Second-harmonic generation-based Mueller matrix polarization analysis of collagen-rich tissues

Quantitative assessment of the properties of fibrillar collagen in tissue can yield deeper insight into structure-function correlations of the cell and its surrounding matrix. Second-harmonic generation (SHG) microscopy is especially well-suited as an image acquisition technique, due to its specificity to the non-centrosymmetric structure of collagen, and inherent confocality which enables three-dimensional sectioning. SHG imaging can be undertaken in a quantifiable manner, or combined with other techniques that highlight desired properties. A powerful property for characterizing collagenous tissue microstructure is the Mueller matrix polarization response. Two polarimetric imaging approaches are demonstrated for robust Mueller matrix characterization of collagenous tissue. One approach, called the two-photon Mueller matrix second-harmonic generation (MMSHG) microscopy, involves the generalization of Mueller matrix to the case of two-photon excitation. This 4-by-9 two-photon Mueller matrix is extracted using second-harmonic generation microscopy and analyzed for quantitative collagen assessment. The matrix and associated degree-of-polarization parameter from different sample types and thicknesses are also investigated. It was observed that the polarization-dependent degree-of-polarization distribution shape changes and a model-based bimodal mean difference metric increases with sample thickness. The second polarization technique which we developed, called second-harmonic patterned polarization-analyzed reflection confocal (SPPARC) microscopy, uses the conventional linear polarimetry of confocal images, delineated with a second-harmonic mask. This latter approach, combining the metric richness of linear polarimetry with the specificity of SHG imaging, is used for assessing collagen, as well as non-collagenous regions, in porcine tendon and ligament. We observed differences in depolarization and circular degree-of-polarization parameters, that have potential for dfferentiating tissues in varying states. Next, we present the results of SPPARC microscopy and analysis of collagen on varying pathologies of breast tissues. Experiments were conducted on a breast tissue microarray having benign tissues (BT), malignant invasive lobular carcinoma (ILC), and benign stroma adjacent to the malignant tissues (called the benign adjacent tissue, or BAT). We observed that stroma in BAT and ILC exhibits the largest parameter differences, with collagen readings in ILC showing lower depolarization, lower diattenuation and higher linear degree-of-polarization values than stromal collagen in BAT. This result suggests that the optical properties of collagen change most in the vicinity of tumors. A similar trend is also exhibited in the non-collagenous extrafibrillar matrix plus cells (EFMC) region. We finally discuss additional work involving polarization modeling, setup optimization, and implementation of other decomposition techniques