bluuebunny/crossref_metadata_2025 huggingface dataset:
Real‐time confocal microscopy of keratocyte activity in wound healing after cryoablation in rabbit corneas
Authors: Chew S. J. , Beuerman R. W. , Kaufman H. E.
Published: June 2010

A modified tandem scanning confocal microscope was used for real‐time in vivo examination of the rabbit cornea following a cryogenic injury. The corneas of New Zealand white rabbits were frozen with aprobe that had been cooled by immersion in liquid nitrogen, effectively destroying keratocytes in a central 5 mm diameter zone throughout the total thickness of the cornea. In these eyes, keratocyte repopulation and corneal stromal wound healing proceeded similarly to that which occurs after epikeratophakia, a refractive surgical procedure designed to change the curvature and optical power of the cornea. In epikeratophakia, a cryolathed donor corneal stroma lenticule is sutured onto the bare stroma of the recipient cornea. The collagen tissue lenticule is repopulated by keratocytes (corneal fibroblasts) that migrate in from the host cornea. In our study, the confocal microscope permitted sequential, noninvasive examination of the corneal stroma in the treated animals. Necrosis of the keratocytes, followed by activation of the remaining viable cells in the corneal periphery, was observed in the first 2 to 3 days after cryo injury. A fine stromal fibrous network was seen to develop; in three eyes, this network progressed to the development of a retrocorneal fibrous membrane and dense stromal fibrosis, both of which resulted in significant loss of corneal clarity. Our results suggest that the confocal microscope may be a valuable tool to provide much needed information on wound healing processes at the cellular level after corneal surgery and injury.
Improvement of thermally induced bending of cantilevers used for atomic force microscopy
Authors: Radmacher M. , Cleveland J. P. , Hansma P. K.
Published: June 2010

The microfabricated silicon nitride cantilevers that are used for atomic force microscopy (AFM) are, unfortunately, sensitive thermometers. They bend with ambient temperature changes and those due to laser heating. The bend can result in displacements for the silicon nitride cantilevers of an order several hundred nanometers at the tip of the cantilever. If, however, the silicon nitride cantilevers are treated by removing the metallization and annealing at 500°C for 30 min, these displacements can be reduced by one or two orders of magnitude. Silicon cantilevers have approximately a one order of magnitude smaller drift than silicon nitride cantilevers as received from vendors and are improved less by treatment.
Effects of brefeldin‐A on Golgi morphology in human cultured fibroblasts observed in three‐dimensional stereo scanning electron microscopy
Authors: So Derek Y. F. , Temkin Robert J. , Lea Peter J.
Published: June 2010

Brefeldin A (BFA) has been reported to cause disassembly of the Golgi. We have used three‐dimensional (3‐D) high‐resolution scanning electron microscopy (HRSEM) to investigate these effects in human skin fibroblast cells. The spontaneous reassembly during prolonged exposure to BFA and some effects of forskolin were observed. A BFA concentration of 5μg/ml caused Golgi complexes to become vesicular, resulting in a progressive decrease in the size of the Golgi. Morphologic changes were visible within 2 min of BFA incubation, and by 30 min no identifiable Golgi could be found. Spontaneous reassembly of the Golgi apparatus upon the removal of the BFA or with continued long‐term exposure with BFA could not be confirmed. Preliminary experiments with forskolin were not effective in reversing or inhibiting the effects of BFA in human fibroblast cells grown in culture. This inability for spontaneous reassembly and nonreversal by forskolin may reflect a differential effect of BFA in various cell types. HRSEM has proven to be useful for observing 3‐D morphologic effects of BFA in Golgi.
Topographic and material contrast in low‐voltage scanning electron microscopy
Authors: Hejna J.
Published: June 2010

Two scintillation backscattered electron (BSE) detectors with a high voltage applied to scintillators were built and tested in a field emission scanning electron microscope (SEM) at low primary beam energies. One detector collects BSE emitted at low take‐off angles, the second at high takeoff angles. The low take‐off detector gives good topographic tilt contrast, stronger than in the case of the secondary electron (SE) detection and less sensitive to the presence of contamination layers on the surface. The high take‐off detector is less sensitive to the topography and can be used for detection of material contrast, but the contrast becomes equivocal at the beam energy of 1 keV or lower.
An annular toroidal backscattered electron energy analyser for use in scanning electron microscopy
Authors: Rau E. I. , Robinson V. N. E.
Published: June 2010

A backscattered electron energy spectrometer, based on a toroidal energy analyser and an annular detector, has been devised and adapted for use in a scanning electron microscope. Computer simulations have been carried out for equipotentials and trajectories of electrons in the toroidal deflector, which permit the optimisation of the energy analyser characteristics for special applications. Based upon these results, a device has been built and its efficiency is demonstrated by selected images of a multilayered structure and a series of recorded backscattered electron spectra.
Three‐dimensional laser scanning two‐photon fluorescence confocal microscopy of polymer materials using a new, efficient upconverting fluorophore
Authors: Bhawalkar J. D. , Swiatkiewicz J. , Pan S. J. , Samarabandu J. K. , Liou W. S. , He G. S. , Berezney R. , Cheng P. C. , Prasad P. N.
Published: June 2010

