bluuebunny/crossref_metadata_2025 huggingface dataset:
High‐resolution scanning electron microscopy study of sputtered nanolaminated Ti/TiN multilayers
Authors: le Paven‐Thivet C. , Sant C. , Grillon F. , Houdy P.
Published: June 2010

This work presents the morphologic and structural study of nanolaminated Ti/TiN multilayers using high‐resolution scanning electron microscopy (HR‐SEM), coupled to x‐ray reflectometry (XRR). The multilayers have been deposited by reactive rf‐sputtering on silicon substrates. For large period thickness (λ=40 nm, 10 periods), in XRR, the low number of interfaces makes the interference less structured. An experimental pattern with broad and weakly intense Braggs peaks is obtained, but is difficult to simulate. On the other hand, HR‐SEM observation of cross sections gives excellent pictures of the multilayer, so that precise measurements of the thickness can be achieved: a 42 nm thick period is observed, formed with 17 nm of Ti and with 25 nm of TiN. For small (Ti+TiN) period thickness (λ=2.5 nm, 120 periods), the XRR pattern exhibits intense and narrow Bragg peaks: the number of interfaces is sufficient to structure the interference and an intense signal is obtained. The best fit of simulation is obtained for a 2.6 nm thin period, made of 0.9 nm of Ti and 1.7 nm of TiN. No laminated structure has been observed by cross‐section HR‐SEM observation because its resolution (around 2 nm at 10 kV) is larger than the layer thickness in a period. High‐resolution SEM and XRR are thus two complementary techniques for the routine characterization of multilayers.
Correlative high‐resolution morphologic analysis of the three‐dimensional organization of human chromosomes
Authors: Gobbi Pietro , Thalhammer Stefan , Falconi Mirella , Stark Robert W. , Heckl Wolfgang M. , Mazzotti Giovanni
Published: June 2010

A correlative morphologic analysis was carried out on isolated metaphase chromosomes by means of field emission in‐lens scanning electron microscopy (FEISEM) and atomic force microscopy (AFM). Whereas FEISEM provides ultra‐high resolution power and allows the surface analysis of biological structures free of any conductive coating, the AFM allows imaging of biological specimens in ambient as well as in physiologic conditions. The analysis of the same samples was made possible by the use of electrical conductive and light transparent ITO glass as specimen holder. Further preparation of the specimen specific for the instrumentation was not required. Both techniques show a high correlation of the respective morphologic information, improving their reciprocal biological significance. In particular, the biological coat represents a barrier for surface morphologic analysis of chromosome spreads and it is sensitive to protease treatment. The chemical removal of this layer permits high‐resolution imaging of the chromatid fibers but at the same time alters the chromosomal dimension after rehydration. The high‐resolution level, necessary to obtain a precise physical mapping of the genome that the new instruments such as FEISEM and AFM could offer, requires homogeneously cleaned samples with a high grade of reproducibility. A correlative microscopical approach that utilizes completely different physical probes provides complementary useful information for the understanding of the biological, chemical, and physical characteristics of the samples and can be applied to optimize the chromosome preparations for further improvement of the knowledge about spatial genome organization.
Visualization of artificial lignin supramolecular structures
Authors: Mičič Miodrag , Jeremič Milorad , Radotič Ksenija , Mavers Melissa , Leblanc Roger M.
Published: June 2010

In this paper we are presenting the results of our environmental scanning electron microscopy (ESEM) investigation of the lignin model compound—enzymatically polymerized coniferyl alcohol, also known as dehydrogenate polymer (DHP). The goals of this study were to visualize the supramolecular organization of DHP polymer on various substrates, namely graphite, mica, and glass, and to explore the influence of substrate surface properties and associated collective phenomena on the lignin self‐assembled supramolecular structure. Based on results obtained with ESEM, combined with previously published results based on scanning tunneling microscopy (STM) and electron spin resonance (ESR) technique, we looked at lignin structure ranging from a monomer on a fraction of nanometer scale to a large aggregate on a fraction of millimeter scale, therefore using six orders of magnitude range of size. Herein, we are presenting evidence that there are at least four different levels of the supramolecular structure of lignin, and that its supramolecular organization is well dependent on the substrate surface characteristics, such as hydrophobicity, delocalized orbitals, and surface‐free energy.
Atomic force microscopy of human hair
Authors: Swift J. Alan , Smith James R.
Published: June 2010

