Semi-automated imaging system to quantitate estrogen and progesterone receptor immunoreactivity in human breast cancer

A semi‐automated imaging system is described to quantitate estrogen and progesterone receptor immunoreactivity in human breast cancer. The system works for any conventional method of image acquisition using microscopic slides that have been processed for immunohistochemical analysis of the estrogen receptor and progesterone receptor. Estrogen receptor and progesterone receptor immunohistochemical staining produce colorimetric differences in nuclear staining that conventionally have been interpreted manually by pathologists and expressed as percentage of positive tumoral nuclei. The estrogen receptor and progesterone receptor status of human breast cancer represent important prognostic and predictive markers of human breast cancer that dictate therapeutic decisions but their subjective interpretation result in interobserver, intraobserver and fatigue variability. Subjective measurements are traditionally limited to a determination of percentage of tumoral nuclei that show positive immunoreactivity. To address these limitations, imaging algorithms utilizing both colorimetric (RGB) as well as intensity (gray scale) determinations were used to analyze pixels of the acquired image. Image acquisition utilized either scanner or microscope with attached digital or analogue camera capable of producing images with a resolution of 20 pixels /10 μ. Areas of each image were screened and the area of interest richest in tumour cells manually selected for image processing. Images were processed initially by JPG conversion of SVS scanned virtual slides or direct JPG photomicrograph capture. Following image acquisition, images were screened for quality, enhanced and processed. The algorithm‐based values for estrogen receptor and progesterone receptor percentage nuclear positivity both strongly correlated with the subjective measurements (intraclass correlation: 0.77; 95% confidence interval: 0.59, 0.95) yet exhibited no interobserver, intraobserver or fatigue variability. In addition the algorithms provided measurements of nuclear estrogen receptor and progesterone receptor staining intensity (mean, mode and median staining intensity of positive staining nuclei), parameters that subjective review could not assess. Other semi‐automated image analysis systems have been used to measure estrogen receptor and progesterone receptor immunoreactivity but these either have required proprietary hardware or have been based on luminosity differences alone. By contrast our algorithms were independent of proprietary hardware and were based on not just luminosity and colour but also many other imaging features including epithelial pattern recognition and nuclear morphology. These features provide a more accurate, versatile and robust imaging analysis platform that can be fully automated in the near future. Because of all these properties, our semi‐automated imaging system ‘adds value’ as a means of measuring these important nuclear biomarkers of human breast cancer.     

Detecting changes in arthritic fibroblast‐like synoviocytes using atomic force microscopy

The morphological and quantitative differences between arthritic fibroblast‐like synoviocytes (FLS) and normal FLS were determined as an effective diagnostic tool for rheumatoid arthritis (RA), and confirmed using atomic force microscopy (AFM). Collagen‐induced arthritic (CIA) mice and normal mice were prepared and FLS were isolated by enzymatic digestion from the synovial tissue of sacrificed mice at 5‐week and 8‐week pathogenesis periods. Analysis of cell morphology using AFM revealed that the surface roughness around the nucleus and around the branched cytoplasm was significantly higher in CIA FLS (P < 0.05) than that in normal FLS. In addition, the roughness of two different sites on the arthritic FLS increased with an increase in the duration of pathogenesis. These results strongly suggest that AFM can be widely used as a diagnostic tool in cytopathology to detect the early signs of RA and various others diseases at the intercellular level. Microsc. Res. Tech. 78:982–988, 2015. © 2015 Wiley Periodicals, Inc.     

Investigation on the properties of gamma irradiated of polytetrafluoroethylene fibers

The physical, thermal, and chemical properties of gamma‐irradiated polytetrafluoroethylene (PTFE) fibers were investigated using X‐ray diffraction (XRD), differential scanning calorimetry (DSC), and Fourier transform infrared (FTIR) spectroscopy. Scanning electron microscopy (SEM) was also used to analyze the surface morphology of irradiated fiber samples. PTFE fiber samples were irradiated by gamma radiation doses ranging from 3 kGy to 40 kGy. The XRD analyses and DSC measurements showed the improvement of crystallinity by gamma irradiation with dose up to 25 kGy reflecting the induced crosslinking with irradiation for PTFE fibers. The crystallinity was found to decrease with higher dose of 40 kGy, reflecting induced amorphization of the polymer sample at the high radiation dose. The calculated crystallite size and XRD parameters showed obvious variations with sample irradiation. The FTIR results showed the liberation of CF₂ groups and the formation of some new chemical bonding with crosslinking‐induced irradiation. The SEM micrographs revealed no variation in the surface morphology of the irradiated fiber samples than the pristine fiber.     

