Automated segmentation of wood fibres in micro‐CT images of paper

A novel algorithm has been developed and validated to isolate individual papermaking fibres in micro‐computed tomographic images of paper handsheets as a first step to characterize the structure of the paper. The three‐step fibre segmentation algorithm segments the papermaking fibres by (i) tracking the hollow inside the fibres via a modified connected component methodology, (ii) extracting the fibre walls using a distance transform and (iii) labelling the fibres through collapsed sections by a final refinement step. Furthermore, postprocessing algorithms have been developed to calculate the length and coarseness of the segmented fibres. The fibre segmentation algorithm is the first ever reported method for the automated segmentation of the tortuous three‐dimensional morphology of papermaking fibres within microstructural images of paper handsheets. The method is not limited to papermaking fibres, but can be applied to any material consisting of tortuous and hollow fibres.     

Modified fabrication process for aperture probe cantilevers

We report the development of cantilever- and fibre-based probes for scanning near-field optical microscopy. Both probe concepts rely on the integration of a microfabricated aperture tip with reproducible optical and mechanical properties. Numerical calculations were carried out using a finite integration code to investigate the polarization-sensitive transmission behaviour of aperture tips. In order to establish technological guidelines for the optimization of the properties of the optical tip the distinct influence of the tip geometry on the intensity distribution in the vicinity of the aperture is studied in detail.     

Moisture and gamma‐ray irradiation effects on the mechanical properties of carbon fibre–reinforced plastics

The effects of γ‐irradiation and moisture absorption on the mechanical properties of carbon fibres–epoxy resin composites were studied. The properties dominated by the matrix and fibre–matrix interface (interlaminar and in‐plane shear strength) were measured at room temperature using standard tests. These tests were carried out before and after exposures to gamma irradiation and before and after immersion in water at 80°C during 21 days. The dosage of gamma irradiation was up to 11.7 MGy. The micrographs of surfaces fractured in performed tests were observed on a scanning electron microscope. They were analyzed with consulting the stated effects on mechanical properties and the measured values of the glass transition temperature of tested coupons before and after irradiation and immersion in water. The obtained results show that moisture and irradiation, if they act one after the other, have a significant influence on the degradation of matrix‐dominated mechanical properties of the tested carbon–epoxy composite.     

High‐resolution wide‐field microscopy with adaptive optics for spherical aberration correction and motionless focusing

Live imaging in cell biology requires three‐dimensional data acquisition with the best resolution and signal‐to‐noise ratio possible. Depth aberrations are a major source of image degradation in three‐dimensional microscopy, causing a significant loss of resolution and intensity deep into the sample. These aberrations occur because of the mismatch between the sample refractive index and the immersion medium index. We have built a wide‐field fluorescence microscope that incorporates a large‐throw deformable mirror to simultaneously focus and correct for depth aberration in three‐dimensional imaging. Imaging fluorescent beads in water and glycerol with an oil immersion lens we demonstrate a corrected point spread function and a 2‐fold improvement in signal intensity. We apply this new microscope to imaging biological samples, and show sharper images and improved deconvolution.     

Correlative scanning electron and confocal microscopy imaging of labeled cells coated by indium‐tin‐oxide

Confocal microscopy imaging of cells allows to visualize the presence of specific antigens by using fluorescent tags or fluorescent proteins, with resolution of few hundreds of nanometers, providing their localization in a large field‐of‐view and the understanding of their cellular function. Conversely, in scanning electron microscopy (SEM), the surface morphology of cells is imaged down to nanometer scale using secondary electrons. Combining both imaging techniques have brought to the correlative light and electron microscopy, contributing to investigate the existing relationships between biological surface structures and functions. Furthermore, in SEM, backscattered electrons (BSE) can image local compositional differences, like those due to nanosized gold particles labeling cellular surface antigens. To perform SEM imaging of cells, they could be grown on conducting substrates, but obtaining images of limited quality. Alternatively, they could be rendered electrically conductive, coating them with a thin metal layer. However, when BSE are collected to detect gold‐labeled surface antigens, heavy metals cannot be used as coating material, as they would mask the BSE signal produced by the markers. Cell surface could be then coated with a thin layer of chromium, but this results in a loss of conductivity due to the fast chromium oxidation, if the samples come in contact with air. In order to overcome these major limitations, a thin layer of indium‐tin‐oxide was deposited by ion‐sputtering on gold‐decorated HeLa cells and neurons. Indium‐tin‐oxide was able to provide stable electrical conductivity and preservation of the BSE signal coming from the gold‐conjugated markers. Microsc. Res. Tech. 78:433–443, 2015. © 2015 Wiley Periodicals, Inc.     

