Freeze-fracturing for conventional and field emission low-temperature scanning electron microscopy: the scanning cryo unit SCU 020

A dedicated cryopreparation system, the SCU 020 (Balzers), is introduced and described in detail for use in low-temperature scanning electron microscopy (LTSEM). The basic unit consists of two parts: (i) a high-vacuum preparation chamber equipped with a cold-stage, motor-driven fracturing microtome, planar magnetron (PM) sputter source, quartz-crystal thin-film monitor, Meissner cold trap, and turbo molecular pump stand; and (ii) a second part (separated from the first by a sliding, high-vacuum valve) residing in the SEM chamber. This is equipped with an anti-contamination cold trap, a fully movable goniometer cold stage (having motor drives for x, y, and rotation) and replaces the SEM's original stage (Raith). The SCU 020 is entirely self contained allowing independence from, and synchroneity with, the SEM of choice. LTSEM micrographs of specimen (that are fully frozen hydrated or partially freeze-dried) surfaces or fracture faces, without or with various metal coatings, can be examined over a broad temperature range (-150 to +50°C). This is made possible by the combined application of the two, independently controlled, cold stages and the on-line, high-vacuum, specimen cryo transfer between them. In-situ etching is simple and straightforward. Intramembranous particles and membrane fracture steps, typically imaged in transmission electron microscopy (TEM), are resolved by PM sputtering with platinum at low specimen temperature and high-resolution LTSEM in a field emission microscope.     

Artefacts arising during preparation of hydrated paper pulp samples for low-temperature SEM

Beating, a pulp treatment widely used in the paper industry, causes disruption of cell wall layers and fibrillation. Previous studies of the effects of beating on fibre morphology have used conventional methods of specimen preparation, with all the attendant problems of shrinkage and distortion during dehydration. Low-temperature scanning electron microscopy (LTSEM) therefore seemed to offer an ideal method for examining fully hydrated wood pulp fibres. Cryofixation of pulp followed by sublimation of superficial ice, however, is shown to generate artefacts indistinguishable from structures present in the samples. Fibrillar and membranous structures were generated in LTSEM-prepared sugar solutions; their presence in pulp samples was therefore attributed to the dissolved carbohydrates inherent in pulp suspensions. Since artefact and fact are currently impossible to distinguish in LTSEM-prepared pulp samples, it seems that the technique should be applied to wet paper or pulp samples with considerable circumspection.     

An automated image alignment system for the scanning electron microscope

We have developed an automated image alignment system for the scanning electron microscope (SEM). This system enables specific locations on a sample to be located and automatically aligned with submicron accuracy. The system comprises a sample stage motorization and control unit together with dedicated imaging electronics and image processing software. The standard SEM sample stage is motorized in the X and Y axes with stepping motors which are fitted with rotary optical encoders. The imaging electronics are interfaced to beam deflection electronics of the SEM and provide the image data for the image processing software. The system initially moves the motorized sample stage to the area of interest and acquires an image. This image is compared with a reference image to determine the required adjustments to the stage position or beam deflection. This procedure is repeated until the area imaged by the SEM matches the reference image. A hierarchical image correlation technique is used to achieve submicron alignment accuracy in a few seconds. The ability to control the SEM beam deflection enables the images to be aligned with an accuracy far exceeding the positioning ability of the SEM stage. The alignment system may be used on a variety of samples without the need for registration or alignment marks since the features in the SEM image are used for alignment. This system has been used for the automatic inspection of devices on semiconductor wafers, and has also enabled the SEM to be used for direct write self-aligned electron beam lithography.     

Use of low-temperature scanning electron microscopy to compare and characterize three classes of snow cover

This study, which uses low-temperature scanning electron microscopy (LTSEM), systematically sampled and characterized snow crystals that were collected from three unique classes of snow cover: prairie, taiga, and alpine. These classes, which were defined in previous field studies, result from exposure to unique climatic variables relating to wind, precipitation, and air temperature. Snow samples were taken at 10 cm depth intervals from the walls of freshly excavated snow pits. The depth of the snow pits for the prairie, taiga, and alpine covers were 28, 81, and 110 cm, respectively. Visual examination revealed that the prairie snow cover consisted of two distinct layers whereas the taiga and alpine covers had four distinct layers. Visual measurements were able to establish the range of crystal sizes that occurred in each layer, the temperature within the pit, and the snow density. The LTSEM observations revealed the detailed structures of the types of crystals that occurred in the snow covers, and documented the metamorphosis that transpired in the descending layers. Briefly, the top layers from two of the snow covers consisted of freshly fallen snow crystals that could be readily distinguished as plates and columns (prairie) or graupel (taiga). Alternatively, the top layer in the alpine cover consisted of older dendritic crystal fragments that had undergone early metamorphosis, that is, they had lost their sharp edges and had begun to show signs of joining or bonding with neighboring crystals. A unique layer, known as sun crust, was found in the prairie snow cover; however, successive samplings from all three snow covers showed similar stages of metamorphism that led to the formation of depth hoar crystals. These changes included the gradual development of large, three-dimensional crystals having clearly defined flat faces, sharp edges, internal depressions, and facets. The study, which indicates that LTSEM can be used to enhance visual data by systematically characterizing snow crystals that are collected at remote locations, is important for understanding the physics of snowpacks and the metamorphosis that leads to potential avalanche situations. In addition, the metamorphosis of snow crystals must be considered when microwave radiometry is used to estimate the snow water equivalent in the winter snowpack, because large snow crystals more effectively scatter passive microwave radiation than small crystals.     

