In situ three-dimensional monitoring of collagen fibrillogenesis using SHG microscopy.

International audienceWe implemented in situ time-lapse Second Harmonic Generation (SHG) microscopy to monitor the three-dimensional (3D) self-assembly of collagen in solution. As a proof of concept, we tuned the kinetics of fibril formation by varying the pH and measured the subsequent exponential increase of fibril volume density in SHG images. We obtained significantly different time constants at pH = 6.5 ± 0.3 and at pH = 7.5 ± 0.3. Moreover, we showed that we could focus on the growth of a single isolated collagen fibril because SHG microscopy is sensitive to well-organized fibrils with diameter below the optical resolution. This work illustrates the potential of SHG microscopy for the rational design and characterization of collagen-based biomaterials

Fibrillogenesis from nanosurfaces: multiphoton imaging and stereological analysis of collagen 3D self-assembly dynamics

International audienceThe assembly of proteins into fibrillar structures is an important process that concerns different biological contexts, including molecular medicine and functional biomaterials. Engineering of hybrid biomaterials can advantageously provide synergetic interactions of the biopolymers with an inorganic component to ensure specific supramolecular organization and dynamics. To this aim, we designed hybrid systems associating collagen and surface-functionalized silica particles and we built a new strategy to investigate fibrillogenesis processes in such multicomponents systems, working at the crossroads of chemistry, physics and mathematics. The self-assembly process was investigated by bimodal multiphoton imaging coupling second harmonic generation (SHG) and 2 photon excited fluorescence (2PEF). The in-depth spatial characterization of the system was further achieved using the three-dimensional analysis of the SHG/2PEF data via mathematical morphology processing. Quantitation of collagen distribution around particles offers strong evidence that the chemically induced confinement of the protein on the silica nanosurfaces has a key influence on the spatial extension of fibrillogenesis. This new approach is unique in the information it can provide on 3D dynamic hybrid systems and may be extended to other associations of fibrillar molecules with optically responsive nano-objects

Ex vivo multiscale quantitation of skin biomechanics in wild-type and genetically-modified mice using multiphoton microscopy

International audienceSoft connective tissues such as skin, tendon or cornea are made of about 90% of extracellular matrix proteins, fibrillar collagens being the major components. Decreased or aberrant collagen synthesis generally results in defective tissue mechanical properties as the classic form of Elhers-Danlos syndrome (cEDS). This connective tissue disorder is caused by mutations in collagen V genes and is mainly characterized by skin hyperextensibility. To investigate the relationship between the microstructure of normal and diseased skins and their macroscopic mechanical properties, we imaged and quantified the microstructure of dermis of ex vivo murine skin biopsies during uniaxial mechanical assay using multiphoton microscopy. We used two genetically-modified mouse lines for collagen V: a mouse model for cEDS harboring a Col5a2 deletion (a.k.a. pN allele) and the transgenic K14-COL5A1 mice which overexpress the human COL5A1 gene in skin. We showed that in normal skin, the collagen fibers continuously align with stretch, generating the observed increase in mechanical stress. Moreover, dermis from both transgenic lines exhibited altered collagen reorganization upon traction, which could be linked to microstructural modifications. These findings show that our multiscale approach provides new crucial information on the biomechanics of dermis that can be extended to all collagen-rich soft tissues

Determination of collagen fiber orientation in histological slides using Mueller microscopy and validation by second harmonic generation imaging.

International audienceWe studied the azimuthal orientations of collagen fibers in histological slides of uterine cervical tissue by two different microscopy techniques, namely Mueller polarimetry (MP) and Second Harmonic Generation (SHG). SHG provides direct visualization of the fibers with high specificity, which orientations is then obtained by suitable image processing. MP provides images of retardation (among other polarimetric parameters) due to the optical anisotropy of the fibers, which is enhanced by Picrosirius Red staining. The fiber orientations are then assumed to be those of the retardation slow axes. The two methods, though fully different from each other, provide quite similar maps of average fiber orientations. Overall, our results confirm that MP microscopy provides reliable images of dominant fiber orientations at a much lower cost that SHG, which remains the "gold standard" for specific imaging of collagen fibers using optical microscopy

Analyse multi-échelles des propriétés biomécaniques de la peau de souris saine et malade

