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      Maturation of the Meniscal Collagen Structure Revealed by Polarization-Resolved and Directional Second Harmonic Generation Microscopy

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          Abstract

          We report Polarization-resolved Second Harmonic Generation (P-SHG) and directional SHG (forward and backward, F/B) measurements of equine foetal and adult collagen in meniscus, over large field-of-views using sample-scanning. Large differences of collagen structure and fibril orientation with maturation are revealed, validating the potential for this novel methodology to track such changes in meniscal structure. The foetal menisci had a non-organized and more random collagen fibrillar structure when compared with adult using P-SHG. For the latter, clusters of homogeneous fibril orientation (inter-fibrillar areas) were revealed, separated by thick fibers. F/B SHG showed numerous different features in adults notably, in thick fibers compared to interfibrillar areas, unlike foetal menisci that showed similar patterns for both directions. This work confirms previous studies and improves the understanding of meniscal collagen structure and its maturation, and makes F/B and P-SHG good candidates for future studies aiming at revealing structural modifications to meniscus due to pathologies.

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          Live tissue intrinsic emission microscopy using multiphoton-excited native fluorescence and second harmonic generation.

          Multicolor nonlinear microscopy of living tissue using two- and three-photon-excited intrinsic fluorescence combined with second harmonic generation by supermolecular structures produces images with the resolution and detail of standard histology without the use of exogenous stains. Imaging of intrinsic indicators within tissue, such as nicotinamide adenine dinucleotide, retinol, indoleamines, and collagen provides crucial information for physiology and pathology. The efficient application of multiphoton microscopy to intrinsic imaging requires knowledge of the nonlinear optical properties of specific cell and tissue components. Here we compile and demonstrate applications involving a range of intrinsic molecules and molecular assemblies that enable direct visualization of tissue morphology, cell metabolism, and disease states such as Alzheimer's disease and cancer.
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            Dynamic imaging of collagen and its modulation in tumors in vivo using second-harmonic generation.

            The content and structure of collagen is essential in governing the delivery of therapeutic molecules in tumors. Thus, simple histological staining of tumor tissue biopsies for collagen could be used to assess the accessibility of molecular therapeutics in tumors. Here we show that it is possible to optically image fibrillar collagen in tumors growing in mice using second-harmonic generation (SHG). Using this noninvasive technique, we estimated relative diffusive hindrance, quantified the dynamics of collagen modification after pharmacologic intervention and provided mechanistic insight into improved diffusive transport induced by the hormone relaxin. This technology could offer basic scientists and clinicians an enhanced ability to estimate the relative penetrabilities of molecular therapeutics.
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              Polarization-modulated second harmonic generation in collagen.

              Collagen possesses a strong second-order nonlinear susceptibility, a nonlinear optical property characterized by second harmonic generation in the presence of intense laser beams. We present a new technique involving polarization modulation of an ultra-short pulse laser beam that can simultaneously determine collagen fiber orientation and a parameter related to the second-order nonlinear susceptibility. We demonstrate the ability to discriminate among different patterns of fibrillar orientation, as exemplified by tendon, fascia, cornea, and successive lamellar rings in an intervertebral disc. Fiber orientation can be measured as a function of depth with an axial resolution of approximately 10 microm. The parameter related to the second-order nonlinear susceptibility is sensitive to fiber disorganization, oblique incidence of the beam on the sample, and birefringence of the tissue. This parameter represents an aggregate measure of tissue optical properties that could potentially be used for optical imaging in vivo.
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                Author and article information

                Contributors
                sheila.laverty@umontreal.ca
                legare@emt.inrs.ca
                Journal
                Sci Rep
                Sci Rep
                Scientific Reports
                Nature Publishing Group UK (London )
                2045-2322
                5 December 2019
                5 December 2019
                2019
                : 9
                : 18448
                Affiliations
                [1 ]ISNI 0000 0000 9582 2314, GRID grid.418084.1, Institut National de la Recherche Scientifique, Centre Énergie Matériaux Télécommunications (INRS-EMT); 1650 Boul. Lionel-Boulet, ; Varennes (QC), J3X 1S2 Canada
                [2 ]ISNI 0000 0001 2292 3357, GRID grid.14848.31, Comparative Orthopedic Research Laboratory, Department of Clinical Sciences, Faculté de médecine vétérinaire, , Université de Montréal, ; Saint-Hyacinthe (QC), J2S 2M2 Canada
                [3 ]ISNI 0000 0004 0370 2251, GRID grid.503294.9, Laboratoire d’Optique et Biosciences (LOB), École Polytechnique, CNRS, Inserm, Institut Polytechnique de Paris, ; F-91128 Palaiseau, France
                Article
                54942
                10.1038/s41598-019-54942-0
                6895152
                31804577
                43f44e94-f6d3-4584-a272-74295687e810
                © The Author(s) 2019

                Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

                History
                : 11 July 2019
                : 12 November 2019
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                © The Author(s) 2019

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                microscopy,imaging
                Uncategorized
                microscopy, imaging

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