection accelerated fin regeneration, moderate or severe infection delayed this processby reducing blastemal cell proliferation and impeding tissue morphogenesis. This wascorrelated with impaired macrophage recruitment at the wound of the larvae receivinghigh infectious doses. Macrophage activation characterized, in part, by a high expressionlevel of tnfa was exacerbated in severely infected fish during the early phase of theregeneration process, leading to macrophage necrosis and their complete absence inthe later phase. Our results demonstrate how a mycobacterial infection influences themacrophage response and tissue regenerative processes.