A spectroscopic protocol is proposed to implement confocal microfluorescence imaging to the analysis of microinhomogeneity in the nanocrystallization of the core of fibers belonging to a new kind of broadband fiber amplifier based on glass with embedded nanocrystals. Nanocrystallization, crucial for achieving an adequate light emission efficiency of transition metal ions in these materials, has to be as homogeneous as possible in the fiber to assure optical amplification. This requirement calls for a sensitive method for monitoring nanostructuring in oxide glasses. Here we show that mapping microfluorescence excited at 633 nm by a He-Ne laser may give a useful tool in this regard, thanks to quasi-resonant excitation of coordination defects typical of germanosilicate materials, such as nonbridging oxygens and charged Ge-O-Ge sites, whose fluorescence are shown to undergo spectral modifications when nanocrystals form into the glass. The method has been positively checked on prototypes of optical fibers—preventively characterized by means of scanning electron microscopy and energy dispersive spectroscopy—fabricated from preforms of Ni-doped Li2O-Na2O-Sb2O3-Ga2O3-GeO2-SiO2 glass in silica cladding and subjected to heat treatment to activate gallium oxide nanocrystal growth. The method indeed enables not only the mapping of the crystallization degree but also the identification of drawing-induced defects in the fiber cladding.

The combination of integrated focused ion beam–scanning electron microscope (FIB-SEM) serial sectioning and imaging techniques with image analysis provided quantitative characterization of three-dimensional (3D) pigment dispersion in dried paint films. The focused ion beam in a FIB-SEM dual beam system enables great control in slicing paints, and the sectioning process can be synchronized with SEM imaging providing high quality serial cross-section images for 3D reconstruction. Application of Euclidean distance map and ultimate eroded points image analysis methods can provide quantitative characterization of 3D particle distribution. It is concluded that 3D measurement of binder distribution in paints is effective to characterize the order of pigment dispersion in dried paint films.

This article reports the microstructural characteristics of various petroleum and pitch based nuclear graphites (IG-110, NBG-18, and PCEA) that are of interest to the next generation nuclear plant program. Bright-field transmission electron microscopy imaging was used to identify and understand the different features constituting the microstructure of nuclear graphite such as the filler particles, microcracks, binder phase, rosette-shaped quinoline insoluble (QI) particles, chaotic structures, and turbostratic graphite phase. The dimensions of microcracks were found to vary from a few nanometers to tens of microns. Furthermore, the microcracks were found to be filled with amorphous carbon of unknown origin. The pitch coke based graphite (NBG-18) was found to contain higher concentration of binder phase constituting QI particles as well as chaotic structures. The turbostratic graphite, present in all of the grades, was identified through their elliptical diffraction patterns. The difference in the microstructure has been analyzed in view of their processing conditions.

The aim was to study the effect of solvents on the phase separation of four commercial dental adhesives. Four materials were tested: ClearfilTM SE Bond (CSE), Clearfil Protect Bond (CPB), Clearfil S3 Bond (CS3), and One-Up Bond F Plus (OUB). Distilled water or ethanol was used as a solvent (30 vol%) for microphase separation studies, by stereoscopy. For nanophase images, the mixtures were formulated with two different solvent concentrations (2.5 versus 5 vol%) and observed by atomic force microscopy. Images were analyzed by using MacBiophotonics ImageJ to measure the area of bright domains. Macrophase separations, identified as a loss of clarity, were only observed after mixing the adhesives with water. Nanophase separations were detected with all adhesive combinations. The area of bright domains ranged from 132 to 1,145 nm2 for CSE, from 15 to 285 nm2 for CPB, from 149 to 380 nm2 for CS3, and from 26 to 157 nm2 for OUB. In water-resins mixtures, CPB was the most homogeneous and OUB showed the most heterogeneous phase formation. In ethanol-resin mixtures, CSE attained the most homogeneous structure and OUB showed the most heterogeneous phase. Addition of 5 vol% ethanol to resins decreased the nanophase separation when compared with the control materials.

Our aim was to test the hypothesis that the use of mouthwashes, consumption of soft drinks, as well as the type of light curing unit (LCU), would change the surface roughness (Ra) and morphology of a nanofilled composite resin (Z350® 3M ESPE). Samples (80) were divided into eight groups: Halogen LCU, group 1, saliva (control); group 2, Pepsi Twist®; group 3, Listerine®; group 4, Colgate Plax®; LED LCU, group 5, saliva; group 6, Pepsi Twist®; group 7, Listerine®; group 8, Colgate Plax®. Ra values were measured at baseline, and after 7 and 14 days. One specimen of each group was prepared for scanning electron microscopy analysis after 14 days. The data were subjected to multifactor analysis of variance at a 95% confidence followed by Tukey's honestly significant difference post-hoc test. All the treatments resulted in morphological changes in composite resin surface, and the most significant change was in Pepsi Twist® groups. The samples of G6 had the greatest increase in Ra. The immersion of nanofilled resin in mouthwashes with alcohol and soft drink increases the surface roughness. Polymerization by halogen LCU (reduced light intensity) associated with alcohol contained mouthwash resulted in significant roughness on the composite.