Three‐dimensional confocal imaging of polymer samples was achieved by the use of two‐photon excited fluorescence in both positive and negative contrast modes. The fluorophore was a new and highly efficient two‐photon induced upconverter, resulting in improved signal strength at low pumping power. Because of the relatively long wavelength of the excitation source (798 nm from a mode‐locked Ti:Sap‐phire laser), this technique shows a larger penetration depth into the samples than provided by conventional single‐photon fluorescence confocal microscopy. Single‐photon and two‐photon images of the same area of each sample show significant differences. The results suggest the possibility of using two‐photon confocal microscopy, in conjunction with highly efficient fluorophores, as a tool to study the surface, interface, and fracture in material science applications.
High‐Resolution Lithography with Near‐Field Optical Microscopy
Authors: Naber A. , Kock H. , Fuchs H.
Published: June 2010

Scanning near‐field optical microscopy (SNOM) is used for lithography to avoid the resolution limiting diffraction of conventional optical methods. We have expanded a commercial SNOM for writing even complex structures on the nanometer scale. Scanning near‐field optical lithography (SNOL) has been applied to conventional resists to explore its potential and the possible combination with conventional optical lithography (mix and match technique).
Ultrastructure of Tracheal Surface Liquid: Low‐Temperature Scanning Electron Microscopy
Authors: Wu D. X‐Y. , Lee C. Y. C. , Widdicombe J. H. , Bastacky J.
Published: June 2010

A layer of liquid lines the airways in the lung. Previous microscopic studies have suggested that it is in two phases, with a mucous gel lying above a periciliary sol. However, shrinkage artifacts due to chemical fixation, dehydration, and drying have prevented reliable estimates of the depth of these layers. To avoid such problems, we have studied the surface liquid of bovine trachea by low‐temperature scanning electron microscopy (LTSEM). A polished copper probe cooled to liquid nitrogen temperature was applied to the mucosal surface of sheets of excised tracheal epithelium to effect rapid freezing of surface liquid. Tissue sheets were then mounted in an LTSEM (AMRay 1000A with Biochamber) which maintains samples at ‐180°C with a Joule‐Thompson refrigerator built into the stage. Tissues were fractured at right angles to the epithelial surface, coated with gold, and viewed, all at 10−5 to 10−6 torr without transfer through air. The sample was stable under the electron beam at accelerating voltages up to 20 kV. Epithelial features (nuclei, cilia, microvilli, mucous granules) were well preserved. The mucosal surface of the cells was covered with material on the order of 8 μm in depth. The mucous gel and periciliary sol could be seen as distinct layers and could be distinguished by the size and pattern of ice crystal voids generated by radiant‐etching of the fractured surface of the sample.
CASINO: A new monte carlo code in C language for electron beam interaction —part I: Description of the program
Authors: Hovington Pierre , Drouin Dominique , Gauvin Raynald
Published: June 2010

This paper is a guide to the ANSI standard C code of CASINO program which is a single scattering Monte CArlo SImulation of electroN trajectory in sOlid specially designed for low‐beam interaction in a bulk and thin foil. CASINO can be used either on a DOS‐based PC or on a UNIX‐based workstation. This program uses tabulated Mott elastic cross sections and experimentally determined stopping powers. Function pointers are used for the most essential routine so that different physical models can easily be implemented. CASINO can be used to generate all of the recorded signals (x‐rays, secondary, and backscattered) in a scanning electron microscope either as a point analysis, as a linescan, or as an image format, for all the accelerated voltages (0.1–30 kV). As an example of application, it was found that a 20 nm Guinier‐Preston Mg2Si in a light aluminum matrix can, theoretically, be imaged with a microchannel backscattered detector at 5 keV with a beam spot size of 5 nm.
Real‐time white light reflection confocal microscopy using a fibre‐optic bundle
Authors: Juškattis R. , Wilson T. , Watson T. F.
Published: June 2010

We describe a real‐time white light reflection con‐focal microscope incorporating an optical fibre bundle and characterise the optical performance of the bundle. The use of an incoherent light source enables us, for the first time, to present speckle‐free endoscopic reflected light confocal images. The system has potential application for in vivo studies.
CASINO: A new monte Carlo code in C language for electron beam interactions—part III: Stopping power at low energies
Authors: Hovington Pierre , Drouin Dominique , Gauvin Raynald , Joy David C. , Evans Neal
Published: June 2010

This paper is a description of the stopping power routine utilized in the CASINO program that is based on the experimental measurement of the energy loss function (ELF). In addition, we present an ANSI C standard program that can be used to generate the data needed for the stopping power routine. Both optical and energy loss spectrum (ELS) measurements of the ELF can be used as input to compute the stopping power. For ELS, only the single scattering spectrum is needed. Hence, measurement of the stopping power for a given element or compound of interest can easily be performed and used in the CASINO program. The resulting effect of using these stopping powers in Monte Carlo simulations is generally to increase the backscattering coefficient. Except for carbon, the change of stopping power for pure elements so far compiled is relatively small. In some compounds (i.e., Al2O3 and ZnSe), the discrepancy with the Joy and Luo (1989) expression is significant.
Assessment and correction of geometric distortions in low‐magnification scanning electron microscopy images
Authors: Marschallinger Robert , Topa Dan
Published: June 2010