The atomic force microscope (AFM) was used to investigate the surface architecture of the entire lengths of cleaned human head hairs. Many features previously seen with the scanning electron microscope (SEM) were identified. However, the AFM has provided much greater detail and, in particular, the hair's cuticular surfaces appear not to be as smooth as had been previously supposed. A consistent feature was of step discontinuities or “ghosts” on the scale surfaces. These delineated the original location of each overlying scale before its edge had been chipped away. There was a change in the longitudinal angular presentation of the surfaces about each ghost. This means the distal ends of each cuticle cell have been syn‐thesised in the follicle to be thicker than where that same cuticle cell is bounded on both sides by other cuticle cells. The undamaged outer cuticular surfaces at the root end of each hair were covered everywhere by longitudinal ridges (striations). Where the hair surface was worn, the striations terminated at a scale edge ghost. The ridges were approximately 9 nm high and were in parallel array with a lateral repeat spacing of about 350 nm. The striations are evidently formed on the outer surface of each cuticle cell following earlier contact in the hair follicle with the inner root sheath. The study of stained transverse sections of hairs in the transmission electron microscope (TEM) is suggested as a means for throwing some light on the underlying structure and chemistry of the striations. Finally, our AFM studies have revealed that the surface of the freshly emergent hair gradually changes over a distance of about 20 mm and that the surface of the hair for most of its length is quite different from that near the root. This is likely to be of import to those engaged in the hair toiletries industry.
Electron microscopy of squamous cell carcinoma of the head and neck
Authors: Leek Håkan , Albertsson Maria
Published: June 2010

Squamous cell carcinoma of the head and neck carries a bad prognosis. In order to achieve cure, the most important thing to attain is local tumour control. The main therapy available is external radiotherapy, which can be supplemented when necessary, with interstitial radiotherapy, chemotherapy and surgery.

In this paper we have evaluated specimens, taken before therapy, from 35 patients with squamous cell carcinoma of the head and neck. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) analyses were made. With SEM, the parameters analysed were the amount and appearance of microvilli, filaments, and blood vessels. From TEM, scoring was made of the filaments, desmosomes, nuclei, nucleoli, mitochondria, and blood vessels. Scoring of the samples showed a difference between the group with recurrent disease (n = 10, Group 1) and the group with local tumor control (n = 25, Group 2) in regard to both blood vessels and intracellular filaments. No differences of the nuclei, nucleoli, or the mitochondria were observed.
Correlative light and backscattered electron microscopy of bone —Part II: Automated image analysis
Authors: Goldman Haviva M. , Blayvas Aron , Boyde Alan , Howell Peter G. T. , Clement John G. , Bromage Timothy G.
Published: June 2010

Detailed studies of biological phenomena often involve multiple microscopy and imaging modes and media. For bone biology, various forms of light and electron microscopy are used to study the microscopic structure of bone. Integrating information from the different sources is necessary to understand how different aspects of the bone structure interact. To accomplish this, methods were developed to prepare and image thin sections for cor‐relative light microscopy (LM) and backscattered electron imaging in the scanning electron microscope (BSE‐SEM). Images of the same fields of view may then be analyzed for degrees of relationships between specimen features not observed by LM or SEM alone. These methods are applied here to study possible associations between the degree of bone mineralization and pattern of collagen fiber orientation in the mid‐shaft of the human femur. The “relational images” obtained allow us to examine the relationship between these two variables, both objectively and quantitatively.
An anomalous contrast in scanning electron microscopy of insulators: The pseudo‐mirror effect
Authors: Belhaj M. , Jbara O. , Odof S. , Msellak K. , Rau E. I. , Andrianov M. V.
Published: June 2010

In a scanning electron microscope, electron‐beam irradiation of insulators may induce a strong electric field due to the trapping of charges within the specimen interaction volume. On one hand, this field modifies the trajectories of the beam of electrons subsequently entering the specimen, resulting in reduced penetration depth into the bulk specimen. On the other hand, it leads to the acceleration in the vacuum of the emitted secondary electrons (SE) and also to a strong distortion of their angular distribution. Among others, the consequences concern an anomalous contrast in the SE image. This contrast is due to the so‐called pseudo‐mirror effect. The aim of this work is first to report the observation of this anomalous contrast, then to give an explanation of this effect, and finally to discuss the factors affecting it. Practical consequences such as contrast interpretations will be highlighted.
The role of induced contrast in images obtained using the environmental scanning electron microscope
Authors: Toth M. , Phillips M. R.
Published: June 2010

Generation of contrast in images obtained using the environmental scanning electron microscope (ESEM) is explained by interpretation of images acquired using the gaseous secondary electron detector (GSED), ion current, and the Everhart‐Thornley detector. We present a previously unreported contrast component in GSED and ion current images attributed to signal induction by changes in the concentration of positive ions in the ESEM chamber during image acquisition. Changes in positive ion concentration are caused by changes in electron emission from the sample during image acquisition and by a discrepancy between the drift velocities of negative and positive charge carriers in the imaging gas. The proposed signal generation mechanism is used to explain contrast reversal in images produced using the GSED and ion current signals and accounts for discrepancies in contrast observed, under some conditions, in these types of images. Combined with existing models of signal generation in the ESEM, the proposed model provides a basis for correct interpretation of ESEM images.
Immobilization of proteins on self‐assembled monolayers
Authors: Wadu‐Mesthrige Kapila , Amro Nabil A. , Liu Gang‐Yu
Published: June 2010

