A portable cryo-storage system for low-temperature scanning electron microscopy,suitable for international transport of cryo-specimens

A cryo-specimen storage system for low-temperature scanning electron microscopy (LTSEM) specimens is described, which: liberates multi-specimen experiments from sampling restrictions imposed by the rate at which LTSEM specimens can be examined in the SEM; provides security against experiment loss resulting from breakdown of the SEM or cryo-system; enables collection of specimens in the field or in laboratories remote from the SEM laboratory; and facilitates international air transport of LTSEM specimens. The components of the system, which has a capacity of 98 stub-mounted specimens, are readily made in a laboratory workshop. The details of the design may be altered to suit particular specimen types or experimental approaches.     

Mechanical scanning of the specimen in the scanning electron microscope

A scanning electron microscope (SEM) system equipped with a motor drive specimen stage fully controlled with a personal computer (PC) has been utilized for obtaining ultralow magnification SEM images. This modem motor drive stage works as a mechanical scanning device. To produce ultra-low magnification SEM images, we use a successful combination of the mechanical scanning, electronic scanning, and digital image processing techniques. This new method is extremely labor and time saving for ultra-low magnification and wide-area observation. The option of ultra-low magnification observation (while maintaining the original SEM functions and performance) is important during a scanning electron microscopy session.     

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.     

High-resolution scanning electron microscopy of frozen-hydrated and freeze-substituted kidney tissue

Inner surfaces and fracture faces of rabbit kidney tissue were investigated with high-resolution scanning electron microscopy using two different cryopreparation techniques: (i) for the observation of fracture faces, cryofixed tissue was fractured and coated in a cryopreparation chamber dedicated to SEM, vacuum transferred onto a cold stage and observed in the frozen-hydrated state; (ii) for the observation of inner surfaces of the nephron, water was removed after freezing and fracturing by freeze substitution and critical-point drying of the tissue. By both methods, macromolecular structures such as intramembranous particles on fracture faces and particles on inner surfaces were imaged. The latter method was used to investigate in more detail surface structures of cells in the cortical collecting duct. These studies revealed a heterogeneity of intercalated cells not described thus far.     

Low-temperature scanning electron microscopy of frozen hydrated mouse lung

A method is presented by which water is preserved as ice during examination of the lung in the scanning electron microscope (SEM). The lung need only be inflated, frozen, transferred to the microscope and examined with the electron beam. Chemical fixation, solvent dehydration, and drying are not necessary. The low-temperature SEM of Pawley and Norton [11] maintains lung at ?180° C, nearly liquid nitrogen temperature, for extended periods with a Joule-Thomson refrigerator built into the stage. It has an integral high-vacuum preparation chamber attached to the microscope column which allows serial fracture, low-magnification stereo light microscopy, radiant etching, and evaporative coating with gold or carbon. The stage can be tilted from 0° to 45° and rotated a full 360°. It is demonstrated that the air-liquid interface in the lung can be examined and that low-temperature SEM can be used to investigate the shape of alveoli, the patency of the pores of Kohn in the hydrated state, and the shrinkage and distortion of lung with drying.     

Low-temperature SEM for detection of fungicide activity

Low-temperature scanning electron microscopy (LTSEM) combined with cryopreparation methods provided images of well-preserved biological surfaces and structures on a routine basis. Fractures of wheat leaves revealed epidermal and parenchymatous cells and masses of fungal hyphae growing in intercellular spaces. Freeze-fractured plant cells contained haustoria of the brown rust fungus Puccinia triticina. Extrahaustorial matrices were clearly distinguishable and at infection sites granular material was found. Activity of the triazole fungicide cyproconazole was mainly directed towards fungal hyphae and sporogenic tissue, resulting in a stronger branching and swelling of hyphal tips and collapse of fungal cells. Cryofixation methods combined with the use of a cryopreparation unit (Balzers SCU 020) were more reliable in interpreting the observed biological events through easier discrimination between evidence and artefacts. More advantages in the use of the cryopreparation unit are described in detail.     

An approach to the reduction of hydrocarbon contamination in the scanning electron microscope

The deposition of electron beam-induced specimen contamination in both the transmission (TEM) and scanning electron microscopes (SEM) has remained a problem since the beginning of these forms of microscopy. Generally, sources of SEM contamination can be attributed to one or a combination of three major contributors: (1) the pumping system; (2) outgassing of other internal SEM component parts (i.e., specimen stage, stage lubricants, O-rings, etc.), or (3) the sample (including its preparation and handling). Generally, because of the nature of SEM, specimen contamination can be minimized but is difficult to avoid fully. This work outlines three approaches taken with instruments at NIST to reduce the deposition of contamination in high-resolution cold-field emission SEMs. With some modification these techniques could be applied to any SEM. These approaches have been in successful operation for several years, resulting in a reduction in electron beam-induced hydrocarbon contamination.     

Application of environmental scanning electron microscopy to determine biological surface structure

The use of environmental scanning electron microscopy in biology is growing as more becomes understood about the advantages and limitations of the technique. These are discussed and we include new evidence about the effect of environmental scanning electron microscopy imaging on the viability of mammalian cells. We show that although specimen preparation for high-vacuum scanning electron microscopy introduces some artefacts, there are also challenges in the use of environmental scanning electron microscopy, particularly at higher resolutions. This suggests the two technologies are best used in combination. We have used human monocyte-derived macrophages as a test sample, imaging their complicated and delicate membrane ruffles and protrusions. We have also explored the possibility of using environmental scanning electron microscopy for dynamic experiments, finding that mammalian cells cannot be imaged and kept alive in the environmental scanning electron microscopy. The dehydration step in which the cell surface is exposed causes irreversible damage, probably via loss of membrane integrity during liquid removal in the specimen chamber. Therefore, mammalian cells should be imaged after fixation where possible to protect against damage as a result of chamber conditions.     

