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Both ovarian cancer and idiopathic pulmonary fibrosis (IPF) are accompanied by significant collagen remodeling in the respective extracellular matrix (ECM). These diseases have similar attributes and collagen alterations can be probed with the same methods. Remodeling can be reflected in increased collagen concentration, changes in alignment within fibrils and fibers and/or up-regulation of different collagen isoforms. We used pixel-based SHG polarization analyses to discriminate the macro/supramolecular collagen structure in human tissues by: i) determination of the helical pitch angle via the single axis molecular model, ii) dipole alignment within fibrils via anisotropy, and iii) chirality via SHG circular dichroism (SHG-CD). For ovarian cancer, the largest differences were between normal stroma and benign tumors, consistent with gene expression showing Col III is up-regulated in the latter. The tissues also displayed differing SHG anisotropies and SHG-CD responses, consistent with randomization of Col I alignment in fibrils in all tumors. These results collectively indicate the fibril assemblies are distinct in all ovarian tissues and likely result from synthesis of new collagen rather than remodeling of existing collagen. For IPF, the largest change was in the SHG-CD response, indicating the fibrotic collagen has different helical structure than that of normal tissues. Interestingly, for both diseases, no increase in Col IIII was found, in contrast to previous reports by immunostaining. We suggest these polarization-based metrics could form the basis of a new classification scheme and complement conventional classification based on genetic profiles and conventional histology for these diseases as well as other cancers and fibroses.

Remodeling of the extracellular matrix in human ovarian cancer, can be reflected in increased collagen concentration, changes in alignment and/or up-regulation of different collagen isoforms, including Col III. Using fibrillar gel models, we demonstrate that Col I and Col III can be quantitatively distinguished by 3 distinct SHG polarization specific metrics: i) determination of helical pitch angle via the single axis molecular model, ii) dipole alignment via anisotropy, and iii) chirality via SHG circular dichroism (SHG-CD). These sub-resolution differentiations are possible due to differences in the α helix angles of the two isoforms, which co-mingle in the same fibrils. We also investigated the mechanism of the SHG-CD response and show that unlike conventional CD, it is dominated by electric dipole interactions and is consistent with the two state SHG model. We further applied these 3 polarization resolved analyses to human normal, high risk, benign tumors, and malignant human ovarian tissues. We found that these tissues could all be differentiated by these metrics, where high grade tissues had analogous α-helical pitch angles to the in the Col I/Col III gel model. This confirms literature suggestions based on immunofluorescence and gene expression that Col III is up-regulated in high grade ovarian cancers. The different tissues also displayed differing anisotropies, indicating the fibril assemblies are distinct and likely do not result from remodeling of existing collagen but synthesis of new collagen. Importantly, these SHG polarization methods provide structural information not otherwise possible and can serve as label-free biomarkers for ovarian and other cancers.

Previous studies have shown that bone growth acceleration can occur in many animal species after periosteal resection (removal of a strip of periosteum) with minimum morbidity. This has numerous clinical applications, including treatment of limb length differences. Here we use Second Harmonic Generation (SHG) imaging microscopy to evaluate changes in collagen architecture reflective of the different strains the periosteum may encounter during bone growth. Specifically, we image rabbit tibial periosteum strips at -20%, 0%, 5%, and 10% strains. We first quantify these changes using the SHG creation ratio (Forward/Backward) or the initially emitted SHG directionality to provide information on the fibril level of assembly. The in situ (i.e. physiological) strain had the highest creation ratio compared to the non-in situ strains of -20%, 5%, and 10%, which were shown to be significantly different via RCBD statistical analysis. These trends are consistent with SHG phasematching considerations, where more organized fibrils/fibers result in primarily forward emitted components, which here is the physiological strain. We further use the relative SHG conversion efficiency to assess the tissue structure under strain, where this results from the combination of collagen concentration and organization. The 0% strain SHG conversion efficiency was significantly higher than all other strains, where this is expected as the fibers have the highest local density and organization, and is consistent with the emission directionality results. Importantly, due to the underlying physical process, the label-free SHG imaging modality can non-invasively monitor the effect of treatments for bone growth and other orthopedic disorders.