An electron beam lithography and digital image acquisition system for scanning electron microscopes

A low‐cost microcontroller based control and data acquisition unit for digital image recording of scanning electron microscope (SEM) images and scanning electron microscope based electron beam lithography (EBL) is described. The developed microcontroller low‐level embedded software incorporates major time critical functions for image acquisition and electron beam lithography and makes the unit an intelligent module which communicates via USB with the main computer. The system allows recording of images with up to 4096 × 4096 pixel size, different scan modes, controllable dwell time, synchronization with main power frequency, and other user controllable functions. The electron beam can be arbitrary positioned with 12‐bit precision in both dimensions and this is used to extend the scanning electron microscope capabilities for electron beam lithography. Hardware and software details of the system are given to allow its easy duplication. Performance of the system is discussed and exemplary results are presented.     

Quasi-cleavage fracture planes in spheroidized A533B steel

Electron backscattered diffraction (EBSD) has been used to acquire crystal orientation information around unusual microcracks induced by tensile deformation of notched specimens of spheroidized A533B steel. This unusual fracture mode has been called quasi‐cleavage and occurs at relatively low temperatures with fracture energies below that of the upper shelf. EBSD measurements on sectioned samples showed that the quasi‐cleavage cracks were intragranular. A two‐dimensional analysis technique was used in which EBSD measured crystal orientations were combined with secondary electron imaging to obtain the trace of the crack facet on the section plane. The measurements revealed that the observed crack facets were consistent with crack propagation along the {001} and {011} planes.     

In vivo remineralization of acid-etched enamel in non-brushing areas as influenced by fluoridated orthodontic adhesive and toothpaste

This study aimed to evaluate the in vivo remineralization of acid-etched enamel in non-brushing areas as influenced by fluoridated orthodontic adhesive and toothpaste. One hundred and twenty teeth from 30 volunteers were selected. The teeth were assigned to four treatments: no treatment (negative control); 37% phosphoric acid-etching (PAE) (positive control); PAE + resin-modified glass ionomer cement (RMGIC); and, PAE + composite resin. Patients brushed teeth with fluoridated (n = 15) or non-fluoridated (n = 15) toothpastes, so that etched enamel was protected with screens and it was not in contact with the brush bristles. Remineralization was evaluated by means of laser fluorescence (LF), environmental scanning electronic microscopy, and energy dispersive spectrometry after extraction. The LF means were compared by means of Wilcoxon and Mann Whitney tests. Environmental scanning electron microscopy scores were compared among the groups using a Kruskal Wallis test, whereas the Ca/P ratio was evaluated by means of an Analysis of Variance with subparcels (treatments) and Tukey's post-hoc test. There were no statistically significant differences between the tooth pastes and between the orthodontic adhesives evaluated. Most teeth presented only partial enamel remineralization. Therefore, the fluoride released by the RMGIC was not enough to cause increased crystal regrowth in the acid-etched enamel. The use of fluoridated toothpaste did not provide positive additional effect.     

The Atmospheric Scanning Electron Microscope with open sample space observes dynamic phenomena in liquid or gas

Although conventional electron microscopy (EM) requires samples to be in vacuum, most chemical and physical reactions occur in liquid or gas. The Atmospheric Scanning Electron Microscope (ASEM) can observe dynamic phenomena in liquid or gas under atmospheric pressure in real time. An electron-permeable window made of pressure-resistant 100 nm-thick silicon nitride (SiN) film, set into the bottom of the open ASEM sample dish, allows an electron beam to be projected from underneath the sample. A detector positioned below captures backscattered electrons. Using the ASEM, we observed the radiation-induced self-organization process of particles, as well as phenomena accompanying volume change, including evaporation-induced crystallization. Using the electrochemical ASEM dish, we observed tree-like electrochemical depositions on the cathode. In silver nitrate solution, we observed silver depositions near the cathode forming incidental internal voids. The heated ASEM dish allowed observation of patterns of contrast in melting and solidifying solder. Finally, to demonstrate its applicability for monitoring and control of industrial processes, silver paste and solder paste were examined at high throughput. High resolution, imaging speed, flexibility, adaptability, and ease of use facilitate the observation of previously difficult-to-image phenomena, and make the ASEM applicable to various fields.     

