Polarized Light Microscopy Technique Research Articles

VALIDATION OF ANALYTICAL METHOD BY UV SPECTROPHOTOMETRIC QUANTIFICATION OF GEMFIBROZIL INCORPORATED IN THE MICROEMULSIONS

The present study aims to validate an analytical method by UV spectrophotometric for the quantification of gemfibrozil incorporated into microemulsions. Experimental development by constructing a ternary phase diagram for the identification of microemulsions. The characterization of the microemulsions was carried out by the polarized light microscopy technique. The analytical method was validated according to the parameters described by the United State Pharmacopeia (USP) 39 NF34 and International Conference on Harmonization (ICH) (2005), which evaluated linearity, robustness, intra and inter day accuracy, specificity accuracy, limits of detection and quantification. The developed method demonstrated linearity with a coefficient of determination (R2) of 0.9898, in addition to precision, accuracy and robustness for each of the tests with values that presented a coefficient of variation of less than 5 %. The results obtained were favorable, since they comply with the parameters stipulated by USP 39-NF34 (2016) and ICH (2005), indicating that the method is effective for the intended purpose.

Novel quantitative signature of tumor stromal architecture: polarized light imaging differentiates between myxoid and sclerotic human breast cancer stroma.

As a leading cause of death in women, breast cancer is a global health concern for which personalized therapy remains largely unrealized, resulting in over- or under-treatment. Recently, tumor stroma has been shown to carry important prognostic information, both in its relative abundance and morphology, but its current assessment methods are few and suboptimal. Herein, we present a novel stromal architecture signature (SAS) methodology based on polarized light imaging that quantifies patterns of tumor connective tissue. We demonstrate its ability to differentiate between myxoid and sclerotic stroma, two pathology-derived categories associated with significantly different patient outcomes. The results demonstrate a 97% sensitivity and 88% specificity for myxoid stroma identification in a pilot study of 102 regions of interest from human invasive ductal carcinoma breast cancer surgical specimens (20 patients). Additionally, the SAS numerical score is indicative of the wide range of stromal characteristics within these binary classes and highlights ambiguous mixed-morphology regions prone to misclassification. The enabling polarized light microscopy technique is inexpensive, fast, fully automatable, applicable to fresh or embedded tissue without the need for staining and thus potentially translatable into research and/or clinical settings. The SAS metric yields quantifiable and objective stromal characterization with promise for prognosis in many types of cancers beyond breast carcinoma, enabling researchers and clinicians to further investigate the emerging and important role of stromal architectural patterns in solid tumors.

Quantification of the regional bioarchitecture in the human aorta.

Regional variance in human aortic bioarchitecture responsible for the elasticity of the vessel is poorly understood. The current study quantifies the elements responsible for aortic compliance, namely, elastin, collagen and smooth muscle cells, using histological and stereological techniques on human tissue with a focus on regional heterogeneity. Using donated cadaveric tissue, a series of samples were excised between the proximal ascending aorta and the distal abdominal aorta, for five cadavers, each of which underwent various staining procedures to enhance specific constituents of the wall. Using polarised light microscopy techniques, the orientation of collagen fibres was studied for each location and each tunical layer of the aorta. Significant transmural and longitudinal heterogeneity in collagen fibre orientations were uncovered throughout the vessel. It is shown that a von Mises mixture model is required accurately to fit the complex collagen fibre distributions that exist along the aorta. Additionally, collagen and smooth muscle cell density was observed to increase with increasing distance from the heart, whereas elastin density decreased. Evidence clearly demonstrates that the aorta is a highly heterogeneous vessel which cannot be simplistically represented by a single compliance value. The quantification and fitting of the regional aortic bioarchitectural data, although not without its limitations, including mean cohort age of 77.6years, facilitates the development of next-generation finite element models that can potentially simulate the influence of regional aortic composition and microstructure on vessel biomechanics.