Low-temperature scanning electron microscopy in biology

A review of low-temperature scanning electron microscopy (LTSEM) with regard to preparation protocols, specimen preservation, experimental approaches, and high-resolution studies, is provided. Preparative procedures are described and recent developments in methodologies highlighted. It is now well established that LTSEM, for most biological specimens, provides superior specimen preservation than does ambient-temperature SEM. This is because frozen-hydrated samples retain most or all of their water, are rapidly immobilized and stabilized by cryofixation, and are not exposed to chemical modification or solvent extraction. Nevertheless, artefacts in LTSEM are common and most arise because frozen-hydrated specimens contain water. LTSEM can be used as a powerful experimental tool. Advantages of employing LTSEM for this purpose and ways in which it can be used for novel experimentation are discussed. The most exciting development in recent years has been high-resolution LTSEM. The advantages, problems and requirements for this approach are defined.     

Image comparisons of snow and ice crystals photographed by light (video) microscopy and low-temperature scanning electron microscopy

Light (video) microscopy and low-temperature scanning electron microscopy (SEM) were used to examine and record images of identical precipitated and metamorphosed snow crystals as well as glacial ice grains. Collection procedures enabled numerous samples from distant locations to be shipped to a laboratory for storage and/or observation. The frozen samples could be imaged with a video microscope in the laboratory at ambient temperatures or with the low-temperature SEM. Stereo images obtained by video microscopy or low-temperature SEM greatly increased the ease of structural interpretations. The preparation procedures that were used for low-temperature SEM did not result in sublimation or melting. However, this technique did provide far greater resolution and depth of focus over that of the video microscope. The advantage of resolution was especially evident when examining the small particles associated with rime and graupel (snow crystals encumbered with frozen water droplets), whereas the greater depth of focus provided clearer photographs of large crystals such as depth hoar, and ice. Because the SEM images contained only surface information while the video images were frequently confounded by surface and internal information, the SEM images also clarified the structural features of depth hoar crystals and ice grains. Low-temperature SEM appears to have considerable promise for future investigations of snow and ice.     

Calibration of an electron back-scattering pattern set-up

The determination of lattice orientations from electron back-scattering patterns (EBSPs) in a scanning electron microscope (SEM) requires accurate knowledge of the position of the pattern centre and the source point to screen distance. This paper outlines a new procedure that enables the determination of these parameters for any given set-up of the EBSP/SEM system. The calibration procedure simply requires the positions and indices of at least four poles in a pattern obtained from an arbitrary specimen, and eliminates the need for standard specimens or special attachments to the EBSP/SEM system. The pattern centre is shown to be located with a precision of approximately 0·5° and the source point to screen distance can be determined with a relative precision of approximately 0·5%.     

Ultrastructure of Tracheal Surface Liquid: Low-Temperature Scanning Electron Microscopy

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.     

Simple modifications for accurate high-temperature gas exposures using scanning electron microscopy

Relatively low-cost modifications to standard commercial scanning electron microscopes (SEM) that allow accurate exposure of sample(s) to noncorrosive gases at ambient and high temperatures are outlined. Energy-dispersive spectroscopic analysis of sample(s) exposed to noncorrosive gases at high temperatures is demonstrated. Gas exposure is limited to pressures of less than 10^?4 torr (1.33 × 10^?2 Pa) as a result of limitations on SEM system operation and SEM safety interlocks. Gases are limited to noncorrosive types as a result of potential damage to system detection devices and internal mechanical parts.     