The immobilization of protein molecules on self‐assembled monolayers (SAM) by physical interactions and chemical bonding has been studied using atomic force microscopy (AFM). The proteins used for our investigation are bovine serum albumin (BSA), lysozyme (LYZ), and normal rabbit immunoglobulin G (IgG). The surfaces are methyl‐, hydroxyl‐, carboxylic acid‐ and aldehyde‐terminated SAMs. We found that BSA and LYZ can be readily immobilized on SAMs at their isoelectric point (IEP). The detailed surface morphology of adsorbed proteins varies with the functionality of the SAMs. The strong hydrophobic interaction at the IEP is attributed to immobilization. If the solution pH is deviated from the IEP, proteins may be attached onto the surface via electrostatic interactions. Covalent binding between the aldehyde‐terminated SAM and the H2N‐groups in the protein results in immobilization of all three proteins. The individual proteins and their orientations on SAMs are clearly resolved from high‐resolution AFM images. The stability and bioactivity of these immobilized proteins are also studied.
Multiphoton confocal microscopy using a femtosecond Cr:Forsterite laser
Authors: Liu Tzu‐Ming , Chu Shi‐Wei , Sun Chi‐Kuang , Lin Bai‐Ling , Cheng Ping‐Chin , Johnson I.
Published: June 2010

With its output wavelength covering the in‐ frared penetrating window of most biological tissues at 1200–1250 nm, the femtosecond Cr:forsterite laser shows high potential to serve as an excellent excitation source for the multiphoton fluorescence microscope. Its high output power, short optical pulse width, high stability, and low dispersion in fibers make it a perfect replacement for the currently widely used Ti: sapphire laser. In this paper, we study the capability of using a femtosecond Cr: forsterite laser in multiphoton scanning microscopy. We have performed the multiphoton excited photolumines‐cence spectrum measurement on several commonly used bioprobes using the 1230 nm femtosecond pulses from a Cr:forsterite laser. Efficient fluorescence can be easily observed in these bioprobes through two‐photon or three‐photon excitation processes. These results will assist in the selection of dichroic beam splitter and band pass filters in a multiphoton microscopic system. We have also performed the autofluorescence spectrum measurement from chlorophylls in live leaves of the plant Arabidopsis thaliana excited by 1230 nm femtosecond pulses from the Cr:forsterite laser. Bright luminescence from chlorophyll, centered at 673 and 728 nm, respectively, can be easily observed. Taking advantage of the bright two‐photon photoluminescence from chlorophyll, we demonstrated the two‐photon scanning paradermal and cross‐sectional images of palisade mesophyll cells in live leaves of Arabidopsis thaliana.
High‐temperature oxidation of nickel and chromium studied with an in‐situ environmental scanning electron microscope
Authors: Schmid B. , Aas N. , Grong Ø. , Ødegård R.
Published: June 2010

Investigations of the morphology of metal oxide scales formed at high temperatures in oxidative environments are usually undertaken after exposure of the samples is completed. In this study, an environmental scanning electron microscope (ESEM) was used as a tool for the in‐situ observation of oxide scale formation. Pure nickel and chromium samples were oxidized at a temperature of 973 K in either pure oxygen or water vapour at a pressure of 667 Pa. The evolution of an oxide scale was followed in‐situ for up to 3 h. The morphology of the developing oxide scales was found to be a function of the metal substrate and the gaseous species. The growth mechanisms of the different metal oxide scales are reviewed and related to the analysed in‐situ images. Emphasis is placed on the relationship between oxidation mechanism and scale morphology. Nickel is seen to oxidise by outward diffusion of nickel probably on oxide grain boundaries when exposed to oxygen. Water vapour changes the scale morphology and a duplex‐type scale arises due to preferential overgrowth. The scale which develops due to chromium oxidation in oxygen is a fine‐grained, thin, and dense layer. In contrast, water vapour leads to whisker growth on chromium and an open, felt‐like structure forms. The applicability of the ESEM to the study of such systems is demonstrated, and its limitations are outlined. The results are encouraging examples of the possibilities which the in‐situ ESEM technique possesses.
Manipulating biological samples for environmental scanning electron microscopy observation
Authors: Tai Susan S. W. , Tang Xue Ming
Published: June 2010

Biological samples having different characteristics were observed by environmental scanning electron microscopy (ESEM). The environmental conditions for untreated biological samples was determined by optimizing sample temperature and chamber pressure. When the temperature was at 4° – 6°C and chamber pressure was 5.2–5.9 Torr, the relative humidity in the specimen chamber was about 85%. Under these conditions, the surface features of the sample were completely exposed and did not exhibit charging. The images obtained from the untreated samples at different ESEM conditions were also compared with fixed and coated samples observed under high vacuum.
Confocal laser scanning photoelasticity: Improvement of precision for quantitative photoelastic studies on model composite materials
Authors: Lütze S.
Published: June 2010