Fractal features and surface micromorphology of diamond nanocrystals

This paper analyses the three‐dimensional (3‐D) surface texture of growing diamond nanocrystals on Au thin films as catalyst on p‐type Si substrate using hot filament chemical vapour deposition (HFCVD). Rutherford backscattering spectrometry (RBS), atomic force microscopy (AFM), Raman, X‐ray diffraction (XRD) and scanning electron microscopy (SEM) analyses were applied also to characterize the 3‐D surface texture data in connection with the statistical, and fractal analyses. This type of 3‐D morphology allows a deeper understanding of structure/property relationships and surface defects in prepared samples. Our results indicate a promising way for preparing high‐quality diamond nanocrystals on Au thin films as catalyst on p‐type Si substrate via HFCVD method.     

Characteristics of biofilm attaching to carriers in moving bed biofilm reactor used to treat vitamin C wastewater

In order to investigate characteristics of biofilm attaching firmly to carriers in the moving bed biofilm reactor (MBBR) used for vitamin C wastewater treatment, experiments were undertaken with instrumental analysis methods. Scanning electron microscopy (SEM) micrographs of MBBR biofilms revealed that there were rod‐shaped microbes and cocci in the biofilm, and microbes were embedded within medium substances and the biofilm matrix adhered firmly to carriers, leading to the formation of a smooth compacted surface at the base of the biofilm. Transmission electron microscopy (TEM) analysis revealed that extracellular polymeric substances (EPS) layer surrounded cell, sequestered inorganics to form a mixed structure, which ensured firm attachment of the biofilm to the carrier. X‐ray diffraction (XRD) experiments and thermogravimetry analysis revealed that (i) the biofilm contained many inorganic substances, about 70.5%, and the inorganic substances contained multiple classes of inorganic with a high boiling point; (ii) inorganic elements such as calcium and phosphorous were selectively absorbed and accumulated in the biofilm as insoluble compounds with amorphous phases, rendering the biofilm highly resistant to detachment. Fourier‐transform infrared (FTIR) spectroscopy showed carbohydrates were the main EPS. SCANNING 35:283‐291, 2013. © 2012 Wiley Periodicals, Inc.     

Ultrastructural study of mouse adipose‐derived stromal cells induced towards osteogenic direction

We investigated the ultrastructural characteristics of mouse adipose‐derived stem/stromal cells (ASCs) induced towards osteogenic lineage. ASCs were isolated from adipose tissue of FVB‐Cg‐Tg(GFPU)5Nagy/J mice and expanded in monolayer culture. Flow cytometry, histochemical staining, and electron microscopy techniques were used to characterize the ASCs with respect to their ability for osteogenic differentiation capacity. Immunophenotypically, ASCs were characterized by high expression of the CD44 and CD90 markers, while the relative content of cells expressing CD45, CD34 and CD117 markers was <2%. In assays of differentiation, the positive response to osteogenic differentiation factors was observed and characterized by deposition of calcium in the extracellular matrix and alkaline phosphatase production. Electron microscopy analysis revealed that undifferentiated ASCs had a rough endoplasmic reticulum with dilated cisterns and elongated mitochondria. At the end of the osteogenic differentiation, the ASCs transformed from their original fibroblast‐like appearance to having a polygonal osteoblast‐like morphology. Ultrastructurally, these cells were characterized by large euchromatic nucleus and numerous cytoplasm containing elongated mitochondria, a very prominent rough endoplasmic reticulum, Golgi apparatus and intermediate filament bundles. Extracellular matrix vesicles of variable size similar to the calcification nodules were observed among collagen fibrils. Our data provide the ultrastructural basis for further studies on the cellular mechanisms involved in osteogenic differentiation of mouse adipose‐derived stem/stromal cells. Microsc. Res. Tech. 79:557–564, 2016. © 2016 Wiley Periodicals, Inc.     