Phosphorus detection in vitrified bacteria by cryo‐STEM annular dark‐field analysis

Bacterial cells often contain dense granules. Among these, polyphosphate bodies (PPBs) store inorganic phosphate for a variety of essential functions. Identification of PPBs has until now been accomplished by analytical methods that required drying or chemically fixing the cells. These methods entail large electron doses that are incompatible with low‐dose imaging of cryogenic specimens. We show here that Scanning Transmission Electron Microscopy (STEM) of fully hydrated, intact, vitrified bacteria provides a simple means for mapping of phosphorus‐containing dense granules based on quantitative sensitivity of the electron scattering to atomic number. A coarse resolution of the scattering angles distinguishes phosphorus from the abundant lighter atoms: carbon, nitrogen and oxygen. The theoretical basis is similar to Z contrast of materials science. EDX provides a positive identification of phosphorus, but importantly, the method need not involve a more severe electron dose than that required for imaging. The approach should prove useful in general for mapping of heavy elements in cryopreserved specimens when the element identity is known from the biological context.     

Optimized temporal response in multichannel two-photon fluorescence lifetime microscopy using a photonic crystal fibre

The integration of fibre optics into an imaging system for the convenient delivery and collection of light has resulted in many hybrid forms of novel biomedical optical instrumentation. Although it is extremely robust and cost effective, fibre integration requires special consideration in a time‐domain fluorescence lifetime imaging schema where multipath propagation in the fibre causes significant spread in photon transit times. In this study, we investigated the effect of the length of a multimode collection fibre on the temporal performance of a multichannel fluorescence lifetime microscope and demonstrated the effectiveness of a photonic crystal fibre as a means of optimizing the collection and delivery of emitted fluorescence in terms of temporal resolution. The findings are pertinent to all studies that employ a multimode optical fibre to collect and deliver an emitted fluorescence signal from a sample to a remote detector for measurement of the characteristic fluorescence lifetime.     

A procedure for identifying textile bast fibres using microscopy: Flax,nettle/ramie,hemp and jute

Identifying and distinguishing between natural textile fibres is an important task in both archaeology and criminology. Wool, silk and cotton fibres can readily be distinguished from the textile bast fibres flax, nettle/ramie, hemp and jute. Distinguishing between the bast fibres is, however, not easily done and methods based on surface characteristics, chemical composition and cross section size and shape are not conclusive. A conclusive method based on X-ray microdiffraction exists, but as the method requires the use of a synchrotron it is not readily available. In this paper we present a simple procedure for identifying the above mentioned textile bast fibres. The procedure is based on measuring the fibrillar orientation with polarised light microscopy and detecting the presence of calcium oxalate crystals (CaC₂O₄) in association with the fibres. To demonstrate the procedure, a series of fibre samples of flax, nettle, ramie, hemp and jute were investigated. The results are presented here. An advantage of the procedure is that only a small amount of fibre material is needed.     

Condenser‐free contrast methods for transmitted‐light microscopy

Phase contrast microscopy allows the study of highly transparent yet detail‐rich specimens by producing intensity contrast from phase objects within the sample. Presented here is a generalized phase contrast illumination schema in which condenser optics are entirely abrogated, yielding a condenser‐free yet highly effective method of obtaining phase contrast in transmitted‐light microscopy. A ring of light emitting diodes (LEDs) is positioned within the light‐path such that observation of the objective back focal plane places the illuminating ring in appropriate conjunction with the phase ring. It is demonstrated that true Zernike phase contrast is obtained, whose geometry can be flexibly manipulated to provide an arbitrary working distance between illuminator and sample. Condenser‐free phase contrast is demonstrated across a range of magnifications (4–100×), numerical apertures (0.13–1.65NA) and conventional phase positions. Also demonstrated is condenser‐free darkfield microscopy as well as combinatorial contrast including Rheinberg illumination and simultaneous, colour‐contrasted, brightfield, darkfield and Zernike phase contrast. By providing enhanced and arbitrary working space above the preparation, a range of concurrent imaging and electrophysiological techniques will be technically facilitated. Condenser‐free phase contrast is demonstrated in conjunction with scanning ion conductance microscopy (SICM), using a notched ring to admit the scanned probe. The compact, versatile LED illumination schema will further lend itself to novel next‐generation transmitted‐light microscopy designs. The condenser‐free illumination method, using rings of independent or radially‐scanned emitters, may be exploited in future in other electromagnetic wavebands, including X‐rays or the infrared.     

