Manipulating biological samples for environmental scanning electron microscopy observation

Biological samples having different characteristics were observed by environmental scanning electron microscopy (ESEM). The environmental conditions for untreated biological samples was determined by optimizing sample temperature and chamber pressure. When the temperature was at 4 degrees - 6 degrees C and chamber pressure was 5.2-5.9 Torr, the relative humidity in the specimen chamber was about 85%. Under these conditions, the surface features of the sample were completely exposed and did not exhibit charging. The images obtained from the untreated samples at different ESEM conditions were also compared with fixed and coated samples observed under high vacuum.     

Review and outline of environmental SEM at present

The environmental scanning electron microscope (ESEM) allows the examination of specimens in a gaseous environment. It is based on an integration of efficient differential pumping with a new design of electron optics and detection systems. Backscattered, cathodoluminescence and X-ray detectors can be designed to fit and to perform optimally in the ESEM. The secondary electron signal can be detected with the gaseous detector device, which is a new multipurpose detector. Insulating, uncoated, wet and generally both treated or untreated specimens can be studied.     

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.     

Controlled dehydration of a biological sample using an alternative form of environmental SEM

In this study a non-conductive biological sample is observed free of charging artefacts when placed on a cooled Peltier stage in the specimen chamber of an alternative form of the environmental scanning electron microscope, equipped with a specially designed hydration system. This system was used to create dynamically changing surrounding conditions leading to controlled dehydration of the sample enabling us to visualize the topographical structure of a rat tongue in the transition region between the liquid and the gas state of water in the microscope specimen chamber.     

Liquid substitution: A versatile procedure for SEM specimen preparation of biological materials without drying or coating

Certain liquids with a very low vapour pressure, such as glycerol or triethylene glycol, can be used to infiltrate biological specimens so that they may be observed in the scanning electron microscope (SEM) without drying. The conductive properties of the fluids allow specimens to be examined either uncoated or with very thin coatings. The advantages of liquid substitution include the retention of lipids, waxes, loose particles, and surface contaminants. Since the procedure does not require expensive equipment, it offers an alternative to critical point drying or cryo-preparation. For certain types of specimens, liquid substitution may represent the best preparation procedure. In addition, the fluids themselves may be imaged directly in the SEM, or indirectly by cathodoluminescence following labelling with fluorochromes.     

Figure of merit for environmental SEM and its implications

A recently introduced figure of merit for environmental and low vacuum scanning electron microscopes has now been computed in the full operational pressure range for one commercial instrument. The direct simulation Monte Carlo method has been used in lieu of experimental measurements. The theory of this figure of merit is further consolidated. It is shown that a thin pressure limiting aperture can indeed be used as an optimum reference system for all instruments employing differential pumping in the transfer of an electron beam from high vacuum to high pressure. The implications of the results obtained are discussed both in relation to existing commercial instruments and associated literature to pave the way for future progress in the field.     

Solutions to difficulties encountered in low-temperature SEM of some frozen hydrated specimens: Examination of Penicillium nalgiovense cultures

Penicillium nalgiovense cultures, which are used in the food industry, were found to be collapsed when prepared by standard procedures for scanning electron microscopy. Neither freeze-drying nor critical point-drying preserved the structure of cultures grown on agar media. Cryofixation and preparation of frozen hydrated samples using the Hexland Cryotrans CT 1000 attachment in conjunction with an AMR 1000A scanning electron microscope yielded micrographs of uncollapsed structures which could be used for morphological characterization. Several additional steps had to be used in sample preparation to achieve satisfactory results. Samples were held in a humid chamber prior to freezing; growth substrate was trimmed as thinly as possible (less than 1 mm above the support); the sides of samples were painted with a conductive cement to their upper edge; and frozen samples were coated intermittently with gold sputtered in several 2-min bursts.     

Novel sample preparation method of polymer emulsion for SEM observation

The aim of this study was to design a simple and reliable method for obtaining the detailed information about the average size, size distribution, and the surface morphology of particles with variation of the sample preparation of a polymer emulsion. In this work, the characteristic features of the particles of rosin size with high viscosity were first described by scanning electron microscopy (SEM). The morphologies of polymer emulsion of solid lipid nanoparticles and of the microspheres were observed. The advantage of the method is that not only the true size and shape of emulsion particles can be shown, but the problem of high-viscosity emulsion that prevents there study with SEM is solved. Using this new method, the micromorphology and size distribution of the emulsion particles with different viscosities have been clearly observed.     

Optimizing environmental scanning electron microscopy of spheroidal reaggregated neuronal cultures

Electrophysiological recordings from hen embryo brain spheroidal reaggregates on penetrating 3D multielectrode arrays could be understood more easily if the surface structure was known in more detail. Electrophysiological activity, as grouped spikes in trains, is acquired from spheroids, indicating the inner formation a neuronal network. To this end, spheroids can be observed by environmental scanning electron microscopy. Live spheroids collapse when the supporting water is evaporated. By careful adjustment of the chamber pressure it is possible to observe fully hydrated fixed spheroids. A thin film of water tends to prevent a clear view of the surface detail. This can be evaporated to reveal the surface while taking steps to avoid both inadvertent shrinkage and rewetting. Conventional SEM shows a very different surface that is rich in protruding cell bodies and fibers. The images are compared and interpreted with some images of the surface using transmission electron microscopy.     