Photoelastic studies on model composite materials with different model materials and load applications have been performed to understand the various failure mechanisms in real composites. Optical investigations of the stress fields have been conducted using a polarising, confocal laser scanning microscope (Zeiss LSM 410, Zeiss, Jena, Germany), where the phase of the isochromatic fringes has been determined from the data. Two different methods are presented to improve the quantitative evaluation of the isochromatic phase. By combining manual and automatic evaluation of fringes, even samples with a high stress gradient can be analysed. A method has been developed for the correction of the integrated through‐thickness birefringence, in the case of rotationally symmetric stress fields. This technique permits the inverse calculation of stress at various depths in the sample, in particular in the case of a nonlinear dependence of optical birefringence on stress. Both methods are demonstrated in the evaluation of tensile tests on multifibre model samples (glass fibres in polycarbonate).
Investigation of human hair fibers using lateral force microscopy
Authors: McMullen Roger L. , Kelty Stephen P.
Published: June 2010

Atomic force microscopy (AFM) and lateral force microscopy (LFM) were used to investigate the morphologic and surface changes associated with various surface modifications to human hair. These included extraction with a series of solvents, bleaching, and treatment with a cationic copolymer. The study assessed the ability of these techniques to distinguish the changes in surface properties, including morphology and friction coefficient, as manifested in changes brought about by the indicated surface modifications. While topographic morphology can easily be investigated with contact AFM, LFM offers an additional tool for probing the surface distribution of oils and waxes. The removal of surface lipids from the fiber surface was accomplished using soxhlet extraction with t‐butanol and n‐hexane, while the free internal lipids (within the fiber structure) were removed by extraction with a mixture of chloroform and methanol (70:30, v/v). In addition, the surface of hair was modified with the cationic polymer, co(vinyl pyrrolidone‐methacry lamidopropyl trimethylam‐monium chloride [PVP/MAPTAC]), and its distribution on the surface was monitored. Ambient AFM and LFM studies of surface modified and native fibers clearly indicate that when investigated as a function of tip loading force, the different modifications result in changes of the friction coefficient, which increase in this order: native, bleached, solvent extracted, and polymer‐treated hair. Friction images show surface variations that are interpreted as areas of varying lipid film coverage. In addition, topographic images of the fibers show the presence of small pores, which become increasingly prevalent upon solvent extraction.
High‐resolution analysis of engineered type I collagen nanofibers by electron microscopy
Authors: Huang Lei , Apkarian Robert P. , Chaikof Elliot L.
Published: June 2010

Collagen nanofibers were generated at ambient temperature and pressure by electrospinning a 1wt% solution of type I collagen and polyethylene oxide. Products were imaged with high‐resolution scanning electron microscopy (HRSEM) at medium (∼30,000 ×) and high magnifications (∼100,000 ×) and with transmission electron microscopy (TEM). The capacity to produce collagen nanofibers may lead to the generation of extracellular matrix‐based fabrics with applications in the fields of wound healing and tissue engineering.
Convolution and correlation: A case study of scanning imaging and analysis systems
Authors: Yan H. , Joy D. , Lei M.
Published: June 2010

The relationship between convolution/correlation operation and the data acquisition process of the scanning microscope and spectrometer families is analyzed. It is shown that a coordinate or event sensitive detector, and the intrinsic or extrinsic property of the specimen response, are two important factors in distinguishing the data acquisition mode of such systems. Four types of convolution‐ and correlation‐based modes are extracted to illuminate the physical characteristics of scanning imaging and analysis systems by focusing on the probe, specimen, detector, and their relationships. Criteria for identifying these modes are explored. In addition, the physical meanings of general existing coefficients between the independent variables of convolution and correlation are investigated.
Three‐dimensional reconstruction of microstructures in gibbsite using charge contrast images
Authors: Baroni Travis C. , Lincoln Frank J. , Griffin Brendan J. , Cornell John B. , Roach Gerald I. D.
Published: June 2010

An iterative process of serial sectioning and charge contrast imaging has been applied to gibbsite particles embedded in epoxy resin. The features observed in the two‐dimensional sections have been reconstructed into three‐dimensional (3‐D) objects, so that the volumetric relationships in the microstructure are visualised. The 3‐D objects confirm that the image detail present in charge contrast images of gibbsite is related to the microstructure and the processes occurring during the crystallisation of gibbsite. Speculative explanations for what these microstructures may represent are presented.
An empirical energy loss equation of electrons
Authors: Zhenyu Tan , Yancai He
Published: June 2010

A modified Love‐Cox‐Scott (1978) equation of electron energy loss has been suggested. The stopping powers predicted by the modified Love‐Cox‐Scott equation are compared with those by the Tung et al. (1979) model, the Joy and Luo (1989) equation, and the experimental data given in database of Joy at: http://web.ukt.edu/∼scrutk. In the energy range of E0⩽5keV, the Monte Carlo simulations of the electron scattering in Al, Ag, and Au have been performed, applying the Mott cross section for elastic scattering and the modified Love‐Cox‐Scott equation (1978) and the equations by Love et al. (1978) and Joy and Luo (1989), respectively, for the inelastic scattering. The calculated results on the backscattering coefficients, the energy distributions of the backscattered electrons, and the energy dissipation of the electron based on the three equations are compared.
Cosmetic assessment of the human hair by confocal microscopy
Authors: Hadjur Christophe , Daty Gérard , Madry Geneviève , Corcuff Pierre
Published: June 2010

