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.
Polarization-second harmonic microscopy was utilized to investigate whether collagen ultrastructure in thyroid due to four carcinoma types and Graves' disease could be differentiated in human histopathology samples. Three parameters were extracted, revealing that the degree of linear polarization and χ(2)zzz/χ(2)zxx were effective in differentiating some diseases, while the parameter χ(2)xyz/χ(2)zxx was less effective.
Abstract
The extracellular matrix (ECM) collagen undergoes major remodeling during tumorigenesis. However, alterations to the ECM are not widely considered in cancer diagnostics, due to mostly ...uniform appearance of collagen fibers in white light images of hematoxylin and eosin-stained (H&E) tissue sections. Polarimetric second-harmonic generation (P-SHG) microscopy enables label-free visualization and ultrastructural investigation of non-centrosymmetric molecules, which, when combined with texture analysis, provides multiparameter characterization of tissue collagen. This paper demonstrates whole slide imaging of breast tissue microarrays using high-throughput widefield P-SHG microscopy. The resulting P-SHG parameters are used in classification to differentiate tumor from normal tissue, resulting in 94.2% for both accuracy and F1-score, and 6.3% false discovery rate. Subsequently, the trained classifier is employed to predict tumor tissue with 91.3% accuracy, 90.7% F1-score, and 13.8% false omission rate. As such, we show that widefield P-SHG microscopy reveals collagen ultrastructure over large tissue regions and can be utilized as a sensitive biomarker for cancer diagnostics and prognostics studies.
Collagen (type I) fibers are readily visualized with second harmonic generation (SHG) microscopy though the molecular origin of the signal has not yet been elucidated. In this study, the molecular ...origin of SHG from type I collagen is investigated using the time-dependent coupled perturbed Hartree–Fock calculations of the hyperpolarizibilities of glycine, proline, and hydroxyproline. Two effective nonlinear dipoles are found to orient in-the-plane of the amino acids, with one of the dipoles aligning close to the pitch orientation in the triple-helix, which provides the dominant contribution to the SHG polarization properties. The calculated hyperpolarizability tensor element ratios for the collagen triple-helix models: (Gly3) n 3, (Gly–Pro2) n 3, and (Gly–Pro–Hyp) n 3, are used to predict the second-order nonlinear susceptibility ratios, χ zzz (2)/χ iiz (2) and χ zii (2)/χ iiz (2) of collagen fibers. From SHG microscopy polarization in, polarization out (PIPO) measurements of type I collagen in human lung tissue, a theoretical method is used to extract the triple-helix orientation angle with respect to the collagen fiber. The study shows the dominant role of amino acid orientation in the triple-helix for determining the polarization properties of SHG and provides a method for determining the triple-helix orientation angle in the collagen fibers.
Personalized cancer theranostics has a potential to increase efficiency of early cancer diagnostics and treatment, and to reduce negative side-effects. Protein-stabilized gold nanoclusters may serve ...as theranostic agents. To make gold nanoclusters personalized and highly biocompatible, the clusters were stabilized with human plasma proteins. Optical properties of synthesized nanoclusters were investigated spectroscopically, and possible biomedical application was evaluated using standard cell biology methods. The spectroscopic investigations of human plasma proteins stabilized gold nanoclusters revealed that a wide photoluminescence band in the optical tissue window is suitable for cancer diagnostics. High-capacity generation of singlet oxygen and other reactive oxygen species was also observed. Furthermore, the cluster accumulation in cancer cells and the photodynamic effect were evaluated. The results demonstrate that plasma proteins stabilized gold nanoclusters that accumulate in breast cancer cells and are non-toxic in the dark, while appear phototoxic under irradiation with visible light. The results positively confirm the utility of plasma protein stabilized gold nanoclusters for the use in cancer diagnostics and treatment.
