Tokarz Danielle, Cisek Richard, Joseph Ariana, Golaraei Ahmad, Mirsanaye Kamdin, Krouglov Serguei, Asa Sylvia L, Wilson Brian C, Barzda Virginijus
Department of Chemistry, Saint Mary's University, Halifax, NS, Canada.
Department of Physics, University of Toronto, Toronto, ON, Canada.
Front Oncol. 2019 Apr 17;9:272. doi: 10.3389/fonc.2019.00272. eCollection 2019.
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 '/χ ', was obtained that serves as a structural parameter for characterizing the tissue. Furthermore, by assuming C symmetry, an additional second-order non-linear optical susceptibility ratio, χ '/χ ', was obtained, which is a measure of the chirality of the collagen fibers. Statistically-significant differences in the χ '/χ ' 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 '/χ ' 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 '/χ ' values, the FWHM of χ '/χ ' values and the DOLP values could potentially be used to aid pathologists in diagnosing pancreatic cancer.
使用多光子激发荧光（MPF）、二次谐波产生（SHG）和三次谐波产生（THG）显微镜对苏木精和伊红染色的正常和肿瘤胰腺组织的薄组织切片进行了研究。由于伊红染色，细胞质、结缔组织、胶原蛋白和细胞外结构可通过MPF可视化，而胶原蛋白则通过不需要染色的内源性SHG对比度成像。由于苏木精染料的聚集，细胞结构，包括膜界面和核成分，可通过THG观察到。通过偏振敏感的SHG显微镜技术，即偏振入、偏振出（PIPO）SHG，研究了胰腺癌中胶原超微结构的变化。这涉及测量SHG信号的线性偏振方向作为入射激光辐射的线性偏振方向的函数。从PIPO SHG数据中，获得了二阶非线性光学极化率比χ '/χ '，它作为表征组织的结构参数。此外，通过假设C对称性，获得了另一个二阶非线性光学极化率比χ '/χ '，它是胶原纤维手性的量度。在导管周围、小叶和实质区域的肿瘤和正常胰腺组织之间，发现χ '/χ '值存在统计学上的显著差异，而在导管周围和实质区域的肿瘤和正常胰腺组织之间，发现χ '/χ '出现直方图的半高宽（FWHM）存在统计学上的显著差异。此外，PIPO SHG数据用于确定SHG信号的线性偏振度（DOLP），它表示信号的相对线性去极化。在导管周围和实质区域的肿瘤和正常胰腺组织之间，发现DOLP值存在统计学上的显著差异。因此，在χ '/χ '值、χ '/χ '值的FWHM和DOLP值中观察到的差异可能有助于病理学家诊断胰腺癌。