A novel double‐image Fizeau system for accurate investigation of anisotropic polymer fibres

This paper introduces a double‐image multiple‐beam Fizeau fringes system. The introduced system can dynamically determine the variations of the refractive indices for both parallel and perpendicular polarization simultaneously. This is achieved by the simultaneous capturing of two multiple‐beam interference patterns during the mechanical processing of isotactic polypropylene fibre. This parallel determination of the refractive indices of both polarization directions allowed us to determine the full‐field distribution of the stress vector, S . To accomplish this, a mathematical model was deduced to calculate the components of the stress vector, S , i.e. parallel stress component, S₁, and perpendicular stress component, S₂. Double‐image Fizeau fringes system and the deduced mathematical model were used to investigate the variation of the refractive index and stress components of the fibre during the stretching process and propagation of necked regions.     

Study of carbon fibres and carbon–carbon composites by scanning thermal microscopy

Scanning thermal microscopy (SThM) is a relatively new technique based on atomic force microscopy in which the tip is replaced by an ultra‐miniature temperature probe. This paper reports on a preliminary investigation of the application of SThM in the characterization of the thermal properties of carbon fibres and carbon–carbon (CC) composites. The technique enabled a comparative study to be made of discrete fibre and matrix thermal properties in a series of model unidirectional composites. The thermal images revealed a marked increase in thermal conductivity of the matrix with increasing temperature of treatment and hence confirmed the development of a highly ordered carbon matrix. The results were in qualitative agreement with previously determined values of thermal conductivity from which the separate values of fibre and matrix thermal conductivity had been derived. The technique was also applied to the characterization of samples of unknown processing history, enabling an estimation to be made of the heat treatment and type of the fibres and matrix present in the composite. It was concluded that SThM promises to be a powerful technique for the study of the thermal properties of CC composites and carbon fibres, as it uniquely enables variations in local thermal conductivity to be detected and resolved. Absolute quantification of the technique remains the key to its future widespread acceptance in materials characterization.     

3D reconstruction of elastin fibres in coronary adventitia

A 3D reconstruction of individual fibres in vascular tissue is necessary to understand the microstructure properties of the vessel wall.  The objective of this study is to determine the 3D microstructure of elastin fibres in the adventitia of coronary arteries.  Quantification of fibre geometry is challenging due to the complex interwoven structure of the fibres.  In particular, accurate linking of gaps remains a significant challenge, and complex features such as long gaps and interwoven fibres have not been adequately addressed by current fibre reconstruction algorithms.  We use a novel line Laplacian deformation method, which better deals with fibre shape uncertainty to reconstruct elastin fibres in the coronary adventitia of five swine. A cost function, based on entropy and Euler Spiral, was used in the shortest path search. We find that mean diameter of elastin fibres is 1.67 ± 1.42 m and fibre orientation is clustered around two major angles of 8.9? and 81.8?.  Comparing with CT‐FIRE, we find that our method gives more accurate estimation of fibre width.  To our knowledge, the measurements obtained using our algorithm represent the first investigation focused on the reconstruction of full elastin fibre length.  Our data provide a foundation for a 3D microstructural model of the coronary adventitia to elucidate the structure–function relationship of elastin fibres.     

Influence of gallium‐doped zinc‐oxide thickness on polymer light‐emitting diode luminescence efficiency

Conducting atomic force microscopy and scanning surface potential microscopy were used to study the local electrical properties of gallium‐doped zinc oxide (GZO) films prepared by pulsed laser deposition (PLD) on a polyimide (PI) substrate. For a PLD deposition process time of 8 min, the root‐mean‐square roughness, coverage percentage of the conducting regions, and mean work function on the GZO surface were 2.33 nm, 96.6%, and 4.82 eV, respectively. When the GZO/PI substrate was used for a polymer light‐emitting diode (PLED), the electroluminescence intensity increased by nearly 20% compared to a standard PLED, which was based on a commercial‐ITO/glass substrate. Microsc. Res. Tech. 76:783–787, 2013. © 2013 Wiley Periodicals, Inc.     

Photonic nanopatterns of gold nanostructures indicate the excitation of surface plasmon modes of a wavelength of 50–100 nm by scanning near-field optical microscopy

Scanning near‐field optical microscopy images of metal nanostructures taken with the tetrahedral tip (T‐tip) show a distribution of dark and bright spots at distances in the order of 25–50 nm. The images are interpreted as photonic nanopatterns defined as calculated scanning near‐field optical microscopy images using a dipole serving as a light‐emitting scanning near‐field optical microscopy probe. Changing from a positive to a negative value of the dielectric function of a sample leads to the partition of one spot into several spots in the photonic nanopatterns, indicating the excitation of surface plasmons of a wavelength in the order of 50–100 nm in metal nanostructures.     