A method is introduced to assess and correct the geometric distortions which frequently occur in low‐magnification scanning electron microscopy (SEM) images. Such images typically exhibit a complex pattern of varying deviations from orthogonality which cannot be adequately corrected by simple geometric transformations such as shifting, scaling, rotation, or shearing. A suitable approach to rectify low‐magnification SEM images is polynomial warping, a correction procedure which also accomplishes rubber sheet transformation. To demonstrate the approach, a reference grid for low magnifications has been scanned at 40‐ and 55‐fold magnifications by means of a microanalyzer. Calculated geometric distortions range from 1.5 to 3.5% of the image dimensions; applying polynomial warping, distortions could be reduced to approximately 0.1% of the image dimensions. Because of its easy application and the widespread availability in image processing packages, polynomial warping can be recommended as a routine procedure for rectifying low‐magnification SEM images.
Chicken erythrocyte nucleosomes have a defined orientation along the linker DNA—A scanning force microscopy study
Authors: Fritzsche Wolfgang , Henderson Eric
Published: June 2010

The orientation of nucleosomes was investigated using scanning force microscopy (SFM) of hypotonically spread chicken chromatin. A virtual cross section parallel to the substrate at half maximum height of the nucleosomal structure revealed an elliptical shape. The orientation of the major axis of this ellipse was investigated in reference to the direction of the axis of the nucleosomal chain. An alignment of the nucleosomes along the nucleosomal chain was observed, with more than 50% of the nucleosomes aligned with the long axis of the chain within ≤ 30° deviation. The alignment distribution peaked at 10–20°. The application of SFM‐based image processing for the structural investigation of a protein‐DNA complex demonstrates the potential for this approach in structural molecular biology.
Environmental scanning electron microscopy investigations of biodeterioration
Authors: Ray Richard , Little Brenda , Wagner Patricia , Hart Kevin
Published: June 2010

Case studies will be presented in which environmental scanning electron microscopy (ESEM) has been used to provide unique insight into the role of microorganisms in deterioration processes. ESEM is an excellent tool for demonstrating spatial relationships between microorganisms and substrata because hydrated, nonconducting samples can be viewed with a minimum of manipulation. Copper and iron‐rich deposits associated with bacteria were detected within corrosion layers on copper and steel surfaces, respectively. Fungal mycelia growing on wooden storage spools were shown to penetrate protective grease on carbon steel wire rope in contact with the spool and to cause localized corrosion. Large numbers of marine bacteria were documented within paint blisters and disbonded regions of fiber‐reinforced polymeric composites. In both cases, it appears that microbial gas production resulted in mechanical damage to the substrata.
Characterization of polyox ® granules using environmental scanning electron microscopy
Authors: Roberts R. A. , Shukla A. J. , Rice T.
Published: June 2010

The scanning electron microscope (SEM) has been frequently used in the pharmaceutical industry for studying pharmaceutical products. However, the technique does not allow for the continuous characterization of a product in both dry and hydrated states without processing the product. Through the recent advent of the environmental scanning electron microscope (ESEM), it is now possible to observe a sample in both the dry and hydrated states without extensive product preparation. The ESEM also allows for continual observation during the hydration process from the dry state until the sample is dissolved. In this study, the ESEM was used to characterize the morphologic differences and hydration patterns of granules formulated with a water‐soluble hydrophilic swelling polymer, Polyoxy. Two molecular weights (1, 000, 000 and 7, 000, 000) of the polymer were used in concentrations ranging from 10 to 25% w/w. Visual differences in granule surface morphology and differences in hydration patterns were seen in granules prepared from different polymer concentrations. The morphologic data were corroborated by surface area measurements taken on a surface area analyzer. The rate at which the granule dissolved in the ESEM also correlated with the drug dissolution times determined by the standard USP dissolution method.
A fast and versatile scan unit for scanning probe microscopy
Authors: Knebel D. , Amrein M. , Voigt K. , Reichelt R.
Published: June 2010

In scanning probe microscopy (SPM), the image acquisition time is usually very long because of the limited speed with which the scanning device can trace the topography of the specimen under feed‐back control. This limitation is often brought about by the natural frequency of the scanner in the direction perpendicular to the sample plane that confines the usable bandwidth of the feed‐back loop. In this paper, we present a piezo‐ceramic scanner that provides a large scan range and at the same time allows for adjustment of the probe‐to‐sample distance faster by about one order of a magnitude than a conventional setup. This is achieved through the combination of a large single tube scanner that provides a high‐scan range and a small piezo element for swift motion in the direction perpendicular to the sample plane. The natural frequency in this direction lies at about 275 kHz. We outline the design considerations to avoid disturbing excitation of the scanner through the fast piezo element.
Enhanced scanning control in a confocal scanning laser microscope
Authors: Trepte Oliver
Published: June 2010

