Polarimetric second harmonic generation microscopy of partially oriented fibers II: Imaging study.

Polarimetric second harmonic generation (SHG) microscopy imaging is employed to investigate the ultrastructural organization of biological and biomimetic partially oriented fibrillar structures. The linear polarization-in polarization-out SHG microscopy measurements are conducted with rat tail tendon, rabbit cornea, pig cartilage, and biomimetic meso-tetra(4-sulfonatophenyl)porphine (TPPS) cylindrical aggregates, which represent different two- and three-dimensional (2D and 3D) configurations of C symmetry fibril structures in the focal volume (voxel) of the microscope. The polarization-in polarization-out imaging of rat tail tendon reveals that SHG intensity is affected by parallel/antiparallel arrangements of the fibers, and achiral (R) and chiral (C) susceptibility component ratio values change by tilting the tendon fibers out of image plane.

Polarimetric second-harmonic generation microscopy of partially oriented fibers I: Digital modeling.

Second-harmonic generation (SHG) in biological tissues originates predominantly from noncentrosymmetric fibrillar structures partially oriented within a focal volume (voxel) of a multiphoton excitation microscope. This study is aimed to elucidate fibrillar organization factors influencing SHG intensity, as well as achiral, R, and chiral, C, nonlinear susceptibility tensor component ratios. SHG response is calculated for various configurations of fibrils in a voxel using the digital nonlinear microscope.

High numerical aperture imaging allows chirality measurement in individual collagen fibrils using polarization second harmonic generation microscopy.

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.

Wide-field Stokes polarimetric microscopy for second harmonic generation imaging.

We employ wide-field second harmonic generation (SHG) microscopy together with nonlinear Stokes polarimetry for quick ultrastructural investigation of large sample areas (700 μm × 700 μm) in thin histology sections. The Stokes vector components for SHG are obtained from the polarimetric measurements with incident and outgoing linear and circular polarization states. The Stokes components are used to construct the images of polarimetric parameters and deduce the maps of ultrastructural parameters of achiral and chiral nonlinear susceptibility tensor components ratios and cylindrical axis orientation in fibrillar materials.

Blood Plasma Stabilized Gold Nanoclusters for Personalized Tumor Theranostics.

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.

Second harmonic generation theory for a helical macromolecule with high sensitivity to structural disorder.

Microscopic theory for the second harmonic generation in a helical molecular system is developed in the minimal coupling representation including non-local interaction effects. At the second order to the field we find a compact expression which combines dipolar, quadrupolar and magnetic contributions. A detailed derivation of the response is performed to specifically isolate the quadratic coupling terms, which we denote as the coupling.

Disruption of Drosophila larval muscle structure and function by UNC45 knockdown.

Background: Proper muscle function is heavily dependent on highly ordered protein complexes. UNC45 is a USC (named since this region is shared by three proteins UNC45/CRO1/She4P) chaperone that is necessary for myosin incorporation into the thick filaments. UNC45 is expressed throughout the entire Drosophila life cycle and it has been shown to be important during late embryogenesis when initial muscle development occurs.

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'/χ' and χ'/χ', were obtained, where χ'/χ' is a structural parameter and χ'/χ' is a measure of the chirality of the collagen fibers.

Polarimetric second-harmonic generation microscopy of the hierarchical structure of collagen in stage I-III non-small cell lung carcinoma.

Polarimetric second-harmonic generation (P-SHG) microscopy is used to quantify the structural alteration of collagen in stage-I,-II and -III non-small cell lung carcinoma (NSCLC) tissue. The achiral and chiral molecular second-order susceptibility tensor components ratios ( and , respectively), the degree of linear polarization () and the in-plane collagen fiber orientation () were extracted. Further, texture analysis was performed on the SHG intensity, , , and .

Live imaging of contracting muscles with wide-field second harmonic generation microscopy using a high power laser.

Wide-field second harmonic generation (SHG) microscopy was developed using a high-power (> 4 W) and high-repetition-rate (MHz range) laser oscillator to achieve fast SHG imaging over a large area (400 µm × 400 µm). The microscope was used for high spatial resolution imaging of contracting muscles in live larvae. Anisotropic and isotropic bands of striated muscle were distinguished, allowing accurate determination of sarcomere length and SHG intensity from individual sarcomeres.

Polar organization of collagen in human cardiac tissue revealed with polarimetric second-harmonic generation microscopy.

Polarimetric second-harmonic generation (P-SHG) microscopy is used to characterize the composition and polarity of collagen fibers in various regions of human cardiac tissue. The boundary between the cardiac conduction system and myocardium is shown to possess a distinct composition of collagen compared to other regions in the heart. Moreover, collagen fibers in this region are macroscopically organized in a unipolar arrangement, which may consequently aid in effective propagation of the electrical signal through the cardiac conduction system.