The microstructure of Ti-35Nb-7.2Zr-5.7Ta (TNZT) and Ti-35Nb-7.2Zr-5.7Ta-0.5B (TNZTB) alloys under different heat treatment conditions has been analyzed. The solution-treated and water-quenched TNZT sample consists mainly of β phase with a very small amount of fine athermal ω precipitate. Precipitation of α can be observed when solution-treated samples are directly aged at 580°C for 8 h. The microstructure of the samples subjected to single-stage aging at 300°C or 400°C consists of ω precipitates in equiaxed β grains. Second stage aging at 580°C for 8 h after first stage of aging at 300°C or 400°C results in the replacement of ω precipitates by secondary α. In all of these samples, the amount of ω or α phase was very small, and therefore they could not be detected by X-ray diffraction studies. However, analysis of selected area diffraction patterns obtained from transmission electron microscopy studies confirms their presence. The addition of boron leads to the formation of dispersed precipitates of TiB in the β matrix of the TNZT alloy and also refines the β grains in the microstructure. However, other microstructural features of the TNZTB alloy are similar to those of the TNZT alloy.

In this study, changes produced in aorta and triceps surae muscle of Wistar rats as response to bee venom (BV) envenomation were analyzed by transmission electron microscopy and morphometry. A subchronic treatment of 30 days with daily doses of 700 μg BV/kg and an acute-lethal treatment with a single dose of 62 mg BV/kg were performed. The subchronic treatment resulted in endothelial cell retraction, a thicker subendothelial layer, and thinner elastic laminae and musculoelastic layers in aorta, and thicker endothelium and basal laminae in skeletal muscle. In both tissues polymorphous, swollen mitochondria with disrupted cristae were observed. The acute treatment produced extensive endothelial lesions, breakdown of the collagen layer and migration of muscle cells toward the intima in the aorta, and dilatation of endoplasmic reticulum in the skeletal muscle cells. Mitochondria were almost devoid of cristae or with few circular cristae in the smooth muscle cells while most of the mitochondria presented abnormal circular cristae in the skeletal muscle cells. Degenerative alterations in the aorta were of higher intensity in our experiments—both the intima and media strongly responded to BV, in contrast to those found at the level of the skeletal muscle cells where a moderate degenerative myopathy was recorded.

The final disposal of residues generated at sewage treatment plants (STPs) has become a major problem for cities, due to the increase in the amount of treated sewage. One of the alternatives for the residue, labeled “sewage sludge,” is its reuse in agriculture and in degraded soil. However, not all pathogens and metals present in it are eliminated during treatment. Diplopods have been used as bioindicators in ecotoxicological tests as they are constantly in close contact with the soil. Owing to this fact, the purpose of this study was to expose specimens of the diplopod Rhinocricus padbergi to substrate containing sewage sludge collected at STPs to analyze morphological alterations in their parietal and perivisceral fat body, where substances are stored. The exposures were held for 7, 15, or 90 days at different concentrations of sewage sludge (control, 1%, 10%, and 50%). The parietal fat body showed no alterations in any of the three exposure periods or concentrations. Alterations in the perivisceral fat body were observed for all exposure periods. According to the results, we suggest that the sludge used has toxic agents responsible for changing the animal's perivisceral fat body.

The present study describes the development and use of a new bioconjugate combining targeted quantum dot labeling with an immunoperoxidase method and explores whether these bioconjugates could specifically and effectively label Cu/Zn superoxide dismutase (SOD1). The new bioconjugate is designed for the examination of samples both under fluorescent and bright-field microscopy at the same time. For this purpose chlorobis(2-2′-bipyridyl) methacryloyl tyrosine-ruthenium(II) and bis (2-2′-bipyridyl) methacryloyltyrosine-methacryloyltryptophan-ruthenium (II) photosensitive monomers and photosensitive poly(Bis (2-2′-bipyridyl)) methacryloyltyrosine-methacryloyltryptophan-ruthenium(II) were synthesized and characterized. The anti-SOD1 antibody and horseradish peroxidase (HRD) conjugated quantum dots were prepared by using this polymer. The anti-SOD1 antibody and HRD conjugated quantum dots were used in labeling and imaging of SOD1 in rat liver sections. Quantum dot particles were observed as a bright fluorescence in their specific binding locations inside the hepatocytes. The HRD-diaminobenzidine reaction product was observed as brown-colored particles at the same locations under bright-field microscopy. Structural details of the tissue sections could be examined at the same time. The conjugation protocol is simple; the bioconjugate is applicable for efficient cell labeling and can be adapted for imaging of other targets in different tissues. Also, the prepared nanobioconjugates have mechanic stability and can be used for a long period.