Characterization of the cutting edge of glass knives for ultramicrotomy by scanning force microscopy using cantilevers with a defined tip geometry

The geometry of glass knife edges for ultramicrotomy was studied with nanoscale resolution using scanning force microscopy (SFM) in the contact mode. The local shape of the cutting edge was estimated from single line profiles of the SFM topographic images by taking into account the exact radius of the ultrasharp silicon tip. The tip radius was estimated from secondary electron micrographs recorded at low voltage by field emission scanning electron microscopy (FESEM). The radius of the investigated cutting edges was found to be in range 5–20 nm. The results obtained illustrate that the combination of SFM and high resolution FESEM provides a unique means to determine precisely the radius of glass knives.     

A computerized microspectrophotometer using fibre optics for transmission and detection of light

A computerized microspectrophotometer was developed to provide rapid and accurate sequential measurements on cells, including nuclear DNA content, by fluorescence staining, photometric grain counting from autoradiographs, and nuclear size estimated by measurement aperture area. To alleviate bulk and vibrations caused by additional stepper motors, the excitation light and filters as well as the emission filters and photomultipliers were detached from the main microscope frame. The light was directed to and from the microscope through fibre optic bundles. This configuration provided an excellent light gathering faculty and made optical alignment easier. The machine provided highly accurate sequential measurements on a relatively large number of cells by static photometry.     

Integration of a high‐NA light microscope in a scanning electron microscope

We present an integrated light‐electron microscope in which an inverted high‐NA objective lens is positioned inside a scanning electron microscope (SEM). The SEM objective lens and the light objective lens have a common axis and focal plane, allowing high‐resolution optical microscopy and scanning electron microscopy on the same area of a sample simultaneously. Components for light illumination and detection can be mounted outside the vacuum, enabling flexibility in the construction of the light microscope. The light objective lens can be positioned underneath the SEM objective lens during operation for sub‐10 μm alignment of the fields of view of the light and electron microscopes. We demonstrate in situ epifluorescence microscopy in the SEM with a numerical aperture of 1.4 using vacuum‐compatible immersion oil. For a 40‐nm‐diameter fluorescent polymer nanoparticle, an intensity profile with a FWHM of 380 nm is measured whereas the SEM performance is uncompromised. The integrated instrument may offer new possibilities for correlative light and electron microscopy in the life sciences as well as in physics and chemistry.     

SEM and AFM studies of dip‐coated CuO nanofilms

Cupric oxide (CuO) semiconducting thin films were prepared at various copper sulfate concentrations by dip coating. The copper sulfate concentration was varied to yield films of thicknesses in the range of 445–685 nm by surface profilometer. X‐ray diffraction patterns revealed that the deposited films were polycrystalline in nature with monoclinic structure of (?111) plane. The surface morphology and topography of monoclinic‐phase CuO thin films were examined using scanning electron microscopy (SEM) and atomic force microscopy (AFM), respectively. Surface roughness profile was plotted using WSxM software and the estimated surface roughness was about ～19.4 nm at 30 mM molar concentration. The nanosheets shaped grains were observed by SEM and AFM studies. The stoichiometric compound formation was observed at 30 mM copper sulfate concentration prepared film by EDX. The indirect band gap energy of CuO films was increased from 1.08 to 1.20 eV with the increase of copper sulfate concentrations. Microsc. Res. Tech., 2013. © 2012 Wiley Periodicals, Inc.     

Imaging of various surface properties of fluorescently labelled phospholipid Langmuir–Blodgett films with a combined scanning probe microscope

We present results of phase separation of a single-component system of 1,2-dihexadecanoyl- sn -glycero-3-phospho-[ N -(4-nitrobenz)-2-oxa-1,3-diazolyl]ethanolamine in which a liquid-condensed (LC) phase co-exists with a liquid-expanded (LE) phase. Domain formation in the co-existence region was studied using a newly developed combined scanning near-field optical microscope–atomic force microscope (SNOM–AFM). We demonstrate for the first time that the topographic, friction, fluorescence and surface potential distributions for a phase-separated single-component Langmuir–Blodgett film between the LE and LC phases can be simultaneously observed using the SNOM–AFM with a thin-step etched optical fibre probe.