Quantitative detection of gold nanoparticles on individual, unstained cancer cells by scanning electron microscopy

Gold nanoparticles are rapidly emerging for use in biomedical applications. Characterization of the interaction and delivery of nanoparticles to cells through microscopy is important. Scanning electron microscopes have the intrinsic resolution to visualize gold nanoparticles on cells. A novel sample preparation protocol was developed to enable imaging of cells and gold nanoparticles with a conventional below lens scanning electron microscopes. The negative influence of 'charging' on the quality of scanning electron microscopes' images could be limited by deposition of biological cells on a conductive (gold) surface. The novel protocol enabled high-resolution scanning electron microscopes' imaging of small clusters and individual gold nanoparticles on uncoated cell surfaces. Gold nanoparticles could be counted on cancer cells with automated routines.     

Registration of confocal scanning laser microscopy and quantitative backscattered electron images for the temporospatial quantification of mineralization density in 18-month old thoroughbred racehorse articular calcified cartilage

Combined backscattered electron scanning electron microscopy (BSE SEM) and confocal scanning laser microscopy (CSLM) have been used to put tissue mineralization data into the context of soft tissue histology and fluorescent label information. Mineralization density (Dm) and linear accretion rate (LAR) are quantifiable parameters associated with mineralizing fronts within calcified tissues. Quantitative BSE (qBSE) may be used to determine Dm, while CSLM may be used to detect label fluorescence from which LAR is calculated. Eighteen-month old Thoroughbred horses received single calcein injections 19 and 8 days prior to euthanasia, labeling sites of active mineralization with fluorescent bands. Confocal scanning laser microscopy images of articular calcified cartilage (ACC) from distal third metacarpal condyles were registered to qBSE images of the same sites using an in-house program. ImageJ and Sync Windows enabled the simultaneous collection of LAR and Dm data. The repeatability of the registration and measurement protocols was determined. Dm profiles between calcein labels were explored for an association with time. Dm was 119.7 +/- 24.5 (mean +/- standard deviation) gray levels (where 0 = backscattering from monobrominated and 255 from monoiodinated dimethacrylate standards, respectively), while modal and maximum LAR were 0.45 and 3.45 microm/day, respectively. Coefficients of variation (CV) for Dm were 0.70 and 0.77% with and without repeat registration, respectively; CVs for LAR were 1.90 and 2.26% with and without repeat registration, respectively. No relationship was identified between Dm and time in the 11-day interlabel interval. Registration of CSLM to qBSE images is sufficiently repeatable for quantitative studies of equine ACC.     

Monte Carlo analysis of the detection of clay occlusion of respirable quartz particles using multiple voltage scanning electron microscopy

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.     

Microwave embedment for light and electron microscopy using epoxy resins,LR white,and other polymers

Microwave energy can be used to polymerize embedding polymers used for light and electron microscopy, providing novel and ultrarapid processing methods to accomplish cell and tissue embedments. These quick methods steadily show promise for diagnosis of disease where rapid methods can be crucial. This review is intended to answer some of the questions microscopists may have in attempting to utilize such microwave embedding procedures and to provide a comparison of results using divergent Medcast and LR White.     

Functional and comparative study of lingual papillae in four species of bear (Ursidae) by scanning electron microscopy

The eight current species of bear (Ursidae) are widely distributed throughout Europe, Asia, and America. They are mainly encountered in the northern hemisphere, except for the spectacled bear and the sun bear, which are also found in the south of the equator. Adaptations of the masticatory apparatus (teeth, tongue, and musculature) to diet are one of the factors that imply the greatest structural changes in the cranium. This diet may be carnivorous, herbivorous, melliferous, or insectivorous, with one type of food predominating according to the time of year. The way in which food is eaten determines the morphology of the lingual surface; generally speaking, all bears put their mouth to the food, which, initially, they lick or they let the food stick to their tongue, as occurs when insects are eaten. As in all mammals, a distinction can be made between mechanical and gustatory papillae and the development and distribution of which depend on the species and their eating habits. In this study of the complete tongues of four species of adult bears, we describe the morphology of the lingual surfaces, the different types of papillae, their characteristics, and topographic distribution. It was seen that there were five main types of lingual papillae: filiform, conical, fungiform, foliate, and vallate. Morphology of the microgrooves and pores was similar to that observed in other mammals. In general, there were no great differences among the four species of bears studied, perhaps due to the similarity in the kind of food they consume in captivity.