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

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

Published versionThin 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

Polarimetric Second-Harmonic Generation Microscopy for Histopathology

Second-harmonic generation (SHG) is one of the modalities of nonlinear microscopy, which can provide structural visualization and characterization, as well as 3D optical sectioning of the material without the need for staining. Polarimetric SHG microscopy can reveal an immense amount of structural information about the specimen beyond the diffraction limited spatial resolution of the microscope by extracting the second-order nonlinear susceptibility tensor elements ratios. SHG microscopy as well as polarimetric SHG microscopy have become important imaging techniques, specially in the study of biological materials such as collagen. Collagen is the most abundant protein in human body which plays an important role in a number of biological processes including wound repair, tumour development, and cancer metastasis. This thesis focuses on studying the nonlinear optical properties of collagen using polarimetric SHG microscopy and utilizing these nonlinear optical parameters as new metrics for the field of histopathology.Two polarimetric SHG techniques, double Stokes-Mueller polarimetry (DSMP) and reduced DSMP known as linear polarization-in, polarization-out (PIPO) were utilized to study the nonlinear optical properties of collagen fibres in thin tissue sections. Two most commonly assumed symmetry classes for collagen fibres, C6 and C6v were experimentally investigated. C6 class is a more precise description of collagen fibres, as it accounts for chirality of collagen. Measurement of the chiroptical properties of collagen is shown to be linked to the 3D organization of collagen fibres, the polarity of collagen, as well as the relative phase differences between susceptibility tensor elements. Further, PIPO SHG microscopy provides the molecular susceptibility ratios of collagen. Polarimetric SHG microscopy revealed the structural alteration of collagen in tissues with pathological conditions through three main parameters: the susceptibility components ratio, degree of linear polarization of the outgoing SHG and the angle distribution of collagen fibres in the image plane. Polarimetric SHG microscopy shows great potential for understanding the tissue morphology and function, and ultimately as a diagnostic tool in histopathology. The polarimetric SHG technique was further implemented into a large area scanning system for the first time. A large area polarimetric SHG image can elucidate the morphological changes of collagen in diseased tissues such as cancer and can be used as a complementary method to the currently existing techniques in histopathology.Ph.D

Collagen fibril organization within rat vertebral bone modified with metastatic involvement

Metastatic involvement diminishes the mechanical integrity of vertebral bone, however its specific impact on the structural characteristics of a primary constituent of bone tissue, the collagen-I fibril matrix, has not been adequately characterized. Female athymic rats were inoculated with HeLa or Ace-1 cancer cells lines producing osteolytic or mixed (osteolytic & osteoblastic) metastases respectively. A maximum of 21days was allowed between inoculation and rat sacrifice for vertebrae extraction. Linear polarization-in, polarization-out (PIPO) second harmonic generation (SHG) and transmission electron microscopy (TEM) imaging was utilized to assess the impact of metastatic involvement on collagen fibril organization. Increased observations of deviations in the typical plywood motif or a parallel packing structure and an increased average measured susceptibility ratio (related to relative degree of in-plane vs. out-plane fibrils in the analyzed tissue area) in bone adjacent to metastatic involvement was indicative of change in fibrilar organization compared to healthy controls. In particular, collagen-I fibrils in tumour-induced osteoblastic bone growth showed no adherence to the plywood motif or parallel packing structure seen in healthy lamellar bone, exhibiting a much higher susceptibility ratio and degree of fibril disorder. Negative correlations were established between measured susceptibility ratios and the hardness and modulus of metastatic bone tissue assessed in a previous study. Characterizing modifications in tissue level properties is key in defining bone quality in the presence of metastatic disease and their potential impact on material behaviour.Grant funding for this study was provided by the Canadian Institutes of Health Researc

Dual- and single-shot susceptibility ratio measurements with circular polarizations in second-harmonic generation microscopy

Polarization-resolved second harmonic generation (P-SHG) microscopy is a technique capable of characterizing nonlinear optical properties of noncentrosymmetric biomaterials by extracting the nonlinear susceptibility tensor components ratio χð2Þ0 zzz =χ ð2Þ0 zxx , with z-axis parallel and x-axis perpendicular to the C6 symmetry axis of molecular fiber, such as a myofibril or a collagen fiber. In this paper, we present two P-SHG techniques based on incoming and outgoing circular polarization states for a fast extraction of χ ð2Þ0 zzz =χ ð2Þ0 zxx : A dual-shot configuration where the SHG circular anisotropy generated using incident right- and left-handed circularly-polarized light is measured; and a single-shot configuration for which the SHG circular anisotropy is measured using only one incident circular polarization state. These techniques are used to extract the χ ð2Þ0 zzz =χ ð2Þ0 zxx of myosin fibrils in the body wall muscles of Drosophila melanogaster larva. The results are in good agreement with values obtained from the double Stokes-Mueller polarimetry. The dual- and single-shot circular anisotropy measurements can be used for fast imaging that is independent of the in-plane orientation of the sample. They can be used for imaging of contracting muscles, or for high throughput imaging of large sample areas

Complex susceptibilities and chiroptical effects of collagen measured with polarimetric second-harmonic generation microscopy

Nonlinear optical properties of collagen type-I are investigated in thin tissue sections of pig tendon as a research model using a complete polarimetric second-harmonic generation (P-SHG) microscopy technique called double Stokes-Mueller polarimetry (DSMP). Three complex-valued molecular susceptibility tensor component ratios are extracted. A significant retardance is observed between the chiral susceptibility component and the achiral components, while the achiral components appear to be in phase with each other. The DSMP formalism and microscopy measurements are further used to explain and experimentally validate the conditions required for SHG circular dichroism (SHG-CD) of collagen to occur. The SHG-CD can be observed with the microscope when: (i) the chiral second-order susceptibility tensor component has a non-zero value, (ii) a phase retardance is present between the chiral and achiral components of the second-order susceptibility tensor and (iii) the collagen fibres are tilted out of the image plane. Both positive and negative areas of SHG-CD are observed in microscopy images, which relates to the anti-parallel arrangement of collagen fibres in different fascicles of the tendon. The theoretical formalism and experimental validation of DSMP imaging technique opens new opportunities for ultrastructural characterisation of chiral molecules, in particular collagen, and provides basis for the interpretation of SHG-CD signals. The nonlinear imaging of chiroptical parameters offers new possibilities to further improve the diagnostic sensitivity and/or specificity of nonlinear label-free histopathology