Scanning electron microscopy comparison of the resin–dentin interface using different specimen preparation methods

Microscopy has been widely used to complement the data of studies related to dentin bonding; however, different specimen preparation methods may influence the analysis. Aiming to contribute to the reported scenario, this study evaluated the effect of two specimen‐sectioning methods (cleavage and diamond disk cut) on the quality of the scanning electron microscopy (SEM) images. Four crowns of human molars were selected and had an area of approximately 6 mm² of dentin exposed. They were then divided into two groups according to the universal adhesive application: total and self‐etching modes. Then, composite blocks were built up and the specimens were stored in deionized water to allow the postcuring. The specimens were further divided according to the sectioning method: cleavage or diamond disk cut. Four specimens were obtained from each tooth. They were desiccated, placed on aluminum stubs, sputter‐coated with gold, and observed in a scanning electron microscope, with ×2000 of magnification. The quality of the SEM images were evaluated by two calibrated examiners and classified into four scores (1–4). Mann–Whitney test (p < .05) showed that the diamond disk provided significantly higher scores than cleavage, whereas no significant difference was observed when comparing the total‐etching and self‐etching modes of application. The diamond disk cut method is preferable to the cleavage method to ensure the quality of the SEM analysis in studies involving the resin–dentin interface.     

Metallization and Ar‐O plasma effects on dental enamel roughness evaluated with SEM and MeX™ for 3D reconstruction

The MeX? software is a useful tool for tridimensional data collection for surface evaluation and could be relevant to evaluate the same specimen in different phases of the study, assuming repeated measures of dental enamel roughness. The aim of this study was to evaluate the influence of sample metallization for dental enamel roughness analysis with 3D images reconstructed using MeX? software from Scanning Electron Microscopy (SEM) images. The influence of 74.98% (%mol/mol) argon?oxygen plasma for carbon layer removal on surface roughness of the metallized specimen was also evaluated. Dental enamel specimens were prepared for SEM analysis with and without carbon metallization using conventional or environmental modes. Argon?oxygen plasma for carbon layer removal was used and surface roughness was re‐evaluated. Roughness obtained by SEM and MeX? reconstructed images, with or without metallization, did not differ. No significant alteration on surface roughness after carbon layer removal using plasma was found. SEM baseline evaluation using conventional mode without sample preparation and in environmental mode were not comparable. Roughness of enamel 3D images reconstructed with MeX? software from SEM images, with or without metallization was similar. The 74.98% (%mol/mol) argon?oxygen plasma removed the carbon layer with no effect on enamel roughness.     

The micromorphological characterization of adhesive bond in dentin different locations

The aim of this study was to analyse the interfacial micromorphology of total‐etch adhesives and dentin structures different locations by using SEM. Standardized cylindrical cavities (3mm in diameter, 2mm deep) with all margins in dentin were prepared on occlusal and buccal surfaces of twenty extracted human third molars. A total‐etch dentin adhesive system and a light‐cure flowable composite (Filtek Ultimate Flowable, 3M ESPE, St. Paul, MN, USA) were used in this study. Micro‐morphological SEM analysis of the marginal seal of the original tooth specimens was performed using high magnification of up to 1000×. In this study, we found the difference in interfacial micromorphology in dentin different locations. Also, marginal gap was found in both observed dentin area. Better understanding of complexity and three‐ dimensional variations of the tooth structure is important for prevention of clinical challenges such as postoperative sensitivity, marginal discoloration and secondary caries, which could be prevented by achieving of predictable and long‐lasting adhesive bond.     

Angioarchitecture of the ventral surface of the tongue from Wistar rats

The goal of this study was to describe the angioarchitecture of the ventral surface of the tongue from Wistar rats using a vascular corrosion casting technique associated with scanning electron microscopy (SEM). Six Wistar rats were used for the vascular casting method with Mercox resin. Following the resin polymerization, the tongue of each animal was dissected and corroded in a 5% sodium hydroxide solution. Once the corrosion and drying of the specimens were completed, the specimens were mounted on aluminum stubs, coated with carbon and gold and analyzed under SEM. The results showed that the ventral surface of the tongue presents simple, even and abundant vasculature constituted by a vascular plexus consisting of a superficial vascular network and by the ranine veins. The superficial vascular network, made up of the ascending and descending branches, presents as a loose network, with little morphological variation between the capillary loops.     

Nanotip electron gun for the scanning electron microscope

Experimental nanotips have shown significant improvement in the resolution performance of a cold field emission scanning electron microscope (SEM). Nanotip electron sources are very sharp electron emitter tips used as a replacement for the conventional tungsten field emission (FE) electron sources. Nanotips offer higher brightness and smaller electron source size. An electron microscope equipped with a nanotip electron gun can provide images with higher spatial resolution and with better signal-to-noise ratio. This could present a considerable advantage over the current SEM electron gun technology if the tips are sufficiently long-lasting and stable for practical use. In this study, an older field-emission critical dimension (CD) SEM was used as an experimental test platform. Substitution of tungsten nanotips for the regular cathodes required modification of the electron gun circuitry and preparation of nanotips that properly fit the electron gun assembly. In addition, this work contains the results of the modeling and theoretical calculation of the electron gun performance for regular and nanotips, the preparation of the SEM including the design and assembly of a measuring system for essential instrument parameters, design and modification of the electron gun control electronics, development of a procedure for tip exchange, and tests of regular emitter, sharp emitter and nanotips. Nanotip fabrication and characterization procedures were also developed. Using a "sharp" tip as an intermediate to the nanotip clearly demonstrated an improvement in the performance of the test SEM. This and the results of the theoretical assessment gave support for the installation of the nanotips as the next step and pointed to potentially even better performance. Images taken with experimental nanotips showed a minimum two-fold improvement in resolution performance than the specification of the test SEM. The stability of the nanotip electron gun was excellent; the tip stayed useful for high-resolution imaging for several hours during many days of tests. The tip lifetime was found to be several months in light use. This paper summarizes the current state of the work and points to future possibilities that will open when electron guns can be designed to take full advantage of the nanotip electron emitters.     

