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

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.

Coherent and incoherent SHG in fibrillar cellulose matrices.

Second Harmonic Generation (SHG) microscopy probes the organization of tissue or material structure through morphological and polarization analyses. In terms of diagnostic or analytical potential, it is important to understand the coherent and incoherent aspects of the emission in highly scattering environments. It is also of fundamental importance whether the SHG polarization signatures are retained in such turbid media. We examine these issues for purified cellulose specimens, which, in analogy to structural proteins, comprise highly birefringent and chiral fibrillar structures. In these matrices we observe predominantly coherent forward directed emission as well as backwards contrast consisting of direct, coherent emission and an incoherent component arising from multiply scattered forward directed SHG. These processes display a pronounced depth dependence evidenced by changes in morphology as well in the measured forward-backwards ratio (F/B). Specifically, from regions near the surface the backwards channel displays small fibrils not present in the forward emission. In addition, at depths beyond one mean free path, the fibril morphologies become highly similar, suggesting the observed backwards contrast is also comprised of a component that arises from multiple scattering of the initially forward directed wave. The depth dependence of the forward to backward ratio is consistent with Monte Carlo simulations of photon diffusion based on the measured scattering coefficient mus of 75 cm-1 and anisotropy factor, g=0.94 at the SHG wavelength. Consistent with the experimental observations, these simulations indicate that the backwards channel becomes increasingly incoherent with increasing depth into the specimen. We also demonstrate that the polarization dependence of the SHG can be measured through 500 microm of thickness. Similarly, the SHG signal anisotropy is largely preserved through this depth with only a slight depolarization being observed.