A fast and flexible scanning unit, allowing scanning rates of more than 1 kHz over regions identified in a specimen, has been developed and evaluated. This scanning unit replaces the original scanning unit in the Phoibos confocal scanning laser microscope and features full backward compatibility, while at the same time allowing fast and flexible scanning modes, such as point scanning, line scanning, and scanning along user‐selected closed curves. The scanning unit uses two galvanometer‐mounted mirrors for scanning. A standard procedure for recordings with this scanning unit would be to scan an overview image with conventional raster scanning to identify a region of interest, mark a point, a line, or a closed curve over this region, and to start the scanner. An iterating algorithm then calculates the waveforms needed by the scanner to follow the identified curves with pixel precision. With this scanning unit and its controlling software, experiments demanding time‐resolved recordings within the millisecond range can be performed. Repetition rates up to > 1 kHz for line scanning and curve scanning, and > 100 kHz for point scanning are obtainable. This allows time‐resolved studies of fast reactions in living tissue to be performed with the spatial resolution and signal‐to‐noise ratio obtainable with a point scanning confocal microscope.
Effects of gas pressure on low‐pressure electron‐beam lithography
Authors: Paul B. K.
Published: June 2010

In electron‐beam (EB) lithography, it is common practice to prepare the resist for exposure using a metal over‐layer or an ion shower to eliminate the charging of the resist. Recent efforts to prototype micromechanical structures using EB lithography methods have avoided the need for resist preparation by using low‐pressure EB lithography methods. While a great deal of research has centered around high‐vacuum electron‐beam (EB) lithography, little research has been conducted concerning the effect of gas chamber pressure on linewidth in low‐pressure EB lithography. In this paper, experimental data are presented linking the gas pressure in low‐pressure EB lithography with lithography line profiles. The results show that as gas pressure is varied, a compromise exists between line broadening and the charging‐up effect.
Transmission electron microscopy and scanning force microscopy of poly r(A‐U) and poly r(A‐U)‐ethidium bromide
Authors: Gilloteaux J. , Jamison J. M. , Zenhausern F. , Adrian M. , Summers J. L.
Published: June 2010

Transmission electron microscopy and scanning force microscopy of negative‐stained, carbon‐coated replica and mica‐adsorbed preparations of 200 μM poly r(A‐U) and 50 μM ethidium bromide/200 μM poly r(A‐U) have been employed to evaluate ethidium‐induced changes in poly r(A‐U) topology. Poly r(A‐U) alone exhibits elongated conformations 85–115 nm in length that possess a number of hairpin loops as well as single‐stranded domains. While the double‐stranded domains are found predominately at the base of the hairpin loops (diameter = 5–30 nm), other rod‐like (presumably double‐stranded) regions ranging from 25–80 nm in length are present in other portions of the poly r(A‐U). In contrast with the poly r(A‐U) alone, the EB/poly r(A‐U) combination appears as a heterogeneous population of condensed structures whose lengths and widths vary from 12–88 nm and 15–45 nm, respectively. These conformational changes are due to a number of factors, including the displacement of ordered water surrounding the poly r(A‐U) and charge shielding of the phosphate groups of the poly r(A‐U) upon the binding of the ethidium.
A robust focusing and astigmatism correction method for the scanning electron microscope
Authors: Ong K. H. , Phang J. C. H. , Thong J. T. L.
Published: June 2010

This paper discusses a new approach to focusing and astigmatism correction based on the fast fourier transforms (FFTs) of scanning electron microscopy (SEM) images. From the FFTs, it is possible to obtain information on the severity of the defocus and astigmatism. This information is then processed by an algorithm to perform real‐time focusing and astigmatism correction on the SEM. The algorithm has been tested on defocused and astigmatic images of different samples, including those with highly directional features. Experiments show that the images obtained after running the algorithm can be as good as those that an experienced SEM operator can achieve.
Monte carlo simulation of charging effects in linewidth metrology (II): On insulator substrate
Authors: Ko Yeong‐Uk , Chung Myung‐Sae
Published: June 2010

Charging effects have been investigated quantitatively using Monte Carlo (MC) simulation when the linewidth of polymethylmethacrylate (PMMA) insulator patterns on SiO2 insulator substrate are measured by scanning electron microscope (SEM). We established reference operating and shape conditions for array patterns and we have calculated the offset on linewidth metrology according to the change in each condition. We have used a 50% threshold algorithm for the edge determination, calculated the offsets in those conditions, and compared them with the results in the case of Si substrate. Finally, the question of which factor is the most sensitive in linewidth metrology is discussed.
Evaluation of cyanoacrylate glues for making attached living‐leaves epidermis replicas and its scanning electron microscopy observations (evaluación de pegamentos de cianoacrilato para hacer réplicas epidérmicas en hojas vivas adheridas y su observación al MEB)
Authors: Castilloa Juan Jose , Ferrarotto Maria
Published: June 2010