Light-induced birefringence in the isotropic phase of a lyotropic liquid crystal

In this paper, the light-induced order in the isotropic phase (I) of the KL-DeOH-water mixture is investigated. All mixtures show L1-I-L2 phase transitions being L1 and L2 two optically birefringent phases at low and high temperatures, respectively. The phase transitions were verified by polarized light microscopy technique. In the appropriate conditions of the relative concentration of the mixture and of temperature, an astonishing increase in the optical transmittance of the isotropic mixture can be observed in the presence of an external pulsed laser beam. The best induction effect was obtained with molar relative concentration C = [KL]/[DeOH] = 2.61 at about 2.0 °C from I-L2 phase transition. From each time-domain optical transmittance, the signal was analyzed in the low frequency domain (5 Hz < f < 50 Hz) by means of Discrete Fourier Transform (DFT). DFT analysis revealed two typical frequencies (ν1 and ν2) in the middle of the micellar isotropic phase with pronounced peaks in |F(ω)|. However, for mixtures with C = 2.61 (the lowest concentration), only one well pronounced peak at ν1 = 29.5Hz was found. For C = 2.62, the frequencies were ν1 = 29.5 Hz and ν2 = 36.5 Hz. And, for C = 2.63, ν1 = 36.5 Hz and ν2 = 40.0 Hz. It was verified that for all relative concentrations ν1 and ν2 have no dependence on temperature. These frequencies are being interpreted as an evidence of high correlation between the anisometric micelles (or correlation volumes) of the mixtures.

Accuracy optimization of combined multiparameter measuring systems with application to polarized light microscopy.

We study the accuracy of combined multiparameter measuring systems (CMPMSs) that determine several unknown quantities from measurements of a single variable at different preprogrammed conditions determined by control parameters. To reduce inaccuracies of determined quantities, we propose a mathematical method for selection of control parameters that are optimal for all possible values of determined quantities. Using the submultiplicativity of the spectral and Frobenius matrix norms, we construct the upper bound of the error function and determine the set of control parameters by minimizing this bound. To demonstrate the capability of our method for CMPMSs, we apply it to the polarized light microscopy technique called LC-PolScope, which is used for determining inhomogeneous two-dimensional fields of optical retardation and orientation of the slow optic axis in thin organic and inorganic samples. We compare the computed set of control parameters with other sets, including the one used in the PolScope, and demonstrate that our choice of control parameters works very well even though it does not take into account any specific features of the PolScope. We expect that our method will be successful in various CMPMSs, as it is applicable to any error distribution of the control parameters and measured values.

Mechanisms of Rotational Dynamics of Chiral Liquid Crystal Droplets in an Electric Field

The dynamics of the orientational structure of chiral nematic (CN) droplets in an isotropic medium in dc and ac electric fields is investigated by the polarized light microscopy technique. It is shown theoretically that the dynamics of rotational processes in these kinds of systems is determined by electroconvective processes developing due to the flexoelectric polarization associated with the initial configuration of the director field in droplets. It is established experimentally that the linear and quadratic regions of dependence of the rotational velocity of droplets on the electric field strength are explained by the above-mentioned mechanisms. Numerical simulation on the basis of the approach developed gives good agreement with experimental data.

Crystallization, melting, supermolecular structure and properties of isotactic polypropylene nucleated with dicyclohexyl-terephthalamide

Dicyclohexyl-terephthalamide (DCHT) is an efficient and partially soluble nucleating agent for isotactic polypropylene. Its properties, nucleating efficiency and selectivity were characterized using infrared spectroscopy, calorimetry and polarized light microscopy techniques with special attention to the dissolution and recrystallization of DCHT in the iPP melt. The mechanical properties of the nucleated samples were studied using standard tensile tests. It was found that DCHT possesses dual (α and β) nucleating ability. DCHT is soluble in polypropylene melt and recrystallized from the melt during the cooling. It was observed that a special supermolecular structure is formed in its presence, where micron-sized α-iPP is dispersed finely in the β-iPP matrix. This unique structure promoted good stiffness and toughness simultaneously.

Imaging of Scleral Collagen Deformation Using Combined Confocal Raman Microspectroscopy and Polarized Light Microscopy Techniques.