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.     

Advantages of indium–tin oxide-coated glass slides in correlative scanning electron microscopy applications of uncoated cultured cells

A method of direct visualization by correlative scanning electron microscopy (SEM) and fluorescence light microscopy of cell structures of tissue cultured cells grown on conductive glass slides is described. We show that by growing cells on indium–tin oxide (ITO)-coated glass slides, secondary electron (SE) and backscatter electron (BSE) images of uncoated cells can be obtained in high-vacuum SEM without charging artefacts. Interestingly, we observed that BSE imaging is influenced by both accelerating voltage and ITO coating thickness. By combining SE and BSE imaging with fluorescence light microscopy imaging, we were able to reveal detailed features of actin cytoskeletal and mitochondrial structures in mouse embryonic fibroblasts. We propose that the application of ITO glass as a substrate for cell culture can easily be extended and offers new opportunities for correlative light and electron microscopy studies of adherently growing cells.     

A technique for the examination of polar ice using the scanning electron microscope

The microstructure and location of impurities in polar ice are of great relevance to ice core studies. We describe a reliable method to examine ice in the scanning electron microscope (SEM). Specimens were cut in a cold room and could have their surfaces altered by sublimation either before (pre‐etching) or after (etching) introduction to the cryo‐chamber of the SEM. Pre‐etching was used to smooth surfaces, whilst etching stripped away layers from the specimen surface, aiding the location of particles in situ, and allowing embedded structures to be revealed. X‐ray analysis was used to determine the composition of localized impurities, which in some cases had been concentrated on the surface by etching. Examining uncoated surfaces was found to be advantageous and did not detract from qualitative X‐ray analysis. Imaging uncoated was performed at low accelerating voltages and probe currents to avoid problems of surface charging.     

Optimisation of the environmental scanning electron microscope for observation of drying of matt water-based lacquers

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 degrees 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 drop 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 microm 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.     

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.     

X-ray microanalysis of wet biological specimens in the environmental scanning electron microscope. 1. Reduction of specimen distance under different atmospheric conditions

X-ray microanalysis of non-biological and biological specimens was carried out in the environmental scanning electron microscope (ESEM) under different conditions of specimen distance (the distance travelled by the electron probe within the specimen chamber) and chamber atmosphere. Using both water vapour and argon atmospheres, it was shown that reduction in specimen distance had no effect on atmospheric gas X-ray signal in either case. Unlike water vapour, increased levels of argon (up to 10 torr) caused a marked depression of specimen P/B ratios, with a decrease in both characteristic and background (continuum) counts. These effects in argon were not altered by reduction in specimen distance. Specimen distance was important in relation to beam skirting and elemental analysis. With an extended assembly (short specimen distance), beam skirting in a water-vapour atmosphere was much reduced – leading to enhanced element detectability in a discrete biological specimen (Anabaena cyclindrica).     

A portable scanning electron microscope column design based on the use of permanent magnets

A portable scanning electron microscope (SEM) column design is presented which makes use of permanent magnets. Simulation results predict that such an SEM column is feasible and that it can be compact. The column height is typically less than 12 cm. The column is designed to be modular, so that it can fit onto a wide range of different specimen chamber types, and can also be readily replaced.     

Low-temperature scanning electron microscopy of particle-exposed mouse lung

Inflated frozen mouse lungs were examined using low-temperature scanning electron microscopy (LTSEM) following bulk fracture under vacuum. Various aspects of pulmonary architecture were identified and correlated with structures revealed by SEM following conventional fixation and preparation techniques. Surface etching of selected samples was performed by radiant heating, revealing characteristic cytoplasmic, nuclear and extracellular lattice patterns resulting from ice crystal formation during freezing. These patterns aided in distinguishing between intra- and extracellular spaces. Pulmonary fluids such as mucus and surfactant were identified. Iron oxide particles were introduced into the lungs of some animals by intratracheal instillation and were subsequently identified in frozen-hydrated lung tissue using characteristic X-ray identification and mapping techniques. Particles were observed both intra-and extracellularly and were commonly found in large deposits. These observations confirm the utility of LTSEM techniques for examination of particles within pulmonary tissue. Particle exposure by intratracheal instillation was found to result in a non-uniform distributional pattern.     

Practical method for high-resolution imaging of polymers by low-voltage scanning electron microscopy

Morphologic characterization of polymers by scanning electron microscopy (SEM) is often made difficult by their sensitivity to electron beam damage. We describe here a specimen preparation method for the imaging of polymer blends by low-voltage SEM (LV-SEM) that improves their stability in the electron beam and hence facilitates focusing and recording of high magnification images. Its application to nanosized core-shell latexes embedded in a polymethylmethacrylate matrix and semi-crystalline polypropylene/ethylene-propylene rubber blends is discussed.     

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.     

Better visualization inside the environmental scanning electron microscope through the infrared chamberscope coupled with a mirror

Clearances are tight inside the specimen chamber of the environmental scanning electron microscope (ESEM), and it is difficult to see the relative positions of detectors and specimens through the viewport. For example, the relative placement of the energy-dispersive spectrometer (EDS) and the specimen is critical for attaining reasonable x-ray efficiency while protecting the detector window from damage. An infrared chamberscope and mirror attachment were added to improve the visibility inside the chamber.