Evaluation of demineralized dentin contraction by stereo measurements using environmental and conventional scanning electron microscopy

Numerous investigations of etched human dentin are performed using scanning electron microscopy (SEM). Usually specimens are fractured and cross sections of etched layers with underlying unaffected dentin are observed. Results from this study showed that the edge of the etched layer contracted and became curved after fracture of wet specimens and that tensile stresses were developed in this layer by acid etching. The degree of contraction was determined utilizing profiles of the specimen edges obtained with the help of stereo measurements. Fixation in glutaraldehyde decreased the contraction in wet specimens prepared for environmental scanning electron microscopy (ESEM). Fixation also decreased shrinkage of the demineralized layer due to gradual desiccation in the ESEM during observation. For conventional SEM, the contraction was minimized if specimens of etched and fixed dentin were fractured in the dry condition after dehydration.     

Developing 3D SEM in a broad biological context

When electron microscopy (EM) was introduced in the 1930s it gave scientists their first look into the nanoworld of cells. Over the last 80 years EM has vastly increased our understanding of the complex cellular structures that underlie the diverse functions that cells need to maintain life. One drawback that has been difficult to overcome was the inherent lack of volume information, mainly due to the limit on the thickness of sections that could be viewed in a transmission electron microscope (TEM). For many years scientists struggled to achieve three‐dimensional (3D) EM using serial section reconstructions, TEM tomography, and scanning EM (SEM) techniques such as freeze‐fracture. Although each technique yielded some special information, they required a significant amount of time and specialist expertise to obtain even a very small 3D EM dataset. Almost 20 years ago scientists began to exploit SEMs to image blocks of embedded tissues and perform serial sectioning of these tissues inside the SEM chamber. Using first focused ion beams (FIB) and subsequently robotic ultramicrotomes (serial block‐face, SBF‐SEM) microscopists were able to collect large volumes of 3D EM information at resolutions that could address many important biological questions, and do so in an efficient manner. We present here some examples of 3D EM taken from the many diverse specimens that have been imaged in our core facility. We propose that the next major step forward will be to efficiently correlate functional information obtained using light microscopy (LM) with 3D EM datasets to more completely investigate the important links between cell structures and their functions.     

Primary considerations for image enhancement in high-pressure scanning electron microscopy

The mechanisms of electron beam scattering are examined to evaluate its effect on contrast and resolution in high-pressure scanning electron microscopy (SEM) techniques reported in the literature, such as moist-environment ambient-temperature SEM (MEATSEM) or environmental SEM (ESEM). The elastic and inelastic scattering cross-sections for nitrogen are calculated in the energy range 5–25 keV. The results for nitrogen are verified by measuring the ionization efficiency, and measurements are also made for water vapour. The effect of the scattered beam on the image contrast was assessed and checked experimentally for a step contrast function at 20 kV beam voltage. A considerable degree of beam scattering can be tolerated in high-pressure SEM operation without a significant degradation in resolution. The image formation and detection techniques in high-pressure SEM are considered in detail in the accompanying paper.     

High-pressure scanning electron microscopy of insulating materials: A new approach

A new SEM technique for imaging uncoated non-conducting specimens at high beam voltages is described which employs a high-pressure environment and an electric field to achieve charge neutralization. During imaging, the specimen surface is kept at a stable low voltage, near earth potential, by directing a flow of positive gas ions at the specimen surface under the action of an electric bias field at a pressure of about 200 Pa. In this way charge neutrality is continuously maintained to obtain micrographs free of charging artefacts. Images are formed by specimen current detection containing both secondary electron and backscattered electron signal information. Micrographs of geological, ceramic, and semiconductor materials obtained with this method are presented. The technique is also useful for the SEM examination of histological sections of biological specimens without any further preparation. A simple theory for the charge neutralization process is described. It is based on the interaction of the primary and emissive signal components with the surrounding gas medium and the resulting neutralizing currents. Further micrographs are presented to illustrate the pressure dependence of the charge neutralization process in two glass specimens which show clearly identifiable charging artefacts in conventional microscopy.