The optical sectioning property of the confocal microscope offers a breakthrough from the classic observation of the hair in a scanning electron microscope (SEM). Confocal microscopy requires minimal sampling preparation, and the hair can be observed in its natural environment with less damage than by other microscopic methods such as SEM. While used in the reflection mode, the true morphology of the cuticle and the various exogenous deposits at the surface can be identified and quantified. This relatively noninvasive, nondestructive technique is routinely used by us to monitor the efficiency of cleansing shampoos, to assess the homogeneity of layering polymers, and to evaluate the changes they induce in the optical properties of the hair surface in terms of opacity, transparency, and brilliancy. A second important field of investigation uses the fluorescence channel which reveals the internal structure of the hair. Fluorescent probes (rhodamine and its derivatives) demonstrate the routes of penetration and outline the geometry of cortical cells and of the medulla according to their lipophilic or hydrophilic properties. A volume rendering of a hair cylinder provides a better understanding of the interrelationships between cuticle cells, cortical cells, and the medullar channel. This recent technology is becoming an invaluable tool for the cosmetic assessment of the hair.
Application of the scatter diagram technique to the scanning electron microscope: Preliminary results from diamond
Authors: Patat J. M. , Cazaux J. , Lehuede P. , Durand O.
Published: June 2010

Using primary beam energies E0 ranging from 0.2 to 15 keV and an in‐lens detector, a series of images of the same region of an artificial microstructured diamond sample have been acquired in scanning electron microscopy. Next, the images were analysed by using a scatter diagram technique to underline the topographic contrast change and contrast reversal. The results obtained from 0.5 to 15 keV are discussed with the help of an expression derived from the constant loss model for the secondary electron yield δ of diamond, but including the respective roles of the angle of incidence, i, and of the angle of detection, α. More surprising is the quality of images obtained at a beam energy as low as 0.2 keV, and more difficult to explain is the significant contrast change between 0.2 keV and 0.5 keV. For the first time, scatter diagrams are used as a diagnostic tool in scanning electron microscopy, and after some improvements it is hoped that the experimental approach followed here may lead to quantitative estimates of the local tilts of a specimen surface.
Tracers in vascular casting resins enhance backscattering brightness
Authors: Schraufnagel Dean E. , Ganesan Dhanalakshmi P.
Published: June 2010

Studying cast microvasculature with scanning electron microscopy has expanded our knowledge of many circulations, but need arises to determine the blood source of vascular beds that are supplied by two circulations. One way to do this is to mark the casting resin by adding a tracer compound that can be detected in the scanning electron microscope. A potential method of distinguishing different substances is to detect the backscattered electrons that are emitted from the tracer if the tracer is a heavier element, because heavier elements backscatter more electrons. To explore different tracers, we tested lead, titanium, iron, osmium, and uranium as solutions of different polarity and powders. The tracers were added to 1 ml of methyl methacrylate in log concentrations. Shrinkage, hardness, cast quality, and change in brightness from the tracer were compared with multivariate analysis at scanning electron microscopic working distances of 15 and 39 mm on carbon‐coated and uncoated specimens. Several concentrations caused sedimentation of the tracer and prevented the resin from solidifying. Tetraethyl lead shortened the hardening time; uranyl acetate and osmium tetroxide prolonged it. Most tracers decreased shrinkage. When lead citrate and Reynolds solutions were removed, the brightness correlated with increasing atomic number, concentration of the tracer, and mean atomic number of the specimen (p <0.0001). The substances that increased contrast most were tetraethyl lead and uranium. Backscattering electron detection can distinguish methacrylate casts that have small amounts of heavier elements added to them, but an optimal tracer has not yet been established.
Error rates in dental microwear quantification using scanning electron microscopy
Authors: Grine F. E. , Ungar P. S. , Teaford M. F.
Published: June 2010

There is a degree of correlation between dietary habits and dental microwear in extant primates, and this has enabled inferences to be made about prehistoric diets. Several techniques have been used to quantify microwear, but the comparability of results derived from each has not been demonstrated. Moreover, neither intra‐ nor interobserver error rates in microwear quantification have been documented to date. We here assess intra‐ and interobserver error using Microware 4.0, and evaluate intertechnique comparability using the three methods that have been most widely employed in the field. This study documents an overall intraobserver error rate of about 7%, and an overall interobserver error rate of some 9%. Both intra‐and interobserver error appears to be influenced substantially by the nature of the micrograph being measured. In no instance did the results obtained by different observers using Microware 4.0 differ significantly, and there was a reasonable degree of interobserver consistency in the rank ordering of micrographs in relation to any given parameter. The results obtained through the use of different quantitative techniques differed significantly, with an overall intertechnique error rate of approximately 19%. Several variables, including differences in magnification factor, scanning electron microscope kV settings, and specimendetector relationships undoubtedly contribute to the differences among the three methods, but we were not able to assess their relative importance. Microwear quantification permits distinctions between broad dietary categories, but the margin of intra‐ and interobserver error should be taken into account when defining pattern differences between populations (or species) or when documenting seaseasonally mitigated differences within a taxon. In view of the error introduced by the use of different methods, we suggest that a consistent technique, such as offered by the Microware software package, be adopted by current researchers to establish a common microwear database.
Scanning electron microscopy observation of the substructural evolution of aluminium alloys during cold rolling and partial annealing
Authors: Schouwenaars Rafael , Cerrud Sara , Ortiz Armando
Published: June 2010