This work presents an optospectroscopic characterization technique for soft tissue microstructure using site-matched confocal Raman microspectroscopy and polarized light microscopy. Using the technique, the microstructure of soft tissue samples is directly observed by polarized light microscopy during loading while spatially correlated spectroscopic information is extracted from the same plane, verifying the orientation and arrangement of the collagen fibers. Results show the response and orientation of the collagen fiber arrangement in its native state as well as during tensile and compressive loadings in a porcine sclera model. An example is also given showing how the data can be used with a finite element program to estimate the strain in individual collagen fibers. The measurements demonstrate features that indicate microstructural reorganization and damage of the sclera’s collagen fiber arrangement under loading. The site-matched confocal Raman microspectroscopic characterization of the tissue provides a qualitative measure to relate the change in fibrillar arrangement with possible chemical damage to the collagen microstructure. Tests and analyses presented here can potentially be used to determine the stress-strain behavior, and fiber reorganization of the collagen microstructure in soft tissue during viscoelastic response.

Mueller matrix microscope with a dual continuous rotating compensator setup and digital demodulation

In this paper we describe a new Mueller matrix (MM) microscope that generalizes and makes quantitative the polarized light microscopy technique. In this instrument all the elements of the MU are simultaneously determined from the analysis in the frequency domain of the time-dependent intensity of the light beam at every pixel of the camera. The variations in intensity are created by the two compensators continuously rotating at different angular frequencies. A typical measurement is completed in a little over one minute and it can be applied to any visible wavelength. Some examples are presented to demonstrate the capabilities of the instrument.

Measuring the Maturity of Developing Cotton Fibers using an Automated Polarized Light Microscopy Technique

Cotton fibers are trichome cells composed primarily of cellulose. Mature fibers have more cellulose and a greater degree of cell wall thickening, and perform better than less mature fibers during textile processing. An automated polarized light microscope instrument called Siro-Mat that measures cotton fiber cell wall thickening was employed to assess the maturity of developing fibers from single cotton fruit. Fruit were taken from the first fruiting branch and position on glasshouse grown Gossypium hirsutum L. (Upland) and G. barbadense L. (Pima) plants, sequentially harvested from 24 days postanthesis (dpa) at approximately four-day intervals up until approximately 50 dpa. The instrument assessed an average of 13,000 fiber snippets per fruit. Upland fibers matured at a slower rate than Pima fibers up to 35 dpa. However, after 45 dpa Upland fibers had achieved a higher average maturity (i.e. 0.99 birefringence maturity index (BMI), cf. 0.79 for Pima). For both species the uniformity of fiber maturity increased as fibers matured up until 35 dpa for Upland and 29 dpa for Pima (i.e. the BMI coefficient of variation decreased as BMI increased during fruit development). It is envisaged that SiroMat will be a useful tool in helping to understand and manage fiber maturity by characterizing the maturation dynamics of cultivars with different inherent fiber properties, and for cultivars subjected to different environmental and agronomic conditions.

Anisotropy of collagen fibre alignment in bovine cartilage: comparison of polarised light microscopy and spatially resolved diffusion-tensor measurements

To compare collagen fibre alignment angles obtained from polarised light microscopy (PLM) and diffusion-tensor imaging (DTI) in bovine articular cartilage. Five samples of bovine articular cartilage from five different animals were studied using magnetic resonance imaging and PLM techniques. T(2)-weighted, diffusion-tensor (DT), and PLM images were acquired for each sample and average depth profiles of the PLM and DTI angles, as well as the banding patterns observed in T(2)-weighted magnetic resonance (MR) images, were compared. Statistical properties of the distributions of the DTI and PLM angles were examined. The samples exhibited a range of alignment morphologies. In the samples with the "conventional" three-zone alignment pattern, a correlation between the PLM and DTI alignment zones and the banding in T(2)-weighted MR images was observed. The shapes of the depth profiles of the PLM and DTI alignment angles were qualitatively similar for each sample. Three samples showed good quantitative correlation between the DT and PLM alignment angles. The correlation between the diffusion and PLM alignment angles was best in the regions of low degree of disorder of fibre alignment. This study provides the first quantitative comparison of DTI of cartilage with the more established PLM techniques. The correlation between alignment angles derived from PLM and DTI data was evident across a wide range of alignment morphologies. The results support the use of DTI for the quantitative measurement of collagen fibre alignment. The microscopic-scale (~10 microm) dispersion of fibre alignment angles appears to be an important factor for understanding the extent of quantitative correlation between PLM and DTI results.

