Spinal cord injury is a dramatic disease leading to severe motor, sensitive and autonomic impairments. After injury the axonal regeneration is partly inhibited by the glial scar, acting as a physical and chemical barrier. The scarring process involves microglia, astrocytes and extracellular matrix components, such as collagen, con- structing the fibrotic component of the scar. To investigate the role of collagen, we used a multimodal label-free imaging approach combining multiphoton and atomic force microscopy. The second harmonic generation signal exhibited by fibrillar collagen enabled to specifically monitor it as a biomarker of the lesion. An increase in collagen density and the formation of more tortuous fibers over time after injury are observed. Nano-mechanical investigations revealed a noticeable hardening of the injured area, correlated with collagen fibers' formation. These observations indicate the concomitance of important structural and mechanical modifications during the fibrotic scar evolution.

Spinal cord injury (SCI) is a dramatic disease leading to severe motor, sensitive and autonomic impairments. After the injury, the axonal regeneration is partly inhibited by the glial scar, acting as a physical and chemical barrier[1]. The scarring process involves microglia, astrocytes, and extracellular matrix components, such as collagen, composing the fibrotic part of the scar[2]. To investigate the role of collagen and microglia, we used a multimodal label-free imaging approach combining multiphoton and atomic force microscopies. The second harmonic generation signal exhibited by fibrillar collagen-I enables specifically monitoring it as a biomarker of the lesion. An increase in collagen density and the formation of more curved fibers over time after SCI are observed. Whereas 2-photon excitation microscopy (2PEF) showed the appearance and activation of microglia over millimeters in length near the injured area. Nanomechanical investigations revealed a noticeable hardening of the injured area, correlated with collagen fibers’ development. Additionally, we observed that inhibition of microglial proliferation by oral administration of GW2580 decreased the collagen density at the injured area. These observations indicate the concomitance of relevant structural and mechanical modifications during the fibrotic scar evolution.

Sarcomere length (SL) and its variation along the myofibril strongly regulate integrated coordinated myocyte contraction. It is therefore important to obtain individual SL properties. Optical imaging by confocal fluorescence (for example, using ANEPPS) or transmitted light microscopy is often used for this purpose. However, this allows for the visualization of structures related to Z-disks only. In contrast, second-harmonic generation (SHG) microscopy visualizes A-band sarcomeric structures directly. Here, we compared averaged SL and its variability in isolated relaxed rat cardiomyocytes by imaging with ANEPPS and SHG. We found that SL variability, evaluated by several absolute and relative measures, is two times smaller using SHG vs. ANEPPS, while both optical methods give the same average (median) SL. We conclude that optical methods with similar optical spatial resolution provide valid estimations of average SL, but the use of SHG microscopy for visualization of sarcomeric A-bands may be the “gold standard” for evaluation of SL variability due to the absence of optical interference between the sarcomere center and non-sarcomeric structures. This contrasts with sarcomere edges where t-tubules may not consistently colocalize to Z-disks. The use of SHG microscopy instead of fluorescent imaging can be a prospective tool to map sarcomere variability both in vitro and in vivo conditions and to reveal its role in the functional behavior of living myocardium.

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.

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.