La peau est un tissu complexe composé de 3 couches : l'épiderme, le derme et l'hypoderme. Le derme est responsable de la majeure partie des propriétés mécaniques de la peau. Une modification de la composition du derme entraîne ainsi des modifications drastiques du comportement mécanique de la peau, comme dans la maladie d'Ehlers-Danlos, qui se caractérise par une hyper?élasticité des tissus. Au niveau microstructural, le derme est composé essentiellement de matrice extracellulaire, formée pour la majeure partie d'un réseau désordonné de fibres de collagène. Pour élucider le lien exact entre organisation microstructurale et propriétés mécaniques de la peau, nous réalisons des essais de traction uniaxiaux in situ sous un microscope multiphoton avec détection du signal de génération de seconde harmonique. Ceci nous permet de suivre la réponse de la microstructure du tissu au cours de l'essai mécanique. Des paramètres quantitatifs ont été développés pour caractériser à la fois la réponse mécanique macroscopique du tissu et le réarrangement du réseau de fibres de collagène sous chargement. Nous pouvons ainsi comparer le comportement multi-échelles de peau de souris saine et de peau de souris atteinte d'une mutation affectant la microstructure du derme

Imagerie Quantitative du Collagène par Génération de Seconde Harmonique

Collagen is an ubiquitous protein which plays a central role in the architecture and the mechanical integrity of connective tissues. Synthesized as triple helices, collagen self-assembles into fibrils both in vivo and in vitro to form three-dimensional networks. It is essential to probe this fibrillar organization to characterize tissu remodeling involved in many diseases and guide tissue engineering. Multiphoton microscopy based on second harmonic generation (SHG) is a very specific technique to visualize unstained collagen deep into tissues, with sub-micron resolution. This thesis aims to develop quantitative approaches in SHG imaging of collagen, at both fibrillar and tissular scales. We first showed that SHG microscopy is a relevant tool to measure the dynamics of formation of collagen networks, even at single fibril scale. We also characterized the structure of collagen gels controlled by adding functionalized silica nanoparticles. We then performed correlative electron/SHG imaging on these fibrillar gels. This allowed us to measure the sensitivity of our set-up and to calibrate the response of an isolated fibril as a function of its diameter. In addition, we derived the hyperpolarizability of a triple helix, which validated the additive model used to calculate the nonlinear response of a fibril. Concurrently, we developed specific image analysis to phenomenogically quantify the organization of the fibrillar network at micrometer scale with the view investigate the function/structure relationship in biological tissues. This was validated by characterizing the changes of biomechanical properties in genetically modified mice model of Ehlers-Danlos syndrome.Le collagène est une protéine ubiquitaire qui joue un rôle central dans l'architecture et la tenue mécanique des tissus conjonctifs et est impliqué dans de nombreuses pathologies. Synthétisé sous forme de triples hélices, le collagène s'auto-assemble en fibrilles in vivo et in vitro pour former des réseaux tridimensionnels. La microscopie multiphoton basée sur la génération de seconde harmonique (SHG) est une technique très spécifique, permettant de visualiser, sans marquage, le collagène en profondeur dans des tissus, avec une résolution sub-micrométrique. Ce travail de thèse vise à développer des approches quantitatives en imagerie SHG du collagène, tant à l'échelle fibrillaire que tissulaire. Nous avons montré que la microscopie SHG permet de sonder la dynamique de formation du réseau collagénique jusqu'à l'échelle d'une fibrille unique. En outre, nous avons caractérisé la structuration de gels collagéniques contrôlée par ajout de nanoparticules de silice fonctionnalisées. Nous avons ensuite réalisé de l'imagerie corrélative électronique/SHG sur ces gels pour mesurer la sensibilité de notre microscope et calibrer la réponse d'une fibrille en fonction de son diamètre. De plus, nous avons pu évaluer l'hyperpolarisabilité d'une molécule et valider le modèle additif utilisé pour calculer la réponse d'une fibrille. Enfin, nous avons développé une analyse d'images spécifique permettant de quantifier l'organisation d'un tissu collagénique à l'échelle fibrillaire, dans le but d'explorer la relation fonction/structure d'un tissu. Ceci a été validé en étudiant la modification des propriétés biomécaniques de souris génétiquement modifiées modèle du syndrome d'Ehlers-Danlos

