Objectives During malignant transformation, remodeling of the extracellular matrix creates a stromal tumor microenvironment (TME) essential for cancer progression. Collagen is a main component of this TME and its relevance as an indicator of tumor transformation is highlighted in research on breast and prostate cancer. The most sensitive reported technology to study these collagenic architectural transformations is multiphoton microscopy (MPM), monitoring 2-photon excited fluorescence and second harmonic generation. The main objective of this work is to establish a proof of concept of MPM to better understand the TME processes underlying oral cancer (OC) and to support novel effective therapeutic strategies. The relevance of the topic gains its evidence from the current lack of effective prevention and curative treatment for this devastating cancer. Methods Cryostat-sectioned and paraffin-embedded samples were analyzed and the generated SHG images were recorded using a custom-made MPM based on a Tsunami laser and an upright SliceScope microscope furbished with a multiphoton galvanometer scan head. 38 samples, pertaining to 15 types of lesions representative of all tumor stages, were analyzed. 44% of samples (n=168) were usable. Results Results showed that collagen, normally well organized, deteriorates with tumour progression and gradually transforms into an increasingly disordered network, with fibers firstly dissociating and thinning. Analysis by vectorization of perilesional collagen fibers suggests, in invasive tumors, an organization of these fibers perpendicularly to the tumor, creating "gateways" for migration into surrounding tissues of tumor cells. The identification of the evolution of collagen density and organization seems to allow, as for other tumor localizations, to define a tumor-associated collagen signatures (TACS) gradation scale for OC, statistically correlated with overall and recurrence-free survival. Conclusions MPM could be a reliable tool in the identification of tumor progression markers of OC, which would be complementary to pathological examination and useful for identifying target areas for biopsy or surgical margins.

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.

Aim: With the objective to investigate the mechanisms underlying absence of spontaneous axonal regeneration following spinal cord injury (SCI) we employ a multimodal label-free imaging approach to monitor glial scar in a mice SCI model. Method: To determine the relevant structural signature, and the nanobiomechanical behavior of healthy and injured spinal cord tissue we combine the non-linear, multiphoton microscopy (MPM) technique with force measurements via atomic force microscopy (AFM). The glial scar at different post lesion time-points is investigated with these two techniques to monitor structural and elasticity (Young modulus) changes of the tissue.Results: 2-photon excited fluorescence (2PEF) and second harmonic generation (SHG) signals of excised mice SC injured tissues were recorded in MPM at 72h, 1week and 6 weeks post-lesion. The MPM images revealed the apparition of a strong SHG signal at 1week post injury, due to the formation of fibrillar collagen fibers (collagen type I) by the injury site in the glial scar. At 6 weeks’post-injury, the SHG signal is more intense and a higher number of fibers are detected in average. We further assessed the preferential orientation of the collagen bundles performing polarization dependent measurements of the SHG signal. The AFM based force spectroscopy measurements have been performed at the same post-lesion time-points to map the elastic properties of the healthy (grey and white matters) and injured (lesion) parts in the spinal cord tissue. The results suggested an increase of the lesion area stiffness over time that could be correlated with the apparition of fibrillar collagen observed in MPM, indicating the presence of a fibrotic process seven days after injury, that develops in time. As tissue stiffness is a regulator of neuronal growth, such kind of measurements might help to understand why adult mammalian axons do not regenerate after an injury. Our next step is to investigate the effect of a treatment (pharmacological transient depletion of microglia/macrophage in mice that underwent SCI) on the structure and mechanical properties of the lesion site at 1 week and 6 weeks post injury.

Spinal cord injuries (SCI) affect between 2.5 and 4 million patients worldwide yielding major handicaps and inducing high economical costs. A scar, called glial scar and composed of two main cellular populations i.e. astrocytes and microglia, inhibits axonal regeneration by forming a physical and chemical barrier. Currently, there is no curative treatment on any symptoms associated with SCI. In this context, with the objective to investigate the mechanisms underlying absence of spontaneous axonal regeneration following SCI, we employ a multimodal label-free imaging approach to monitor glial scar in a mice SCI model. Method: To determine the relevant structural signature and the nanobiomechanical behavior of healthy and injured spinal cord tissue, we combine the non-linear, multiphoton microscopy (MPM) technique with force measurements via atomic force microscopy (AFM). The glial scar at different key post lesion time-points is investigated with these two techniques to monitor structural and elasticity (Young modulus) changes of the tissue.Results: 2-photon excited fluorescence (2PEF) and second harmonic generation (SHG) signals of excised mice SC injured tissues were recorded in MPM at 72h,1 week and 6 weeks post-lesion. The MPM images revealed the apparition of a strong SHG signal at 1week post injury, due to the formation of fibrillar collagen fibers (collagen type I) by the injury site in the glial scar. At 6 weeks post-injury, the SHG signal is more intense and a higher number of fibers are detected in average. We further assessed the preferential orientation of the collagen bundles performing polarization dependent measurements of the SHG signal. The AFM based force spectroscopy measurements have been performed at the same post-lesion time-points to map the elastic properties of the healthy (grey and white matters) and injured (lesion) parts in the spinal cord tissue. The results suggested an increase of the lesion area stiffness over time that could be correlated with the apparition of fibrillar collagen observed in MPM, indicating the presence of a fibrotic process seven days after injury, that develops in time. As tissue stiffness is a regulator of neuronal growth, such kind of measurements might help to understand why adult mammalian axons do not regenerate after an injury. Our next step is to investigate the effect of a treatment (pharmacological transient depletion of microglia/macrophage in mice that underwent SCI) on the structure and mechanical properties of the lesion site at 2 weeks and 6 weeks post injury.