Characterization of pathological thyroid tissue using polarization-sensitive second harmonic generation microscopy.

Polarization-sensitive second harmonic generation (SHG) microscopy is an established imaging technique able to provide information related to specific molecular structures including collagen. In this investigation, polarization-sensitive SHG microscopy was used to investigate changes in the collagen ultrastructure between histopathology slides of normal and diseased human thyroid tissues including follicular nodular disease, Grave's disease, follicular variant of papillary thyroid carcinoma, classical papillary thyroid carcinoma, insular or poorly differentiated carcinoma, and anaplastic or undifferentiated carcinoma ex vivo. The second-order nonlinear optical susceptibility tensor component ratios, χ(2)zzz'/χ(2)zxx' and χ(2)xyz'/χ(2)zxx', were obtained, where χ(2)zzz'/χ(2)zxx' is a structural parameter and χ(2)xyz'/χ(2)zxx' is a measure of the chirality of the collagen fibers. Furthermore, the degree of linear polarization (DOLP) of the SHG signal was measured. A statistically significant increase in χ(2)zzz'/χ(2)zxx' values for all the diseased tissues except insular carcinoma and a statistically significant decrease in DOLP for all the diseased tissues were observed compared to normal thyroid. This finding indicates a higher ultrastructural disorder in diseased collagen and provides an innovative approach to discriminate between normal and diseased thyroid tissues that is complementary to standard histopathology.

Characterization of Pancreatic Cancer Tissue Using Multiphoton Excitation Fluorescence and Polarization-Sensitive Harmonic Generation Microscopy.

Thin tissue sections of normal and tumorous pancreatic tissues stained with hematoxylin and eosin were investigated using multiphoton excitation fluorescence (MPF), second harmonic generation (SHG), and third harmonic generation (THG) microscopies. The cytoplasm, connective tissue, collagen and extracellular structures are visualized with MPF due to the eosin stain, whereas collagen is imaged with endogenous SHG contrast that does not require staining. Cellular structures, including membranous interfaces and nuclear components, are seen with THG due to the aggregation of hematoxylin dye. Changes in the collagen ultrastructure in pancreatic cancer were investigated by a polarization-sensitive SHG microscopy technique, polarization-in, polarization-out (PIPO) SHG. This involves measuring the orientation of the linear polarization of the SHG signal as a function of the linear polarization orientation of the incident laser radiation. From the PIPO SHG data, the second-order non-linear optical susceptibility ratio, χ(2) zzz '/χ(2) zxx ', was obtained that serves as a structural parameter for characterizing the tissue. Furthermore, by assuming C6 symmetry, an additional second-order non-linear optical susceptibility ratio, χ(2) xyz '/χ(2) zxx ', was obtained, which is a measure of the chirality of the collagen fibers. Statistically-significant differences in the χ(2) zzz '/χ(2) zxx ' values were found between tumor and normal pancreatic tissues in periductal, lobular, and parenchymal regions, whereas statistically-significant differences in the full width at half maximum (FWHM) of χ(2) xyz '/χ(2) zxx ' occurrence histograms were found between tumor and normal pancreatic tissues in periductal and parenchymal regions. Additionally, the PIPO SHG data were used to determine the degree of linear polarization (DOLP) of the SHG signal, which indicates the relative linear depolarization of the signal. Statistically-significant differences in DOLP values were found between tumor and normal pancreatic tissues in periductal and parenchymal regions. Hence, the differences observed in the χ(2) zzz '/χ(2) zxx ' values, the FWHM of χ(2) xyz '/χ(2) zxx ' values and the DOLP values could potentially be used to aid pathologists in diagnosing pancreatic cancer.