Classical etching techniques for revealing cold deformation and partial recrystallisation in metals have been optimised for optical microscopy, which is limited by its resolution. Detailed studies of the mechanisms involved in recovery and recrystallisation during heat treatment are generally made by transmission electron microscopy. The limitation of this technique, with a few exceptions, is its small field of view and the small fraction of the sample available for inspection. The present article departs from the statement that etching, which is a surface alteration technique, must have effects that are detectable by scanning electron microscope (SEM). It was found that carefully adapted polishing and etching procedures allow for substructural investigations by SEM, resulting in various advantages compared with both optical microscopy and TEM.
High‐temperature electron backscatter diffraction and scanning electron microscopy imaging techniques: In‐situ investigations of dynamic processes
Authors: Seward G. G. E. , Prior D. J. , Wheeler J. , Celotto S. , Halliday D. J. M. , Paden R. S. , Tye M. R.
Published: June 2010

In‐situ heating experiments have been conducted at temperatures of approximately 1200 K utilising a new design of scanning electron microscope, the Cam‐Scan X500. The X500 has been designed to optimise the potential for electron backscatter diffraction (EBSD) analysis with concomitant in‐situ heating experimentation. Features of the new design include an inclined field emission gun (FEG) column, which affords the EBSD geometrical requirement of a high (typically 160°) angle between the incoming electron beam and specimen surface, but avoids complications in heating‐stage design and operation by maintaining it in a horizontal orientation. Our studies have found that secondary electron and orientation contrast imaging has been possible for a variety of specimen materials up to a temperature of at least 900°C, without significant degradation of imaging quality. Electron backscatter diffraction patterns have been acquired at temperatures of at least 900°C and are of sufficient quality to allow automated data collection. Automated EBSD maps have been produced at temperatures between 200°C and 700°C in aluminium, brass, nickel, steel, quartz, and calcite, and even at temperatures >890°C in pure titanium. The combination of scanning electron microscope imaging techniques and EBSD analysis with high‐temperature in‐situ experiments is a powerful tool for the observation of dynamic crystallographic and microstructural processes in metals, semiconductor materials, and ceramics.
Limitations on optical sectioning in live‐cell confocal microscopy
Authors: Pawley James B.
Published: June 2010

In three‐dimensional (3‐D) live‐cell microscopy, it has been common to treat cells as having a constant refractive index (RI). Although the variations in RI associated with the nucleus and other organelles were recognized from phase‐ and differential interference contrast (DIC) images, it was assumed that they were small and would not affect 3‐D fluorescence images obtained using widefield/deconvolution, confocal of multiphoton imaging. This paper makes clear that this confidence was misplaced. Confocal images made using backscattered light (BSL) to image the flat, glass/water interfaces above and below living microscope specimens should reveal these structures as flat and featureless. That the image of the interface on the far side of the cells is neither flat nor featureless indicates that the “optical section” surface can be profoundly distorted by the RI irregularities associated with the presence of nuclei and other subcellar structures. This observation calls into question the reliability of images made using any of the current methods for performing 3‐D light microscopy of living cells.
A monte carlo study of the thickness determination of ultra‐thin films
Authors: Zhenyu Tan , Yueyuan Xia
Published: June 2010

A Monte Carlo method is utilized to simulate the energy spectra for low‐energy electron backscattered from different thin films deposited on different bulk substrates. A method of thickness determination for ultra‐thin films is presented, which is obtained from the analysis of the basic characteristics of the energy spectra of backscattered electrons. This method is predicted to be particularly suitable for determining the thickness of ultra‐thin films, and to be useful for both cases of light film on heavy substrate and heavy film on light substrate. The thickness resolution is estimated to be as high as subnanometer scale, provided the resolution of the electron energy spectrometer used to be high enough.
Monte Carlo analysis of the detection of clay occlusion of respirable quartz particles using multiple voltage scanning electron microscopy
Authors: Hnizdo V. , Wallace W. E.
Published: June 2010

The electron incident‐energy dependence of the relative intensities of Al and Si x‐rays produced in a respirable‐sized quartz particle by scanning electron microscopy is sensitive to the inhomogeneity of the distribution of Al and Si in the particle. Realistic Monte Carlo calculations of this energy dependence validate the proposal to use this effect for the detection of particles in which an aluminosilicate coating occludes the surface of a silica core.
The two‐dimensional and three‐dimensional processing of images provided by conventional microscopes
Authors: Martišek Dalibor
Published: June 2010