A new technique for visualization of internal structure by SEM

A simple, rapid, economic and reliable method was used for visualization of internal structure of soft tissues by fracturing paraffin embedded samples at room temperature. The same sample which was used for light microscopy was then fractured for SEM. Most of the cellular and extracellular details are exposed without any need of special equipment. The technique preserves the architecture of tissues and can be used in routine diagnostic pathology.     

扫描电子显微镜探头新进展

介绍了扫描电子显微镜（SEM）信号探头研究的最新成果。针对常见扫描电子显微镜缺陷而开发的新型探头不仅改善了仪器成像质量,也极大地扩展了仪器的使用范围,简化了样品的准备工艺过程。     

Cryo-SEM method for the observation of entrapped bubbles and degree of water filling in large wet powder compacts

There are generally two problems associated with cryogenic scanning electron microscopy (cryo-SEM) observations of large wet powder compacts. First, because water cannot be vitrified in such samples, formation of artefacts is unavoidable. Second, large frozen samples are difficult to fracture but also to machine into regular pieces which fit in standard holders, especially if made of hard materials like ceramics. In this article, we first describe a simple method for planning hard cryo-samples and a low-cost technique for cryo-fracture and transfer of large specimens. Subsequently, after applying the entire procedure to green pellets of iron ore produced by balling, we compare the influence of plunge- and unidirectional freezing on large entrapped bubbles throughout the samples as well as the degree of water filling at the outer surface of the pellets. By carefully investigating the presence of artefacts in large areas of the samples and by controlling the orientation of the sample during freezing and preparation, we demonstrate that unidirectional freezing enables the observation of large entrapped bubbles with minimum formation of artefacts, whereas plunge freezing is preferable for the characterization of the degree of water filling at the outer surface of wet powder compacts. The minimum formation of artefacts was due to the high packing density of the iron ore particles in the matrix.     

A method for the location of specific points on surfaces in the SEM

A method for precisely locating points on large surfaces during SEM examination and for finding corresponding points on matching surfaces (such as those resulting from the fracture of a solid body) is described. This method also allows for the repeated examination of specific points during subsequent SEM sessions after the specimen has been removed and replaced in the SEM. The method is based on a coordinate transformation between the SEM stage coordinates and specimen coordinate systems defined by arbitrarily chosen reference points on the specimen.     

Method of platinum-carbon coating of ultrathin sections for transmission and scanning electron microscopy: an application for study of biological composites

Many biological materials are composites containing two or more components with different mechanical properties. This study is concerned with the application of a method of platinum-carbon coating (Pt/C) of ultrathin sections for TEM and SEM studies of the design of natural composite materials. The changes in profile of the ultrathin resin-embedded sections during different stages of the preparation reflect the material properties of the various components: stiffer regions deform less than softer ones. Such changes in the section profile can be visualized by the Pt/C method and used as evidence of specific material properties in particular regions of composite materials. The method increases the relief contrast, improves the 3D-view of structures, and in combination with standard TEM and SEM procedures can provide clear demonstrations of material design. The distribution of chitin crystallites in the insect cuticle and the ultrastructure of the pore canal system specialized for the transport of epidermal secretions to the cuticle surface were studied here as examples.     

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.     

Radiation damage of water in environmental scanning electron microscopy

Specimen damage from the electron beam poses a considerable problem with electron microscopy. This damage is particularly acute in environmental scanning electron microscopy (ESEM) for two reasons. Firstly, owing to its ability to stabilise insulating and hydrated specimens, ESEM lends itself to polymeric and biological materials that are typically highly beam-sensitive. Secondly, water acts as a source of small, highly mobile free radicals, which accelerate specimen degradation.
By taking the results of single-particle simulations of electron–water interactions, we determine the concentration of reactive species in a water specimen under ESEM conditions. We consider 12 species, which are produced in a Gaussian distribution, and annihilate according to a second-order reaction scheme. Self-diffusion along the concentration gradient is also modelled.
We find that the dominant reactive species is the hydroxyl (.OH) radical. Annihilation of this species is suppressed due to the lower concentration of reactants. The relatively stable hydrogen peroxide is also found at large concentrations. By comparing two beam energies, 5 and 25 keV, we find a drastic increase in the quantities of reactive species produced with beam energy. The longer range of 25 keV primary electrons spreads reactive species over a wider region, which then decay far more slowly.     

Systematic inquiry in tests of negative/positive replica combinations for SEM

High resolution replica materials are routinely used in scanning electron microscopy. A systematic evaluating procedure for replica combinations is proposed which details fourteen points to be recorded. These points include quantitative and qualitative information useful when assessing a replica combination for a particular research problem. A case study employing one silicone-based impression material and one epoxy resin is performed as an example of the procedure.     

Use of the environmental scanning electron microscope for the observation of the swelling behaviour of cellulosic fibres

We have developed a method for observing transverse swelling of cellulosic fibres in the environmental scanning electron microscope (ESEM). The presence of liquid water in the ESEM specimen chamber allows the observation of in situ hydration without the need for coating, freezing, or drying of the sample. For reproducibility of the hydration and dehydration process, specialised mounting techniques are required and control of the conditions for condensation and evaporation of liquid water is necessary. The sensitivity of these cellulosic materials to the electron beam was investigated, showing that some damage mechanisms are enhanced by the continual presence of water vapour in the chamber. A discussion is presented of the effect of various experimental parameters on the extent and time of onset of the damage, and we outline steps to maximise the amount of useful experimental time for these fibres.