Nonisothermal crystallization kinetics and morphology of self-seeded syndiotactic 1,2-polybutadiene

Subsequent melting behavior after isothermal crystallization at different temperatures from the isotropic melt and nonisothermal crystallization kinetics and morphology of partially melting sPB were carried out by differential scanning calorimetry (DSC), polarized light microscopy (POM), respectively. Triple melting-endothermic peaks were observed for the polymer first isothermally crystallized at temperatures ranging from 141 to 149°C, respectively, and then followed by cooling at 10°C/min to 70°C. Comparing with the nonisothermal crystallization from the isotropic melt, the nonisothermal crystallization for the partially melting sPB characterized the increased onset crystallization temperature, and the sizes of spherulites became smaller and more uniform. The Tobin, Avrami, Ozawa, and the combination of Avrami and Ozawa equations were applied to describe the kinetics of the nonisothermal process. Both of the Tobin and the Avrami crystallization rate parameters (KT and KA, respectively) were found to increase with increase in the cooling rate. The parameter F(T) for the combination of Avrami and Ozawa equations increases with increasing relative crystallinity. The Ziabicki's kinetic crytallizability index GZ for the partially melting sPB was found to be 3.14. The effective energy barrier Δϵ describing the nonisothermal crystallization of partially melting sPB was evaluated by the differential isoconversional method of Friedman and was found to increase with an increase in the relative crystallinity. At the same time, Hoffman-Lauritzen parameters (U and Kg) are evaluated and analyzed from the nonisothermal crystallization data by the combination of isoconversional approach and Hoffman-Lauritzen theory. The Kg value obtained from DSC technique was found to be in good agreement with that obtained from POM technique. © 2006 Wiley Periodicals, Inc. J Appl Polym Sci 100: 1479–1491, 2006

Analytical Techniques for Nucleation Studies in Lipids: Advantages and Disadvantages

ABSTRACT: Crystallization is generally considered a 2‐step process. The 1st step, nucleation, involves the formation of molecular aggregates with a critical size great enough to become stable. During the 2nd step, nuclei grow and develop into crystals. Distinguishing between nucleation and growth constitutes a major challenge in lipid crystallization studies. Thus, it is of great importance to discuss the information obtained from the different techniques that are usually used to study nucleation behavior such as nuclear magnetic resonance (NMR), differential scanning calorimetry (DSC), rheological techniques, light‐scattering techniques such as turbidimetry and scanning diffusive light scattering (SDLS), polarized light microscopy (PLM), and laser polarized optical sets such as laser polarizedlight turbidimetry (LPLT). Techniques to describe the nucleation process must be very sensitive to disregard growth. When crystallization is followed by methods such as DSC, NMR, and rheological measurements, at times, small amounts of crystals are present in the melt before any solids are detected. Clearly, at this stage, well beyond the induction time for nucleation (τ), these methods are measuring crystal growth. Techniques of low sensitivity for solid fat contents lower than 0.1% must not be used to evaluate nucleation effects. Sensitive turbidimeters with detectors that saturate below 0.3% solid fat content give good results as do scanning diffusive light‐scattering equipment. Although the PLM technique is sensitive enough for these kinds of studies, an understanding of important basic concepts is essential. Laser optical sets are the most appropriated methods to study nucleation in fats systems.

Imaging the physical and morphological properties of a multi-zone young articular cartilage at microscopic resolution.