In this paper, I present an interesting processing method of microscopic images. High‐pass type filters are generally used for image focusing. They enhance the high spatial frequencies. They are, however, efficient only in cases when the picture is not sharp because of the low contrast on high frequencies (for example in a TV picture). These filters, are not appropriate if the lack of sharpness has been caused by other factors. In this case, it is not possible to construct a three‐dimensional model of the observed object. Better results and a three‐dimensional model can be obtained by applying the following theory. As part of this paper, an original program based on this theory is described.
Optimisation of the environmental scanning electron microscope for observation of drying of matt water‐based lacquers
Authors: Royall C. P. , Donald A. M.
Published: June 2010

Environmental scanning electron microscopy (ESEM) modifies conventional SEM through the use of a partial gas pressure in the microscope specimen chamber. Like conventional SEM, it has the resolution to image structure on the submicron lengthscale, but can also tolerate hydrated specimens if water vapour is used in the specimen chamber. This ability to image aqueous specimens leaves ESEM uniquely placed to study in situ drying in polymer latexes. However, there are two key practical difficulties associated with in situ drying. First, the size of the latex particles: larger latex particles are typically around 500 nm in diameter. Although ESEM can resolve structure on this lengthscale without difficulty, the magnification required results in radiation damage of the specimen due to the electron beam. This means that a given region can be imaged only once during film formation, so the evolution of particular features cannot be followed. Second, the change from ambient temperature and pressure to the ESEM conditions of 7°C and 7.5 torr (100 Pa) can subject the specimen to a very high evaporation rate, which can disrupt film formation. The inclusion of a drsitionop of water in the specimen chamber is shown largely to alleviate this, enabling successful imaging of film formation in the lacquer. Instead of the polymer latex itself, this work concentrates on a matting lacquer with silica inclusions. The silica matting agent particles are 1‐10 μm in size, allowing for a lower magnification to be used, massively reducing specimen damage. Furthermore, the contrast during drying is much enhanced in the presence of silica. The images reveal the silica as bright regions against a darker background of polymer and water. Film formation shows the transition from a uniform, featureless aqueous solution to a polymer film with silica particles present on the surface. The appearance of individual silica particles can be followed. The particles are generally revealed quite early, after a few minutes of drying time. As film formation progresses, these same particles appear larger and more distinct. Few new particles are revealed at longer film formation times.
The biomass dynamics of cyanobacteria in an annual cycle determined by confocal laser scanning microscopy
Authors: Solé A. , Gaju N. , Esteve I.
Published: June 2010

We recently published a method for estimating cyanobacteria biomass in delta microbial mats from the Ebro river using confocal laser scanning microscopy (CLSM). The present paper uses this method for identifying different groups of cyanobacteria and for determining their biomass dynamics. Microcoleus chthonoplastes and the Lyngbya‐Oscillatoria were the most important contributors to the cyanobacterial biomass throughout the study. Biomass values ranged from 1.29 to 6.55 mgC/cm2 sediment for Microcoleus chthonoplastes, and from 128 μgC/cm2 to 3.16 mgC/cm2 sediment for Lyngbya‐Oscillatoria. This technique is useful for determining biomass and for studying filamentous cyanobacteria as well as ramified eukaryotic cells. Confocal serial sections through the samples can be obtained. Two‐dimensional images from the samples can be used to calculate the biomass of individual cells.
Microstructure of monoplacophora (mollusca) shell examined by low‐voltage field emission scanning electron and atomic force microscopy
Authors: Cruz Renato , Weissmüller Gilberto , Farina Marcos
Published: June 2010

The shell of Micropilina arntzi (Mollusca: Monoplacophora), a primitive molluscan class, was examined by using field emission scanning electron microscopy (FESEM) at low voltage and atomic force microscopy (AFM). The use of these two techniques allowed the observation of fine details of Micropilina arntzi shell and contributed to bring new features concerning the study of molluscan shell microtexture. Imaging with low‐voltage FESEM provided well‐defined edge contours of shell structures, while analyzing the sample with AFM gave information about the step height of stacked internal structures as well as the dimension of the particles present in their surface at a nanometric level. The shell microstructure of Monoplacophora species presents different patterns and may be a taxonomic implication in the systematic studies of the group.
Exploring the structure of a hydrogen cyanide polymer by electron spin resonance and scanning force microscopy
Authors: Eastman M. P. , Helfrich F. S. E. , Umantsev A , Porter T. L. , Weber R.
Published: June 2010