Six brands of cyanoacrylate glue were tested to make inverse replicas of a leaf's epidermis. All of them gave satisfactory results for observations up to 8000x with a Philips XL‐20 scanning electron microscope (SEM), working at 10 kV. Genus of plants used were Xanthosoma sp., Psidium sp., Melicocca sp., Sorghum sp., and Stenotaphrum sp. The main limitations were that the epicuticular wax gets embedded within the glue. Spindle‐ or oval‐shaped artifacts are produced by stomatal apertures. This paper confirms previous findings related to the fact that cyanoacrylate glues are an alternative method for making fast, inexpensive, and sturdy epidermis replicas for scanning electron microscopy observation.
Cancer cell necrosis by autoschizis: Synergism of antitumor activity of vitamin C: Vitamin K3 on human bladder carcinoma T24 cells
Authors: Gilloteaux J. , Jamison J. M. , Arnold D. , Ervin E. , Eckroat L. , Docherty J. J. , Neal D. , Summers J. L.
Published: June 2010

Scanning and transmission electron microscopy and fluorescence light microscopy were employed to characterize the cytotoxic effects of vitamin C (VitC), vitamin K3 (VitK3) or a VitC:VK3 combination on a human bladder carcinoma cell line (T24) following 1‐h and 2‐h vitamin treatment. T24 cells exposed to VitC alone exhibited membranous damage (blebs and endoplasmic extrusions, elongated microvilli). VitK3‐treated cells displayed greater membrane damage and enucleation than those treated with VitC as well as cytoplasmic defects characteristic of cytoskeletal damage. VitC:VitK3‐treated cells showed exaggerated membrane damage and an enucleation process in which the perikarya separate from the main cytoplasmic cell body by self‐excision. Self‐excisions continued for perikarya which contained an intact nucleus surrounded by damaged organelles. After further excisions of cytoplasm, the nuclei exhibited nucleolar segregation and chromatin decondensation followed by nuclear karryorhexis and karyolysis. This process of cell death induced by oxidative stress was named autoschizis because it showed both apoptotic and necrotic morphologic characteristics.
The scanning low‐energy electron microscope: First attainment of diffraction contrast in the scanning electron microscope
Authors: Frank L. , Müllerová I. , Faulian K. , Bauer E.
Published: June 2010

Using small Pb crystals deposited in situ on a partially contaminated Si (100) crystal, we demonstrate that a commercial scanning electron microscope (SEM) can easily be converted into a scanning low‐energy electron microscope (SLEEM). Although the contrast mechanism is much more complicated than that in nonscanning LEEM because not only one diffracted monochromatic beam and its close environment are used for imaging, but several diffracted beams and a wide energy spectrum of electrons of different origin (secondary electrons, inelastically andelastically scattered electrons) are used, SLEEM is a valuable addition to the standard SEM because it provides an additional structure‐ and orientation‐sensitive contrast mechanism in crystalline materials, a low sampling depth, and high intensity at low energies.
Confocal laser scanning microscopy of hamster cerebellum using FM4‐64 as intracellular staining
Authors: Castejón O. , Sims P.
Published: June 2010

The FM4‐64, a member of the family of fluorescent dyes, has been applied to the cerebellar cortex to evaluate its properties as an intracellular stain and intracortical tracer. Slabs of hamster cerebellum, 1–2 mm thick, were incubated in 10, 30, and 100 μm solutions of FM4–64 in sodium phosphate buffer and observed in a slow scan confocal laser scanning microscope. Mossy and climbing fibers were traced in the cerebellar white and gray substances. They exhibited a high fluorescence signal at the level of the myelin sheath. Mossy fibers were identified in the granular layer by their typical rosette formation and dichotomous bifurcation pattern. Climbing fiber bundles were observed crossing the granular layer and giving collateral branches around Golgi cell bodies. They ascend to the Purkinje cell layer on their way to the molecular layer. Cerebellar macroneurons (Golgi and Purkinje cells) and microneurons (granule, basket, and stellate cells) showed optimal intracellular staining of cell soma, axonal, and dendritic processes. The z‐series of stacks of optodigital sections allowed us to explore in depth the cytoarchitectonic arrangement, nerve and glial cell morphology, and the topographic relationship with the afferent fibers.
Tilt dependence of the secondary electron emission at low excitation energy
Authors: Libinson A. G.
Published: June 2010

Influence of the specimen's slope on the secondary electron emission has been experimentally studied. Strong deviations from the inverse cos law have been observed and corresponding phenomenological equation (taking into account this deviation) is suggested. The consequences of the dependence on the topography contrast of low‐ and high‐voltage scanning electron microscopy (SEM) image, especially for three‐dimensional (3‐D) reconstruction, are considered.
Correlative light and backscattered electron microscopy of bone—Part I: Specimen preparation methods
Authors: Goldman Haviva M. , Kindsvater John , Bromage Timothy G.
Published: June 2010

A method for preparing nondecalcified bone and tooth specimens for imaging by both light microscopy (LM) and backscattered electron microscopy in the scanning electron microscope (B SE‐SEM) is presented. Bone blocks are embedded in a polymethylmethacrylate (PMMA) mixture and mounted on glass slides using components of a light‐cured dental adhesive system. This method of slide preparation allows correlative studies to be carried out between different microscopy modes, using the same histologic section. It also represents a large time savings relative to other mounting methods whose media require long cure times.
Time‐lapse scanning electron microscopy for measurement of contamination rate and stage drift
Authors: Vladár András E.
Published: June 2010

Measurements done with scanning electron microscopes (SEMs) may lose their validity due to contamination and charging caused by the primary electron beam. The sample stage and the electron beam also slightly drift during the course of the measurements. Consequently, it is essential to find out the time limit of valid measurements, that is, the maximum time before the sample or its position changes too much.