While negative staining can provide detailed, two-dimensional images of biological structures, the potential of combining tomography with negative staining to provide three-dimensional views has yet to be fully realized. Basic requirements of a negative stain for tomography are that the density and atomic number of the stain are optimal, and that the stain does not degrade or rearrange with the intensive electron dose (∼106 e/nm2) needed to collect a full set of tomographic images. A commercially available, tungsten-based stain appears to satisfy these prerequisites. Comparison of the surface structure of negatively stained influenza A virus with previous structural results served to evaluate this negative stain. The combination of many projections of the same structure yielded detailed images of single proteins on the viral surface. Corresponding surface renderings are a good fit to images of the viral surface derived from cryomicroscopy as well as to the shapes of crystallized surface proteins. Negative stain tomography with the appropriate stain yields detailed images of individual molecules in their normal setting on the surface of the influenza A virus.

The charge-coupled devices used in electron microscopy are coated with a scintillating crystal that gives rise to a severe modulation transfer function (MTF). Exact knowledge of the MTF is imperative for a good correspondence between image simulation and experiment. We present a practical method to measure the MTF above the Nyquist frequency from the beam blocker's shadow image. The image processing has been fully automated and the program is made public. The method is successfully tested on three cameras with various beam blocker shapes.

Variable phase dark-field contrast has been developed as an illumination technique in light microscopy, which promises significant improvements and a higher variability in imaging of several transparent specimens. In this method, a phase contrast image is optically superimposed on a dark-field image so that a partial image based on the principal zeroth-order maximum (phase contrast) interferes with an image that is based on the secondary maxima (dark field). The background brightness and character of the resulting image can be continuously modulated from a phase-contrast-dominated to a dark-field-dominated character. The condenser aperture diaphragm can be used for modulations of the image's appearance. Specimens can either be illuminated concentrically or obliquely (eccentrically) when parts of the illuminating light beams are covered and blocked. Moreover, a bright-field-like partial image can be added. In this way, the illumination can be optimally adjusted to the specific properties of the specimen. The techniques described can lead to improved visual information especially in biological specimens consisting of phase structures and additional light-absorbing or -reflecting components. Moreover, the specimen's three-dimensionality can be accentuated with improved clarity because the illuminating light beams associated with phase contrast and dark field run to the specimen at different angles of incidence.

We use a piezoelectric quartz tuning fork to calibrate the displacement of ceramic piezoelectric scanners that are widely employed in scanning probe microscopy. We measure the static piezoelectric response of a quartz tuning fork and find it to be highly linear, nonhysteretic and with negligible creep. These performance characteristics, close to those of an ideal transducer, make quartz transducers superior to ceramic piezoelectric actuators. Furthermore, quartz actuators in the form of a tuning fork have the advantage of yielding static displacements comparable to those of local probe microscope scanners. We use the static displacement of a quartz tuning fork as a reference to calibrate the three axis displacement of a ceramic piezoelectric scanner. Although this calibration technique is a nontraceable method, it can be more versatile than using calibration grids because it enables characterization of the linear and nonlinear response of a piezoelectric scanner in a broad range of displacements, spanning from a fraction of a nanometer to hundreds of nanometers. In addition, the creep and the speed dependent piezoelectric response of ceramic scanners can be studied in detail.

Atom probe tomography (APT) provides three-dimensional analytical imaging of materials with near-atomic resolution using pulsed field evaporation. The processes of field evaporation can cause atoms to be placed at positions in the APT reconstruction that can deviate slightly from their original site in the material. Here, we describe and model one such process—that of preferential retention of solute atoms in multicomponent systems. Based on relative field evaporation probabilities, we calculate the point spread function for the solute atom distribution in the “z,” or in-depth direction, and use this to extract more accurate solute concentration profiles.