The extracellular matrix (ECM) is amongst many tissue components affected by cancer, however, morphological changes of the ECM are not well-understood and thus, often omitted from diagnostic ...considerations. Polarimetric second-harmonic generation (P-SHG) microscopy allows for visualization and characterization of collagen ultrastructure in the ECM, aiding in better understanding of the changes induced by cancer throughout the tissue. In this paper, a large region of hematoxylin and eosin (H&E) stained human lung section, encompassing a tumor margin, connecting a significant tumor portion to normal tissue was imaged with P-SHG microscopy. The resulting polarimetric parameters were utilized in principal components analysis and unsupervised K-Means clustering to separate normal- and tumor-like tissue. Consequently, a pseudo-color map of the clustered tissue regions is generated to highlight the irregularity of the ECM collagen structure throughout the region of interest and to identify the tumor margin, in the absence of morphological characteristics of the cells.
Nonlinear optical microscopy has become a powerful tool for high‐resolution imaging of cellular and subcellular composition, morphology, and interactions because of its high spatial resolution, deep ...penetration, and low photo‐damage to tissue. Developing specific harmonic probes is essential for exploiting nonlinear microscopic imaging for biomedical applications. We report an organized aggregate of porphyrins (OAP) that formed within lipidic nanoparticles showing fingerprint spectroscopic properties, structure‐associated second harmonic generation, and superradiant third harmonic generation. The OAP facilitated harmonic microscopic imaging of living cells with significantly enhanced contrast. The structure‐dependent switch between harmonic (OAP‐intact) and fluorescence (OAP‐disrupted) generation enabled real‐time multi‐modality imaging of the cellular fate of nanoparticles. Robustly produced under various conditions and easily incorporated into pre‐formed lipid nanovesicles, OAP provides a biocompatible nanoplatform for harmonic imaging.
The assembly and characterization of an organized aggregation of porphyrins (OAP) for nonlinear optical microscopy is reported. Its structure‐dependent photoproperty switch between harmonic (OAP‐intact) and fluorescence (OAP‐disrupted) generation permitted nanoparticle tracking in living cells. Green bars=porphyrin aggregates.
Second harmonic generation (SHG) microscopy is a commonly used technique to study the organization of collagen within tissues. However, individual collagen fibrils, which have diameters much smaller ...than the resolution of most optical systems, have not been extensively investigated. Here we probe the structure of individual collagen fibrils using polarization-resolved SHG (PSHG) microscopy and atomic force microscopy. We find that longitudinally polarized light occurring at the edge of a focal volume of a high numerical aperture microscope objective illuminated with linearly polarized light creates a measurable variation in PSHG signal along the axis orthogonal to an individual collagen fibril. By comparing numerical simulations to experimental data, we are able to estimate parameters related to the structure and chirality of the collagen fibril without tilting the sample out of the image plane, or cutting tissue at different angles, enabling chirality measurements on individual nanostructures to be performed in standard PSHG microscopes. The results presented here are expected to lead to a better understanding of PSHG results from both collagen fibrils and collagenous tissues. Further, the technique presented can be applied to other chiral nanoscale structures such as microtubules, nanowires, and nanoribbons.
Aggregation of 5,10,15,20-tetrakis(4-sulfonatophenyl) porphyrin (TPPS4) molecules as a function of solvent pH is a complex process because of the co-existence of many possible interacting monomeric ...ionic forms that self-assemble into various aggregate structures. Possible seeding elements of aggregates were studied using quantum chemical calculations based on density functional theory to determine favorable ionic forms and their dimers, and the simulated spectral characteristics were compared with the measured absorption spectra. Five different forms of TPPS4 monomers and ten dimers were found as possible candidates for aggregate precursors. It was found that monomeric forms dominate at pH = 12.1, pH = 7.1, pH = 4.1, and pH = 3.0 as well as at pH = −1.0. In contrast, J-type dimers become a dominant form at pH = 1.0.
Changes in collagen ultrastructure between malignant and normal human thyroid tissue were investigated ex vivo using polarization second harmonic generation (SHG) microscopy. The second-order ...nonlinear optical susceptibility tensor component ratio and the degree of linear polarization (DOLP) of the SHG signal were measured. The ratio values are related to the collagen ultrastructure, while DOLP indicates the relative amount of coherent signal and incoherent scattering of SHG. Increase in ratio values and decrease in DOLP were observed for tumor tissue compared to normal thyroid, indicating higher ultrastructural disorder in tumor collagen.
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.
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