Camphor sulfonic acid (CSA) doped polypyrrole (PPy) films: Measurement of microstructural and optoelectronic properties

In this paper, camphor sulfonic acid doped polypyrrole have been successfully prepared using different weight percentages of camphor sulfonic acid (10–50%) dispersed in polypyrrole by solid state synthesis method. Films of CSA doped PPy were prepared by spin coating technique on a glass substrate. The effect of varying concentrations of CSA on the structure, morphology, optical and electrical properties of polypyrrole was explored using X-ray diffraction (XRD), Fourier transform infrared (FTIR) spectroscopy, scanning electron microscopy (SEM) atomic force microscopy (AFM), UV–visible spectroscopy and two probe technique respectively. The presence of CSA in PPy matrix and their interaction was confirmed by using XRD and FTIR techniques. The dramatic change in the surface morphology has been observed with increasing content of CSA in PPy matrix. The AFM study shows porous uniform globular morphology. The UV–vis spectra of CSA doped PPy film was found to shift to a lower wavelength side as compared to those of observed in PPy, indicating synergetic interaction between dopant CSA and PPy. CSA doped PPy not only can maintain a good solubility but also enhance the electrical conductivity. The results of dc electrical conductivity shows that, increase in electrical conductivity of PPy with increasing content of CSA (10–50%) in PPy matrix.     

Short‐Term Response of Human Osteoblast‐Like Cells on Titanium Surfaces With Micro‐ and Nano‐Sized Features

Since the way that human bone cells behave on contact with different surfaces topographies seems to be crucial to osseointegration, the aim of the present study is to evaluate the participation of some micro‐ and nanosized features of Ti surfaces in the short‐term response of primary human osteoblast‐like cells (HOC). Surfaces were prepared as ground (G‐Ti), hydrofluoric acid etched (HF‐Ti), and sandblasted/HF‐etched (SLA‐Ti), and analyzed using both three‐dimensional (3D) profilometer and atomic force microscope (AFM). Cell morphology was assessed using scanning electron microscopy (SEM) after 4 and 24 h in culture. Cell viability, adhesion, and spreading were also evaluated 4 and 24 h after seeding over each surface. Data were compared by analysis of variance (ANOVA) complemented by Duncan test. Cell morphology, cell counting, and membrane integrity (Neutral Red, NR) were not affected by surface treatment at any time. However, HF‐Ti presented the smallest surface area and did not increase tetrazolium hydroxide (XTT) reduction from 4 to 24 h. On the other hand, a higher level of spreading was only found on the rougher and isotropic SLA‐Ti at 4 h. In conclusion, although all evaluated Ti surfaces allowed HOC short‐term adhesion, the finer topography introduced by HF as single treatment did not favor HOC mitochondrial activity and spreading. The rougher and more complex SLA surface seems to provide a better substrate for HOC short‐term response. SCANNING 34: 378‐386, 2012. © 2012 Wiley Periodicals, Inc.     

Candida albicans biofilms: comparative analysis of room‐temperature and cryofixation for scanning electron microscopy

Biofilms are frequently related to invasive fungal infections and are reported to be more resistant to antifungal drugs than planktonic cells. The structural complexity of the biofilm as well as the presence of a polymeric extracellular matrix (ECM) is thought to be associated with this resistant behavior. Scanning electron microscopy (SEM) after room temperature glutaraldehyde‐based fixation, have been used to study fungal biofilm structure and drug susceptibility but they usually fail to preserve the ECM and, therefore, are not an optimised methodology to understand the complexity of the fungal biofilm. Thus, in this work, we propose a comparative analysis of room‐temperature and cryofixation/freeze substitution of Candida albicans biofilms for SEM observation. Our experiments showed that room‐temperature fixative protocols using glutaraldehyde and osmium tetroxide prior to alcohol dehydration led to a complete extraction of the polymeric ECM of biofilms. ECM from fixative and alcohol solutions were recovered after all processing steps and these structures were characterised by biochemistry assays, transmission electron microscopy and mass spectrometry. Cryofixation techniques followed by freeze‐substitution lead to a great preservation of both ECM structure and C. albicans biofilm cells, allowing the visualisation of a more reliable biofilm structure. These findings reinforce that cryofixation should be the indicated method for SEM sample preparation to study fungal biofilms as it allows the visualisation of the EMC and the exploration of the biofilm structure to its fullest, as its structural/functional role in interaction with host cells, other pathogens and for drug resistance assays.     