This paper describes digital time‐lapse SEM, a useful tool for investigating the extent and effects of contamination and stage drift. It works with hardware and software that create a stack of sequential images. Later these images can be viewed as a short movie; it is also possible to apply all the image processing and analysis procedures that are otherwise applied to separate, individual images.

This method gives a reliable way of measuring the rate of contaminant deposition and of stage and electron beam drift of SEMs, and it could be helpful in controlling these problems.
A structural study of the bovine vaginal fluid at estrus
Authors: Rutllant J. , López‐Gatius F. , Camon J. , López‐Béjar M. , López‐Plana C.
Published: June 2010

The present study describes the structural components of the bovine vaginal fluid at estrus by scanning electron microscopy (SEM) following critical point‐ and freezedrying preparation procedures. Confocal scanning laser microscopy (CLSM) was also used to evaluate the structural integrity of samples, and a control sample was assessed by adding sperm to the vaginal fluid. Samples were collected from 10 cows at the time of artificial insemination, prepared for SEM by using critical point‐ and freeze‐drying procedures, gold coated, and observed by SEM. Mesh size and filament thickness were measured with an image analyzer. Of the 10 samples processed, 4 were considered altered following critical point drying. Compaction and lack of filaments were observed in these samples. A small area of one sample showed a honey comb‐like structure when freeze drying was used. Nonoriented filaments with different thicknesses and with a network‐like structure were observed throughout the remainder of the samples. Filaments throughout all samples were also observed by CSLM. After critical point drying, the mesh area ranged from 0.8 to 101.4 μm2; the minor axis from 0.7 to 10.8 μm; and filament thickness from 40 to 442 nm. Using freeze drying, the mesh area ranged from 0.9 to 493.8 μm2; the minor axis from 0.7 to 27.5 μm; and filament thickness from 40 to 800 nm. When samples were freeze dried, mesh values were similar to the interstrand channels observed by CSLM. In sperm‐vaginal fluid samples, following critical point‐ or freeze‐drying procedures, spermatozoa were oriented randomly in the vaginal fluid and did not seem to alter filamentous structure. Our data suggest that the freeze‐drying procedure better preserves the true structural dimensions of the vaginal fluid. Furthermore, the filamentous structure of the vaginal fluid does not appear to impede sperm transport.
Crystallographic orientation assessment by electron backscattered diffraction
Authors: Cléton F. , Jouneau P. H. , Henry S. , Gäumann M. , Buffat P. A.
Published: June 2010

With an angular orientation accuracy of at least 1°, the ability of electron backscattered diffraction (EBSD) to determine and emphasise crystallographic orientation is illustrated. Using the abilities of specially developed software for computing Euler angles derived from the scanned specimen, misorientations are pointed out with acceptable flexibility and graphic output through crystallographic orientation maps or pole figures. This ability is displayed in the particular case of laser cladding of nickel‐based superalloy, a process that combines the advantages of a near net‐shape manufacturing and a close control of the solidification microstructure (E‐LMF: epitaxial laser metal forming).
Three‐dimensional reconstruction of fracture surfaces: Area matching algorithms for automatic parallax measurements
Authors: Hein Luis Rogerio de Oliveira , Silva Fernando de Azevedo , Nazar Ana Maria Martinez , Ammann Jean Jacques
Published: June 2010

This paper describes two solutions for systematic measurement of surface elevation that can be used for both profile and surface reconstructions for quantitative fractography case studies. The first one is developed under Khoros graphical interface environment. It consists of an adaption of the almost classical area matching algorithm, that is based on cross‐correlation operations, to the well‐known method of parallax measurements from stereo pairs. A normalization function was created to avoid false cross‐correlation peaks, driving to the true window best matching solution at each region analyzed on both stereo projections. Some limitations to the use of scanning electron microscopy and the types of surface patterns are also discussed. The second algorithm is based on a spatial correlation function. This solution is implemented under the NIH Image macro programming, combining a good representation for low contrast regions and many improvements on overall user interface and performance. Its advantages and limitations are also presented.
A study of electron beam‐induced conductivity in resists
Authors: Hwu J. J. , Joy D. C.
Published: June 2010