The possibility of scanning electron microscope (SEM) observation and energy dispersive X-ray (EDX) spectrometry analysis in microscale regions of insulating samples using diluted ionic liquid was investigated. It is possible to obtain clear secondary electron images of insulating samples such as a rock and mineral at 5,000 times magnification by dropping 10 μL of 1 wt% of 1-ethyl-3-methylimidazolium acetate (EMI-CH3COO) diluted with ethanol onto the samples. We also obtained EDX spectra of the samples in microscale regions (∼5 μm2) without overlapping EDX spectra of other minerals with different composition. It might be possible to perform quantitative analysis of the samples if a method that does not need standard samples is applied or an X-ray detector sensitive for light elements was attached. The method of dropping 1 wt% EMI-CH3COO diluted with ethanol onto insulating samples is useful for SEM observation, EDX analysis in microscale regions, and the preservation of scarce rock and mineral samples because ionic liquid can be easily removed with acetone.

Electron backscatter diffraction (EBSD) is a powerful technique for surface microstructure analysis. EBSD analysis of cubic yttria-stabilized zirconia (YSZ) is demonstrated. The statistics related to EBSD indexing reliability shows that the probability of accurate grain orientation detection increased significantly when the electron beam energy was increased from 10 to 30 kV. As a result of better sampling with increased interaction volume, a disparity between local and average grain misorientation angle also exhibited the dependence of the electron beam energy to determine the accuracy of grain orientation. To study EBSD indexing reliability as a function of surface roughness and overlayer formation, rapid EBSD measurement tests were performed on (a) YSZ surfaces ion-polished at ion beam energies of 65 nA at 30 kV and 1 nA at 30 kV and (b) carbon-coated versus uncoated YSZ surfaces. The EBSD results at both 10 and 30 kV electron beam energies indicate that EBSD indexing reliability is negatively affected by higher ion beam milling current and amorphous overlayer formation.

Specimen quality is vital to (scanning) transmission electron microscopy (TEM) investigations. In particular, thin specimens are required to obtain excellent high-resolution TEM images. Conventional focused ion beam (FIB) preparation methods cannot be employed to reliably create high quality specimens much thinner than 20 nm. We have developed a method for in situ target preparation of ultrathin TEM lamellae by FIB milling. With this method we are able to routinely obtain large area lamellae with coplanar faces, thinner than 10 nm. The resulting specimens are suitable for low kV TEM as well as scanning TEM. We have demonstrated atomic resolution by Cs-corrected high-resolution TEM at 20 kV on a FIB milled Si specimen only 4 nm thick; its amorphous layer measuring less than 1 nm in total.

The reproducibility of contrast in secondary electron (SE) imaging during continuous electron irradiation, which caused surface contamination, was investigated using SE high-pass energy filtering in low-voltage scanning electron microscopy (SEM). According to high-pass energy-filtered imaging, dopant contrast in an indium phosphide remained remarkably stable during continuous electron irradiation although the contrast in unfiltered SE images decreased rapidly as a contamination layer was formed. Charge neutralization and the SE energy distributions indicate that the contamination layer induces a positive charge. This results in a decrease of low-energy SE emissions and reduced dopant contrast in unfiltered SE images. The retention of contrast was also observed in high-pass energy-filtered images of a gold surface. These results suggest that this imaging method can be widely used when SE intensities decrease under continuous electron irradiation in unfiltered SE images. Thus, high-pass energy-filtered SE imaging will be of a great assistance for SEM users in the reproducibility of contrast such as a quantitative dopant mapping in semiconductors.

Scientific digital imaging in three dimensions such as when using X-ray computed tomography offers a variety of ways to obtain, filter, and quantify data that can produce vastly different results. These opportunities, performed during image acquisition or during the data processing, can include filtering, cropping, and setting thresholds. Quantifying features in these images can be greatly affected by how the above operations are performed. For example, during binarization, setting the threshold too low or too high can change the number of objects as well as their measured diameter. Here, two facets of three-dimensional quantification are explored. The first will focus on investigating the question of how many voxels are needed within an object to have accurate geometric statistics that are due to the properties of the object and not an artifact of too few voxels. These statistics include but are not limited to percent of total volume, volume of the individual object, Feret shape, and surface area. Using simple cylinders as a starting point, various techniques for smoothing, filtering, and other processing steps can be investigated to aid in determining if they are appropriate for a specific desired statistic for a real dataset. The second area of investigation is the influence of post-processing, particularly segmentation, on measuring the damage statistics in high purity Cu. The most important parts of the pathways of processing are highlighted.

In conventional X-ray microtomography (μCT), the three-dimensional (3D) distribution of the attenuation coefficient of X-rays is measured and reconstructed in a 3D volume. As spatial resolution increases, the refraction of X-rays becomes a significant phenomenon in the imaging process. Although this so-called phase contrast was initially a cumbersome feature in lab-based μCT, special phase retrieval algorithms were developed to exploit these effects. Clear advantages in terms of visualization and analysis can be seen when phase retrieval algorithms are applied, including an increased signal-to-noise ratio. In this work, this is demonstrated both on simulated and measured data.