To characterize a number of physical and morphologic properties of young articular cartilage. These properties include the anisotropy of T(2) relaxation, optical retardation, orientation of the collagen fibrils, total thickness of the tissue, number of histologic sub-zones in the tissue, width of individual sub-zones, and correlation between the depths of the local T(2) maxima and the local retardation minima. Microscopic magnetic resonance imaging (mciro MRI) and polarized light microscopy (PLM) were used to examine three side-by-side specimens from a humeral head of a three-month-old beagle that exhibited a unique topographic heterogeneity from three-zones centrally to multi-zone peripherally. The centrally located specimen showed that the collagen fibrils across the tissue depth have a pattern of the classic three histologic sub-zones (tangential, transitional, and radial). A much more complicated multi-zone structure was found in the specimen located peripherally, with a second transitional zone and a second tangential zone located at the deep part of the tissue. We also showed that the orientation of the collagen fibrils that form the cocoon-shaped territorial matrix surrounding the clusters of chondrocytes can be imaged by our PLM technique. The results from the young animal in this report, together with our observations from older animals, demonstrate that MRI and PLM can be used to study the epiphyseal expansion of cartilage in young animals during its growth and subsequent loss in older animals. An illustrative model for the structure of collagen fibrils in a humeral head is suggested as an extension to the classic three-zone model for young articular cartilage.

Practical considerations in nucleation studies: The polarized light microscopy technique

Practical considerations in nucleation studies: The polarized light microscopy technique

Evaluating Fibrous Architecture of Nonwovens with Computer-Assisted Microscopy 1

A new computerized imaging, circular polarized light microscopy technique is de veloped to measure the orientation of fibers in images of nonwovens. The system consists of a modified Olympus BX50 polarized microscope, a Sony AVC-D7 video camera, and a Silicon Graphics Indy computer. There are two methods of image seg mentation : Gaussian distribution' based minimum error thresholding and Poisson dis tribution based minimum error thresholding. Segments of fiber midlines are traced with vertical and horizontal scanlines or, alternatively, whole midlines are identified recur sively with the Hilditch technique. The fiber midlines produced with this technique are traversed by a midline traversal algorithm, and the orientation distribution is obtained by least squares line fitting. The accuracy of the technique is evaluated against synthetic images composed of straight lines and sinuous curves. Kupier's statistic is used to evaluate the consistency of the fiber orientation calculations. A statistical analysis of the results shows that the proposed Hilditch connectivity, which preserves skeletoni zation, produces the most accurate results.

Effects of dynamic fixation on shear behaviour of porcine xenograft valves

To evaluate an alternative valve fixation technique, we measured the ability of glutaraldehyde-fixed valve tissue to undergo internal shearing during bending. Porcine aortic valves were fixed statically using conventional means, and dynamically while opening and closing repeatedly in a pulse tank. Using a polarized light microscopy technique developed previously, we measured shear deformation angles in thin sections of bent leaflet tissue and calculated shear strains. Statically-fixed leaflet tissue sheared only 1.2% ± 2.29% ( Mean ± SD) when bent to curvatures of 2.0 mm −1, while dynamically-fixed tissue sheared 5.1% ± 2.63% (significant at P < 0.05). It is likely that dynamic fixation increases the ability of prosthetic valve leaflets to shear during bending by reducing the number of interfibre cross-links that would otherwise impede such deformations. Because shear strains reduce internal fibre strains and protect the leaflets against fatigue, prosthetic valves constructed from dynamically fixed tissue should experience lower stresses and hence last longer.

Reexamination of the Exoskeletal and Muscular Anatomy of the Honey Bee, Apis mellifera,1 and a Polarized Light Microscopy Technique for Morphological Studies2

A new and improved polarized light microscopy technique is described and applied to a reexamination of the muscular and exoskeletal anatomy of the honey bee (Hymenoptera: Apidae). Differences in the description and interpretation from earlier authors were found in: the muscles of the labial palpi, prothoracic tergo-pleural muscles, the pleuroalar muscle, and the tendon to the pleural muscle inserting on the mesothoracic basalare. Also, two muscles, one in the second abdominal segment and one in the sting, previously recognized in an unpublished thesis, are confirmed. This technique provides a magnificent view of the muscular and exoskeletal system of an arthropod.