The charging of polymeric resist materials during electron beam irradiation leads to significant problems during imaging and lithography processes. Charging occurs because of charge deposition in the polymer and charge generation/trapping due to formation of electron‐hole pairs in the dielectric. The presence of such charge also results in the phenomena of electron beam‐induced conductivity (EBIC). Electron beam‐induced conductivity data have been obtained for three commercial e‐beam resists under a variety of dose rate and temperature conditions. From the observed values of induced conductivity under varying conditions significant information about the generation of electron‐hole pair and the transport of charge in the resist can be obtained. Three electron beam resists, EBR900, ZEP7000, and PBS are examined by an external bias method. The difference in resist chemistry is considered to play the role in the initial state EBIC behaviors among three resists even though the way that it affects the behaviors is not clear. A comparison of the power consumption comparison is proposed as a measure to give a preliminary estimate of the carrier concentration and carrier drift velocity differences among the resists. A simple single trap model with constant activation energy is proposed and provides good agreement with experiment.
Using atomic force microscopy to probe food biopolymer functionality
Authors: Morris V. J. , Kirby A. R. , Gunning A. P.
Published: June 2010

Atomic force microscopes (AFMs) generate images by “feeling” rather than “looking” at samples. This permits a magnification range spanning that associated with both the light and electron microscopes, but under the “natural” imaging conditions normally associated with light microscopes. Molecules and molecular interactions can be imaged at molecular or submolecular resolution in gaseous or liquid environments. By careful design of experiments it is possible to use AFM to probe how food biopolymers determine the structure and texture of food products. This approach will be illustrated through studies on food polysaccharides and proteins.
Morphologic changes of starch granules in the apple cv. Mutsu during ripening and storage
Authors: Kovács Etelka , Eads T. M.
Published: June 2010

Unripe (A), semiripe (B), and ripe (C) apples (Malus domestica cv. Mutsu) stored (2–4 °C, 90–95% RH) for 1 week were studied. The decrease in the starch content was as a function of storage time and ripeness.

The size (area, perimeter and circularity) of starch granules was determined by image analyzer (Semper6, Synoptics) interfaced with a standard light microscope (Olympus, BH2) using a bright field illumination. Area (Ar) (larger than 100 pixel), perimeter (Pe), and circularity (Ci) of the granules were measured. The distribution of Ar, Pe, and Ci was calculated by the Distribution Fitting Statistical program; normality of frequency distribution of data (Chi‐square test), average ± standard deviation, t‐values were presented. All data were analyzed by principal component analysis.

Area and perimeter decreased, circularity increased with the storage time and ripeness. Critical differences were observed in the semiripe apple cortex (CoB0) and the ripe apple skin (SC0) compared with the other samples (unripe skin and cortex) at the harvest. In both samples, most starch granules swelled (19–24%), and the starch contents were significantly less than in others. The stage of ripeness could be more exactly followed by image analysis than by the starch iodine test, or the color of apple skin.

The ultrastructure (SEM) of starch did show typical changes as a function of storage time. More smaller starch granules could be seen in the stored apples than in the fresh ones. Damaged (pits, cavities) starch granules were found in all samples.

Carbon‐13 NMR spectra of isolated apple cortex starch were obtained with cross polarization (CP), magic angle spinning (MAS), and high power proton irradiation (HD). There were shoulders on the C‐1 resonance and on the C‐2, 3, 4, 5 cluster. These shoulders indicate the presence of amorphous starch.
Edible starch films and coatings characterization: scanning electron microscopy, water vapor, and gas permeabilities
Authors: García María A. , Martino Miriam N. , Zaritzky Noemí E.
Published: June 2010

Effects of coating formulation (starch type and plasticizer) on microstructure, water vapor, and gas permeabilities of films and coatings were analyzed. Plasticizer addition was necessary for films and coatings integrity to avoid pores and cracks. A satisfactory correlation between microstructure observations by scanning electron microscopy, electronic thickness measurements, and water vapor and gas permeability determinations was observed. Films and coatings with high amylose content starches showed lower water vapor and gas permeabilities compared with films and coatings with medium amylose content starches. Films and coatings with sorbitol showed lower water vapor and gas permeabilities than those with glycerol, with the most effective plasticizer concentration being 20 g/l.
Comparison of different models for the generation of electron backscattering patterns in the scanning electron microscope
Authors: Wells Oliver C.
Published: June 2010

An electron backscattering pattern (EBSP) is formed on a fluorescent (or other) screen from the faster scattered electrons when a single‐crystal region of a solid sample is illuminated by a finely focused electron beam (EB). The EBSP is very similar in appearance to the electron channeling pattern (ECP) that is obtained in the scanning electron microscope (SEM) by rocking the beam about a point on the surface of a single crystal. It has been suggested that the mechanisms that give rise to EBSP and ECP are related by reciprocity. If this is indeed the case, then the models that are used to explain them should be the same except for the direction in which the electrons travel through the specimen. The two‐event “diffraction model” for EBSP (diffuse scattering followed by diffraction) fails this condition, leading to the conclusion that the “channeling in and channeling out” model for EBSP is more likely to be correct. This has been described rigorously by Reimer (1979, 1985). It is named after the title used by Joy (1994). An attempt is made here to describe this model in a simple way.
Cryo‐planing for cryo‐scanning electron microscopy
Authors: Nijsse Jaap , van Aelst Adriaan C.
Published: June 2010