Aqueous solutions of potassium cyanide and ammonium hydroxide are known to yield a heterogeneous cyanide polymer, containing paramagnetic sites and biologically significant substructures including polypeptides. Here, such solutions were used to prepare various samples of polymer for study by X‐band and W‐band electron spin resonance (ESR), scanning electron microscopy (SEM), and scanning force microscopy (SFM). Elemental composition of a typical sample of the polymer was C‐35.2%, N‐38.47%, O‐14.51%, and H‐ 4.13%, exposing the polymer to 6M HCl hydrolyzed portions of the polymer and released glycine and traces of other amino acids. The X‐band ESR spectra consist of a single slightly asymmetric line centered at g = 2.003; spin concentration measurements made at X‐band using a nitroxide radical standard yield approximate radical concentrations of 1018 spins/gm. W‐band ESR indicates the presence of a single rhombic paramagnetic site with gx = 2.0025, gy = 2.0030, and gz = 2.0048 and the possibility of small 14N hyperfine splittings. The ESR spin echo studies yield a longitudinal relaxation time, T1, of 75 μS and a short‐phase memory relaxation time, Tm, of about 300 nS. Scanning electron microscopy studies of the polymer show that it is made of ellipsoidal particles about one micron in size. The particles tend to clump together when suspended in aqueous solution. The particles disperse and dissolve in dimethyl sulfoxide (DMSO); when these solutions dry on microscope slides, optical microscopy shows a branched island morphology for the polymer. This morphology is reminiscent of snowflakes and is identified as dendritic. Phase contrast SFM of the dendritic arms show a striking segregation and ordering of various components of the polymer. Paramagnetic sites are conserved in the series of steps leading to dendritic structures.
Computer‐assisted stereology: Point fraction of lung parenchyma and alveolar surface density in fetal and newborn sheep
Authors: Davey M. G. , Hedrick H. L. , Bouchard S. , Adzick N. S. , Flake A. W. , Doolin E. J.
Published: June 2010

The aim of this study was to determine the accuracy of a new computer‐assisted stereological technique in obtaining structural information of the lung. We compared the point fraction of lung parenchyma (Pp) and alveolar surface density (Sv) obtained by established manual point/intercept counting methods and compared them with those obtained using a computer‐assisted method. Lung tissues obtained from normally grown fetal sheep (n=6) and from newborn lambs with severe lung hypoplasia (n=5) were inflation fixed via the trachea and processed for light microscopy. In verification‐of‐technique experiments, Pp and Sv correlated well with known values. There was a significant linear correlation between manual and computer‐assisted stereological measurements for values of Pp (r2 = 0.92) and Sv (r2 = 0.98). Our data lead us to believe that the computer‐assisted stereological technique described in this study provides accurate estimates of Pp and Sv and hence may be a valuable tool for evaluating the effects of factors upon structural development of the lung.
Identification of fat, protein matrix, and water/starch on microscopy images of sausages by a principal component analysis‐based segmentation scheme
Authors: Kohler Achim , Høst Vibeke , Enersen Grethe , Ofstad Ragni
Published: June 2010

A color‐based segmentation scheme applied to microscopy images of cryosectioned sausages is proposed. The segmentation scheme is capable of segmenting three different levels on the microscopy images: the fat particles, the protein matrix, and water/starch. The method is based on principal component analysis. A user‐friendly program was developed for the manual segmentation of a selection of image pixels by microscopists. Principal component models based on the manually classified pixels are then used to segment fat, protein matrix, and starch/water on microscopy images. The program can also be used as a training tool for microscopists.
Atomic force microscopic imaging of 30 nm chromatin fiber from partially relaxed plant chromosomes
Authors: Sugiyama S. , Yoshino T. , Kanahara H. , Kobori T. , Ohtani T.
Published: June 2010

We have developed a procedure for partially relaxing the barley metaphase chromosomes and exposing fibrous structures from the chromosomes. The observation by atomic force microscopy (AFM) showed that the fibrous structures are typically 0.5 to 1 μm long and 40 to 50 nm in diameter. In higher magnification imaging, we found the fibrous structures were composed of aligned granules and looked like “knobby fiber.” These observations are consistent with previously reported features of chromatin fiber observed by AFM and scanning electron microscopy, suggesting that the structures correspond to 30 nm chromatin fibers. We observed the chromatin fiber extending straight from the periphery of the chromosomes in most cases, but fibers with different shapes, such as loop and spiral, were also observed. The procedure reported here will provide a new approach for observing the organization of chromatin fiber to higher‐order structures by AFM and other high‐resolution microscopy.
Experimental resolution measurement in critical dimension scanning electron microscope metrology
Authors: Lorusso G. F. , Joy D. C.
Published: June 2010

By applying the basic principles of metrology we discuss how to define the standards that any experimental method to measure resolution has to obey. Our results clearly indicate the need to apply a calibration procedure when designing algorithms to estimate resolution to satisfy accuracy requirements. Similarly, the precision of an algorithm has to be clearly specified. We compare here the performances of a variety of commonly used implementations of published methods, with that of an algorithm based on an approach known to be reliable. Our results confirm that when an algorithm is designed with the clear intent of satisfying metrology requirements, it demonstrates excellent accuracy, precision, and lack of sensitivity to the noise level, as is desirable. As a consequence, the algorithm will have the ability to measure accurately the point spread function convoluted in the image, thus paving the way for quantitative deconvolution techniques.