Investigation on infrared laser desorption of solid matrix using scanning electron microscope and fast photography

Infrared light from a pulsed optical parametric oscillator laser system was used to irradiate succinic acid (SA), a usual solid matrix used in matrix‐assisted laser desorption ionization, under vacuum. Ablated SA particles were trapped using a silica plate mounted 3.0 mm above and parallel to the sample surface. The morphology and particle size of ablated particles at different laser fluences were investigated using a scanning electron microscope (SEM). The dynamics of plume propagation for SA desorption process was studied with fast photography at atmospheric pressure. Plume expanding at 1.12 J/cm² laser fluence was recorded using a high‐speed CMOS camera and corresponding propagation distance was measured. The solid matrix desorption was driven by phase explosion according to plume model fitting, which was consistent with the results of SEM. Microsc. Res. Tech. 76:744–750, 2013. © 2013 Wiley Periodicals, Inc.     

The application of convergent beam electron diffraction (CBED) analysis on transformation‐induced plasticity (TRIP) steels

Convergent beam electron diffraction (CBED) in transmission electron microscopy (TEM) was applied to determine local carbon concentrations in low‐carbon transformation‐induced plasticity (TRIP) steels. High‐order Laue‐zone (HOLZ) lines were experimentally obtained for comparison with simulation results. A new procedure for calculating carbon content is thus proposed. Retained austenite (RA) is classified into three types by morphology; the relationship between the carbon content and the corresponding RA morphology is discussed based on CBED results. Furthermore, results of X‐Ray diffractometry measurements are also used for comparison.     

Technology of creation of an expert system for diagnosing thyroid pathology based on a set of qualitative signs of cell atypia

An expert system for differential diagnosis of thyroid pathology has been developed, in which the function of transforming qualitative signs of cell atypia into the quantitative form is realized. It based on the set of qualitative signs of cell atypia and works in the mode by the question—answer principle. An expert system (software) contained the X‐matrix and six standard S‐matrices. The X‐matrix was designed for filling with the alternative answers yes/no regarding the presence of cells with characteristic signs of atypia in the visual fields of a preparation. S‐matrices characterized the main forms of thyroid diseases, such as papillary and follicular cancer, follicular adenoma, autoimmune thyroiditis, nodular colloid and diffuse toxic goiter, by their set of yes/no answers. The answers entered to the X‐matrix were compared with answers of the S‐matrix. Each element of each S‐matrix, besides the answers yes/no, contained its weighting coefficient corresponding to a qualitative feature of a certain nosologic form. Weighting coefficient of each feature for every nosologic form was calculated by their frequency of occurrence in 30 visual fields. Comparison of X‐matrices with S‐matrices and verification of diagnosis according to the value of a diagnostic index with a percentage probability was performed in the automatic mode. Diagnostic index was determined by the sum of weighting coefficients of features with the coincident yes/no answers, while comparing X‐matrix elements with the corresponding S‐matrix elements. The software displayed on the monitor is the list of nosologic forms in the order of decreasing the diagnostic index values. Microsc. Res. Tech. 73:1091–1100, 2010. © 2010 Wiley‐Liss, Inc.     