In the past decade, investigators of cryo‐planing for low‐temperature scanning electron microscopy (cryo‐SEM) have developed techniques that enable observations of flat sample surfaces. This study reviews these sample preparation techniques, compares and contrasts their results, and introduces modifications that improve results from cryo‐planing. A prerequisite for all successful cryo‐planing required a stable attachment of the specimen to a holder. In most cases, clamping with a screw mechanism and using indium as space‐filler sufficed. Once this problem was solved, any of three existing cryo‐planing methods could be used to provide successful results: cryo‐milling, microtomy in a cold room, and cryo‐ultra‐microtomy. This study introduces modifications to the cryo‐planing technique that produces flat surfaces of any desired plane through a specimen. These flat surfaces of frozen, fully hydrated samples can be used to improve observations from cryo‐SEM as well as to enhance results from x‐ray microanalysis and (digital) image analysis. Cryo‐planing results of chrysanthemum (Dendranthema x grandiflorum Tzvelev) stems, hazel (Corylus avelane L.) stems, and rapeseed (Brassica napus L.) pistils are presented to illustrate the use of the planing method on fibrous, hard, and delicate materials, respectively.
Scanning electron microscopy studies of protein‐functionalized atomic force microscopy cantilever tips
Authors: Micic Miodrag , Chen Aileen , Leblanc Roger M. , Moy Vincent T.
Published: June 2010

Protein‐functionalized atomic force microscopy (AFM) tips have been used to investigate the interaction of individual ligand‐receptor complexes. Herein we present results from scanning electron microscopy (SEM) studies of protein‐functionalized AFM cantilever tips. The goals of this study were (1) to examine the surface morphology of protein‐coated AFM tips and (2) to determine the stability of the coated tips. Based on SEM images, we found that bovine serum albumin (BSA) in solution spontaneously adsorbed onto the surface of silicon nitride cantilevers, forming a uniform protein layer over the surface. Additional protein layers deposited over the initial BSA‐coated surface did not significantly alter the surface morphology. However, we found that avidin‐functionalized tips were contaminated with debris after a series of force measurements with biotinylated agarose beads. The bound debris presumably originated from the transfer of material from the agarose bead. This observation is consistent with the observed deterioration of functional activity as measured in ligand‐receptor binding force experiments.
Glycoproteins and skin‐core structure in Nephila clavipes spider silk observed by light and electron microscopy
Authors: Augsten K. , Mühlig P. , Herrmann C.
Published: June 2010

Microscopical imaging of natural, unstressed draglines or of untreated bulk samples showed two types or threads with diameters of either ∼ 1–2 μm or 4–5 μm, which could be identified as products of the minor or major ampullate glands. The threads had a circular profile in serial cross sections and are surrounded by a thin outer layer of a different material within the section. Such fibrillar configurations were also found in untreated threads or in the same serial sections of transmission electron microscopy (TEM) samples by means of the special technique of laser scanning microscopy. In TEM slides, numerous cavities with the same circular profile were detectable, and the length of these cavities is variable from 40–300 nm. The threads are oriented parallel and twisted around themselves to construct a double thread. In the interface between the two single threads, bridge‐like structures are prominent. The single untreated thread consists of cylindrical fibers with a diameter of approximately 1–1.5 μm. Apparently more than eight fibers are within a thread and each fiber is composed of a great number of fibrils with a diameter of about 150 nm. The surface of threads is coated with a characteristic layer ∼ 150–250 nm thick that contains glycoproteins. These were demonstrated for the first time by labeling with concanavalin A lectin‐gold complex and are dependent on the diameter and length of the thread. The same substances could also be detected inside the single thread. The skin can be removed completely or partially by mechanical treatment, or by washing with phosphate‐buffered saline or trypsin.
Direct measurement of electron beam scattering in the environmental scanning electron microscope using phosphor imaging plates
Authors: Wight Scott A. , Zeissler Cynthia J.
Published: June 2010

Phosphor imaging plate technology has made it possible to directly image the distribution of primary beam electrons and scattered electrons in the environmental scanning electron microscope. The phosphor plate is exposed under electron scattering conditions in the microscope chamber. When processed, the electron intensity distribution is displayed as a digital image. The image is a visual representation of the electron probe and skirt and may provide the basis for a more accurate model.
Charge contrast imaging of material growth and defects in environmental scanning electron miscroscopy—linking electron emission and cathodoluminescence
Authors: Griffin Brendan J.
Published: June 2010

An electron‐based technique for the imaging of crystal defect distribution such as material growth histories in non‐ and poorly conductive materials has been identified in the variable pressure or environmental scanning electron microscope. Variations in lattice coherence at the meso‐scale can be imaged in suitable materials. Termed charge contrast imaging (CCI), the technique provides images that correlate exactly with emitted light or cathodoluminescence in suitable materials. This correlation links cathodoluminescence and an electron emission. The specific operating conditions for observation of these images reflect a complex interaction between the electron beam, the positive ions generated by electron‐gas interactions in the chamber, a biased detector, and the sample. The net result appears to be the suppression of all but very near surface electron emission from the sample, probably from of the order of a few nanometres. Consequently, CCI are also sensitive to very low levels of surface contaminants. Successful imaging of internal structures in a diverse range of materials indicate that the technique will become an important research tool.