Imagerie Quantitative du Collagène par Génération de Seconde Harmonique

Le collagène est une protéine ubiquitaire qui joue un rôle central dans l'architecture et la tenue mécanique des tissus conjonctifs et est impliqué dans de nombreuses pathologies. Synthétisé sous forme de triples hélices, le collagène s'auto-assemble en fibrilles in vivo et in vitro pour former des réseaux tridimensionnels. La microscopie multiphoton basée sur la génération de seconde harmonique (SHG) est une technique très spécifique, permettant de visualiser, sans marquage, le collagène en profondeur dans des tissus, avec une résolution sub-micrométrique. Ce travail de thèse vise à développer des approches quantitatives en imagerie SHG du collagène, tant à l'échelle fibrillaire que tissulaire. Nous avons montré que la microscopie SHG permet de sonder la dynamique de formation du réseau collagénique jusqu'à l'échelle d'une fibrille unique. En outre, nous avons caractérisé la structuration de gels collagéniques contrôlée par ajout de nanoparticules de silice fonctionnalisées. Nous avons ensuite réalisé de l'imagerie corrélative électronique/SHG sur ces gels pour mesurer la sensibilité de notre microscope et calibrer la réponse d'une fibrille en fonction de son diamètre. De plus, nous avons pu évaluer l'hyperpolarisabilité d'une molécule et valider le modèle additif utilisé pour calculer la réponse d'une fibrille. Enfin, nous avons développé une analyse d'images spécifique permettant de quantifier l'organisation d'un tissu collagénique à l'échelle fibrillaire, dans le but d'explorer la relation fonction/structure d'un tissu. Ceci a été validé en étudiant la modification des propriétés biomécaniques de souris génétiquement modifiées modèle du syndrome d'Ehlers-Danlos.Collagen is an ubiquitous protein which plays a central role in the architecture and the mechanical integrity of connective tissues. Synthesized as triple helices, collagen self-assembles into fibrils both in vivo and in vitro to form three-dimensional networks. It is essential to probe this fibrillar organization to characterize tissu remodeling involved in many diseases and guide tissue engineering. Multiphoton microscopy based on second harmonic generation (SHG) is a very specific technique to visualize unstained collagen deep into tissues, with sub-micron resolution. This thesis aims to develop quantitative approaches in SHG imaging of collagen, at both fibrillar and tissular scales. We first showed that SHG microscopy is a relevant tool to measure the dynamics of formation of collagen networks, even at single fibril scale. We also characterized the structure of collagen gels controlled by adding functionalized silica nanoparticles. We then performed correlative electron/SHG imaging on these fibrillar gels. This allowed us to measure the sensitivity of our set-up and to calibrate the response of an isolated fibril as a function of its diameter. In addition, we derived the hyperpolarizability of a triple helix, which validated the additive model used to calculate the nonlinear response of a fibril. Concurrently, we developed specific image analysis to phenomenogically quantify the organization of the fibrillar network at micrometer scale with the view investigate the function/structure relationship in biological tissues. This was validated by characterizing the changes of biomechanical properties in genetically modified mice model of Ehlers-Danlos syndrome.PALAISEAU-Polytechnique (914772301) / SudocSudocFranceF

Cover Image, Volume 69, Issue 6

Cover Illustration: 3D stimulated emission depletion (STED) microscopy image of astrocytic processes labeled with a fluorescent protein (ZsGreen; yellow), which infiltrate the neuropil in a living hippocampal brain slice. The neuropil was visualized by adding a fluorescent dye (ATTO514; grey) to the extracellular solution, outlining all cellular structures. The image was inverted so that cells appear as bright structures, while the extracellular spaces between turn out black. (See Arizono, M, et al, https://doi.org/10.1002/glia.23995.

Evolution of the Skin Microstructural Organization During a Mechanical Assay

International audienceSkin is a complex multi-layered tissue, consisting of three main parts: the epidermis, the dermis and the hypodermis. The dermis is responsible for most of the complex mechanical properties of skin, such as viscoelasticity, non-linearity and anisotropy. At the microscopic level the dermis consists for the greater part of extracellular matrix, compounded mainly of collagen fibers forming an orderless network. The mechanical properties of skin have been studied in the past, but their exact link with the microscopic organization is still an open question. The goal of our study is to measure the evolution of the microstructure during a mechanical assay and to improve existing mechanical models of skin with relevant parameters identified at the microscopic level.We perform uniaxial tensile test on ex vivo mouse skin. The mechanical tests are performed in situ under a second harmonic generation microscope. This allows us to determine quantitatively and simultaneously the mechanical response and the microstructural reorganization of the tissue. This technique can be used to better understand the link between pathological alterations of collagen synthesis, fibers organization, and alteration of the biomechanical properties of skin, as in the Ehlers-Danlos syndrome (EDS)