Resin-tooth interfacial morphology and sealing ability of one-step self-etch adhesives: microleakage and SEM study

OBJECTIVES: To compare microleakage of three self‐etch adhesives and to analyze enamel surface morphology and interfacial morphology of resin–enamel and resin–dentin interface under scanning electron microscope (SEM). EXPERIMENTAL DESIGN: Study was conducted in 65 extracted human premolars. Class V cavities were prepared in 45 teeth and assigned to three groups (n = 15) according to three self‐etch adhesives (OptiBond All‐in‐One, iBond, and Adper Prompt L‐Pop). After restoration, 10 samples from each group were used to assess microleakage at enamel and dentin margin. Five samples from each group were used for analysis of interfacial morphology at resin–enamel and resin–dentin interface under SEM. Remaining 20 teeth were used to prepare flat enamel buccal surfaces to analyze the difference in surface morphology after treatment with three adhesives (n = 5 each) and 36% phosphoric acid treatment (n = 5). PRINCIPAL OBSERVATIONS: At enamel margin, Prompt L‐Pop depicted least leakage of all the three adhesives and also showed best interfacial adaptation under SEM. At dentin margin, OptiBond All‐in‐One showed significant less leakage than iBond and Prompt L‐Pop. On flat enamel surface, phosphoric acid produced the most retentive etching pattern when compared with the three adhesives. CONCLUSION: Prompt L‐Pop showed the best bonding effectiveness in enamel, whereas OptiBond All‐in‐One performed significantly better in dentin. Microsc. Res. Tech. 2012. © 2012 Wiley Periodicals, Inc.     

Synthesis and tribological behaviour of oleic acid‐capped lanthanum borate/graphene oxide nanocomposites

The lanthanum borate/graphene oxide (LB/GO) nanocomposites were obtained by GO decorated by LB using an eco‐friendly hydrothermal method. For improving the oil solubility of the LB/GO nanocomposites, the oleic acid (OA)‐capped LB/GO (OA–LB/GO) nanocomposites were achieved. The as‐prepared nanocomposites were determined by a series of characterisations such as Fourier transform‐infrared spectroscopy (FT‐IR), X‐ray powder diffraction (XRD), Raman, UV‐vis, transmission electron microscopy (TEM) and SEM. The results indicated that most of LB nanoparticles with the size of 50–100 nm were well loaded on the surface of GO nanosheets. In addition, the tribological properties of the as‐prepared nanocomposites were tested by a four‐ball friction machine. The results demonstrated that the OA–LB/GO nanocomposites were of good anti‐wear ability and excellent load‐carrying capacity. Copyright © 2016 John Wiley & Sons, Ltd.     

纳米氧化镧和蛇纹石改性PTFE复合材料淡水环境摩擦学性能预测*

以聚四氟乙烯(PTFE)为基体,改性纳米氧化镧(nano-La_2O_3)、改性纳米蛇纹石(nano-serpentine)为添加剂,采用均匀设计法,制备nano-La_2O_3/nano-serpentine/PTFE复合材料。自制水环境模拟装置,设计并进行淡水环境复合材料摩擦学实验。使用Origin软件对实验数据进行曲线拟合,使用SPSS软件进行多元回归分析,得到摩擦学性能回归方程,通过MATLAB解出回归方程摩擦因数和磨损率理论最优解。以复合材料最优理论配比制作试件,进行摩擦学性能对比实验和磨损表面形貌分析。结果表明:复合材料摩擦学性能实验值与回归分析理论结果基本吻合,摩擦因数误差控制在5%以内,磨损率误差控制在10%以内,证明研究所用方法对复合材料摩擦学性能预测具有可行性。     

Experimental investigation of craze morphology of isotactic polypropylene using computed tomography

In this paper we investigated the morphology of crazes formed during the fracture process of polymeric fibres. Computed tomography technique was applied to reconstruct the image of fibrils‐voids structure of crazes. This allowed us to investigate the initial stages of crazes formation during the fracture process of mechanically stretched isotactic polypropylene fibres. The density and morphology of crazes were studied at different regions inside the stretched sample. Accordingly, we are able to visualize the internal structure of the stretched fibres in three dimensions. This revealed in‐depth information on the fracture process of polymeric fibres.     

