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      Alterations of the extracellular matrix in ovarian cancer studied by Second Harmonic Generation imaging microscopy

      research-article
      1 , 1 , 2 , 1 ,
      BMC Cancer
      BioMed Central

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          Abstract

          Background

          Remodeling of the extracellular matrix (ECM) has been implicated in ovarian cancer, and we hypothesize that these alterations may provide a better optical marker of early disease than currently available imaging/screening methods and that understanding their physical manifestations will provide insight into invasion.

          Methods

          For this investigation we use Second Harmonic Generation (SHG) imaging microcopy to study changes in the structure of the ovarian ECM in human normal and malignant ex vivo biopsies. This method directly visualizes the type I collagen in the ECM and provides quantitative metrics of the fibrillar assembly. To quantify these changes in collagen morphology we utilized an integrated approach combining 3D SHG imaging measurements and bulk optical parameter measurements in conjunction with Monte Carlo simulations of the experimental data to extract tissue structural properties.

          Results

          We find the SHG emission attributes (directionality and relative intensity) and bulk optical parameters, both of which are related to the tissue structure, are significantly different in the tumors in a manner that is consistent with the change in collagen assembly. The normal and malignant tissues have highly different collagen fiber assemblies, where collectively, our findings show that the malignant ovaries are characterized by lower cell density, denser collagen, as well as higher regularity at both the fibril and fiber levels. This further suggests that the assembly in cancer may be comprised of newly synthesized collagen as opposed to modification of existing collagen.

          Conclusions

          Due to the large structural changes in tissue assembly and the SHG sensitivity to these collagen alterations, quantitative discrimination is achieved using small patient data sets. Ultimately these measurements may be developed as intrinsic biomarkers for use in clinical applications.

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          Most cited references38

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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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            Interpreting second-harmonic generation images of collagen I fibrils.

            Fibrillar collagen, being highly noncentrosymmetric, possesses a tremendous nonlinear susceptibility. As a result, second-harmonic generation (SHG) microscopy of collagen produces extremely bright and robust signals, providing an invaluable tool for imaging tissue structure with submicron resolution. Here we discuss fundamental principles governing SHG phase matching with the tightly focusing optics used in microscopy. Their application to collagen imaging yields several biophysical features characteristic of native collagen structure: SHG radiates from the shell of a collagen fibril, rather than from its bulk. This SHG shell may correspond to the supporting element of the fibril. Physiologically relevant changes in solution ionic strength alter the ratio of forward-to-backward propagating SHG, implying a resulting change in the SHG shell thickness. Fibrillogenesis can be resolved in immature tissue by directly imaging backward-propagating SHG. Such findings are crucial to the design and development of forthcoming diagnostic and research tools.
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              Three-dimensional high-resolution second-harmonic generation imaging of endogenous structural proteins in biological tissues.

              We find that several key endogenous protein structures give rise to intense second-harmonic generation (SHG)-nonabsorptive frequency doubling of an excitation laser line. Second-harmonic imaging microscopy (SHIM) on a laser-scanning system proves, therefore, to be a powerful and unique tool for high-resolution, high-contrast, three-dimensional studies of live cell and tissue architecture. Unlike fluorescence, SHG suffers no inherent photobleaching or toxicity and does not require exogenous labels. Unlike polarization microscopy, SHIM provides intrinsic confocality and deep sectioning in complex tissues. In this study, we demonstrate the clarity of SHIM optical sectioning within unfixed, unstained thick specimens. SHIM and two-photon excited fluorescence (TPEF) were combined in a dual-mode nonlinear microscopy to elucidate the molecular sources of SHG in live cells and tissues. SHG arose not only from coiled-coil complexes within connective tissues and muscle thick filaments, but also from microtubule arrays within interphase and mitotic cells. Both polarization dependence and a local symmetry cancellation effect of SHG allowed the signal from species generating the second harmonic to be decoded, by ratiometric correlation with TPEF, to yield information on local structure below optical resolution. The physical origin of SHG within these tissues is addressed and is attributed to the laser interaction with dipolar protein structures that is enhanced by the intrinsic chirality of the protein helices.
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                Author and article information

                Journal
                BMC Cancer
                BMC Cancer
                BioMed Central
                1471-2407
                2010
                11 March 2010
                : 10
                : 94
                Affiliations
                [1 ]Center for Cell Analysis and Modeling, Department of Cell Biology, University of Connecticut Health Center, Farmington, CT 06030, USA
                [2 ]Neag Cancer Center, Division of Gynecologic Oncology, University of Connecticut Health Center, Farmington, CT 06030, USA
                Article
                1471-2407-10-94
                10.1186/1471-2407-10-94
                2841668
                20222963
                5ebd99a8-b3a1-4449-a068-dfa47cbede10
                Copyright ©2010 Nadiarnykh et al; licensee BioMed Central Ltd.

                This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

                History
                : 29 October 2009
                : 11 March 2010
                Categories
                Research Article

                Oncology & Radiotherapy
                Oncology & Radiotherapy

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