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.

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.

Collagen chirality and three-dimensional orientation studied with polarimetric second-harmonic generation microscopy.

Polarization-dependent second-harmonic generation (P-SHG) microscopy is used to characterize molecular nonlinear optical properties of collagen and determine a three-dimensional (3D) orientation map of collagen fibers within a pig tendon. C symmetry is used to determine the nonlinear susceptibility tensor components ratios in the molecular frame of reference and , where the latter is a newly extracted parameter from the P-SHG images and is related to the chiral structure of collagen. The is observed for collagen fibers tilted out of the image plane, and can have positive or negative values, revealing the relative polarity of collagen fibers within the tissue.

Monte Carlo simulation of polarization-sensitive second-harmonic generation and propagation in biological tissue.

Polarization-sensitive second harmonic generation (p-SHG) is a nonlinear optical microscopy technique that has shown great promise in biomedicine, such as in detecting changes in the collagen ultrastructure of the tumor microenvironment. However, the complex nature of light-tissue interactions and the heterogeneity of biological samples pose challenges in creating an analytical and experimental quantification platform for tissue characterization via p-SHG. We present a Monte Carlo (MC) p-SHG simulation model based on double Stokes-Mueller polarimetry for the investigation of nonlinear light-tissue interaction.

Examination of eye development with third harmonic generation microscopy.

Third harmonic generation (THG) microscopy can exploit endogenous harmonophores such as pigment macromolecules for enhanced image contrast, and therefore can be used without exogenous contrast agents. Previous studies have established that carotenoid compounds are ideal harmonophores for THG microscopy; we therefore sought to determine whether THG from endogenous carotenoid-derived compounds, such as retinal in photoreceptor cells, could serve as a new label-free method for developmental studies. Here we study the development of the pupal eye in and determine the localization of rhodopsin using THG microscopy technique.

Third-harmonic generation Stokes-Mueller polarimetric microscopy.

An experimental implementation of the nonlinear Stokes-Mueller polarimetric (NSMP) microscopy in third-harmonic generation modality is presented. The technique is able to extract all eight 2D-accessible χ components for any sample from 64 polarization measurements, and can be applied to noninvasive ultrastructural characterization. The polarization signature of an isotropic glass coverslip is presented, and carotenoid crystallites in the root of orange carrot (Daucus carota) are investigated, showing complex χ components with a significant chiral contribution.

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.

Changes of collagen ultrastructure in breast cancer tissue determined by second-harmonic generation double Stokes-Mueller polarimetric microscopy.

Second-harmonic generation (SHG) double Stokes-Mueller polarimetric microscopy is applied to study the alteration of collagen ultrastructure in a tissue microarray containing three pathological human breast cancer types with differently overexpressed estrogen receptor (ER), progesterone receptor (PgR), and human epidermal growth factor receptor 2 (HER2). Kleinman symmetry is experimentally validated in breast tissue for 1028 nm laser wavelength and it has been shown that measurements with only linearly polarized incoming and outgoing states can determine molecular nonlinear susceptibility tensor component ratio, average in-plane orientation of collagen fibers and degree of linear polarization of SHG. Increase in the susceptibility ratio for ER, PgR, HER2 positive cases, reveals ultrastructural changes in the collagen fibers while the susceptibility ratio increase and decrease in degree of linear polarization for ER and PgR positive cases indicate alteration of the ultrastructure and increased disorder of the collagen fibers within each focal volume.

Second harmonic generation double stokes Mueller polarimetric microscopy of myofilaments.

The experimental implementation of double Stokes Mueller polarimetric microscopy is presented. This technique enables a model-independent and complete polarimetric characterization of second harmonic generating samples using 36 Stokes parameter measurements at different combinations of incoming and outgoing polarizations. The degree of second harmonic polarization and the molecular nonlinear susceptibility ratio are extracted for individual focal volumes of a fruit fly larva wall muscle.

Second harmonic generation microscopy investigation of the crystalline ultrastructure of three barley starch lines affected by hydration.

Second harmonic generation (SHG) microscopy is employed to study changes in crystalline organization due to altered gene expression and hydration in barley starch granules. SHG intensity and susceptibility ratio values (R'SHG ) are obtained using reduced Stokes-Mueller polarimetric microscopy. The maximum R'SHG values occur at moderate moisture indicating the narrowest orientation distribution of nonlinear dipoles from the cylindrical axis of glucan helices.

Organized Aggregation of Porphyrins in Lipid Bilayers for Third Harmonic Generation Microscopy.

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.