Synthesis of silicon carbide ceramics from a polysilastyrene at low temperatures

The conventional route for preparation of silicon carbide ceramics is by the use of pressureless sintering, hot pressing, or hot isostatic pressing of silicon carbide starting powders. High sintering temperatures (2073–2473 K) and the addition of sintering additives are normally used to enhance densification. These sintering additives, however, form second phases at grain boundaries which impair the mechanical properties of the material, particularly at high temperatures. It is therefore desirable that new processing routes are developed that overcome these difficulties. A proposed route is to use a polymeric pressure which can provide a Silicon carbide matrix as binding agent for silicon carbide powders, thus making the requirement for high temperatures and sintering additives unnecessary. This paper reports observations of the direct transformation of a polymeric precursor into amorphous Si–C, and crystalline SiC at low temperatures, and the use of this precursor as a binder for the production of SiC powder/ex-precursor SiC composites.     

Imaging cells on polymer spherulites

Polymers are commonly used to construct tissue‐engineered medical products and the conditions used to process these polymeric materials can affect their biological performance. For semi‐crystalline polymers, thermal processing conditions can induce crystallization of the polymer chains into spherulites, which cause the polymer surface to become roughened. This change in surface topology can influence cell behaviours such as adhesion, morphology and proliferation. Herein, a simple microscope technique is presented, which combines fluorescence microscopy with transmitted white light microscopy using crossed polarizers. This technique allows simultaneous imaging of cells and polymer spherulites, which enables the biomaterials researcher to observe the effects of spherulites on cell alignment and morphology.     

A systematic study of maghemite/PMMA nano-fibrous composite via an electrospinning process: Synthesis and characterization

In this study, maghemite/PMMA nano-fibrous composites have been successfully fabricated by using the electrospinning process. PMMA nano-fibres have been selected to be used as the matrix; the PMMA was dissolved in three diverse solvents (Acetone, THF and DMF) in order to obtain fine PMMA nano-fibres. As a result, the PMMA–DMF proved to be the most appropriate polymer solution among the three solvents, with its impressive defect-free surface morphology results. The production of maghemite using Massart’s procedure resulted in nano-particles with an average diameter of 4.98 ± 0.13 nm (using transmission electron microscopy (TEM)). Maghemite nano-particle were then mixed with a prepared polymer solution in order to fabricate maghemite/PMMA nano-fibrous composite. Furthermore, the investigation of the morphology and structure of the composite was carried out using field emission scanning electron microscopy (FESEM), Energy-dispersion X-ray spectroscopy (EDX), Alternating Gradient Magnetometer (AGM), Fourier transform infrared spectrometer (FTIR), X-ray diffraction (XRD) and tensile strength measurement devices. The results indicated that there was a great amount of maghemite, both in and on the composite’s surface, which can be utilized in the purpose of magnetic applications.     

The olfactory organ of the trout Salmo trutta fario: A novel localization for a progestin receptor

A progestin receptor (PR) has been detected in the olfactory organ of the trout Salmo trutta fario. The specificity of this receptor was high for 17α,20β‐dihydroxy‐4‐pregnen‐3‐one (17α,20β‐DP), but it also bound 17α‐hydroxy‐progesterone (17α‐OHP) and 21‐hydroxyprogesterone (21‐OHP), even when present at low concentrations (10‐fold in relative binding affinity assay). Progesterone (P) competed effectively at much higher concentrations (1,000‐fold in relative binding affinity assay). Immunohistochemical studies carried out with three different monoclonal antibodies against human progesterone receptor (hPR), chicken progesterone receptor hinge region (cPR), and chicken progesterone receptor A/B domain (PR22), revealed that immunoreactivity was present in the epithelium of the olfactory organ of females and males of the trout Salmo trutta fario only against hPR. Western blotting showed two hPR immunoreactive bands of about 62 and 66 kDa. Finally, a portion of the cDNA of about 300 nucleotides extending over the DNA binding domain and the ligand binding domain was cloned and sequenced, revealing a high degree of sequence homology of the PR in Salmo trutta fario with the PR in other teleosts. Microsc. Res. Tech., 2010. © 2009 Wiley‐Liss, Inc.