The Influence of Specimen Preparation on Artefacts in Scanning Electron Microscopy of Respiratory Cilia

The effects of various handling procedures used in preparing specimens of human and pig respiratory mucosa for scanning electron microscopy (SEM) were studied. Using five different washing methods, the percentage area of mucosa covered with extracellular material varied from 1.5 to 53.1%. The best results were achieved when specimens were washed by gently inverting a sample 30 times in a container filled with physiological saline. Fixation and drying of the surface layer caused disorientation of cilia and made examination difficult. Mechanical damage caused loss of cilia and rupture of the cell membrane. For SEM of respiratory cilia it is important to wash the specimen in saline before fixation and to use biopsy forceps as little as possible.     

Glutaraldehyde: Role in electron microscopy

In spite of the inherent limitations of chemical fixation, glutaraldehyde is unsurpassed in its ability to preserve cell ultrastructure. This achievement is due to the introduction of irreversible intra-and intermolecular cross-links into cellular proteins by the dialdehyde. Glutaraldehyde is very effective in stabilizing surface as well as intracellular structures for conventional scanning and transmission electron microscopy and high voltage electron microscopy. Even in immunocytochemical and autoradiographical studies, glutaradehyde plays a dominant role. Furthermore, prior to freeze-substitution, freeze-drying and freeze-fracturing, specimens often are stabilized with this dialdehyde. Glutaraldehyde efficiency can be increased by adding appropriate cross-linking and rapidly penetrating reagents as well as contrast enhancing reagents to this dialdehyde. Improved preservation and staining, for example, of ionic sites, soluble inorganic phosphate, lipids, biogenic amines, actin filaments, spermatozoa and phage-infected bacteria can be accomplished by adding polyethyleneimine, lead acetate, malachite green, potassium dichromate, tannic acid, trinitro compounds and uranyl acetate, respectively, to glutaraldehyde. Other refinements include the use of low concentrations of glutaraldehyde, short durations of cross-linking, minimum radiation exposure, and low temperature electron microscopy. The usefulness of glutaraldehyde in high resolution electron microscopy is limited because chemical fixation inevitably causes chemical and structural alterations in the specimen. However, fixation with glutaraldehyde or its mixture with formaldehyde has served immeasurably the progress in the understanding of cell ultrastructure and function. Preservation of specimens with glutaraldehyde for electron microscopy is expected to continue. Therefore, attempts must continue to be made to interpret the dynamics of the living cell from the static electron micrographs.     

Scanning electron microscopy of hydrated and desiccated mature somatic embryos and zygotic embryos of white spruce (Picea glauca [Moench] Voss.)

Summary Four scanning electron microscope techniques for preparing somatic and zygotic embryos of white spruce (Picea glauca [Moench] Voss.) were compared. Direct sputter coating without critical point drying worked well for desiccated embryos while conventional methods using chemical fixation were appropriate for hydrated somatic embryos. Low temperature scanning electron microscopy and plastic replicas provided excellent specimens of all embryos studied. Plastic replicas were used to document cotyledon formation and growth during maturation of somatic embryos. Apart from some differences in embryo size, orientation of cotyledons and surface wrinkling, the general morphology of mature somatic embryos of white spruce was very similar to zygotic embyros at a similar stage of development.     

A protocol for isolating Xenopus oocyte nuclear envelope for visualization and characterization by scanning electron microscopy (SEM) or transmission electron microscopy (TEM)

This protocol details methods for the isolation of oocyte nuclear envelopes (NEs) from the African clawed toad Xenopus laevis, immunogold labeling of component proteins and subsequent visualization by field-emission scanning electron microscopy (FESEM) and transmission electron microscopy (TEM). This procedure involves the initial removal of the ovaries from mature female X. laevis, the dissection of individual oocytes, then the manual isolation of the giant nucleus and subsequent preparation for high-resolution visualization. Unlike light microscopy, and its derivative technologies, electron microscopy enables 3-5 nm resolution of nuclear structures, thereby giving unrivalled opportunities for investigation and immunological characterization in situ of nuclear structures and their structural associations. There are a number of stages where samples can be stored, although we recommend that this protocol take no longer than 2 d. Samples processed for FESEM can be stored for weeks under vacuum, allowing considerable time for image acquisition.     

Use of Low Temperature Scanning Electron Microscopy to Observe Frozen Hydrated Specimens of Nematodes

Frozen hydrated specimens of Pratylenchus agilis and dauer larvae of Steinernema carpocapsae were observed with low-temperature field emission scanning electron microscopy. This new technique provides information about the surface features of nematodes and also allows specimens to be fractured to reveal their internal structure. Furthermore, both halves of fractured specimens can be retained, examined, and photographed either as two-dimensional micrographs or as three-dimensional images for stereo observation (stereology) or quantitative measurements (stereometry). This technique avoids artifacts normally associated with procedures required to prepare nematodes for examination in the transmission and scanning electron microscopes, such as chemical fixation, dehydration, and sectioning or critical point drying.     

A Standardized Method for the Analysis of Liver Sinusoidal Endothelial Cells and Their Fenestrations by Scanning Electron Microscopy

Liver sinusoidal endothelial cells are the gateway to the liver, their transcellular fenestrations allow the unimpeded transfer of small and dissolved substances from the blood into the liver parenchyma for metabolism and processing. Fenestrations are dynamic structures - both their size and/or number can be altered in response to various physiological states, drugs, and disease, making them an important target for modulation. An understanding of how LSEC morphology is influenced by various disease, toxic, and physiological states and how these changes impact on liver function requires accurate measurement of the size and number of fenestrations. In this paper, we describe scanning electron microscopy fixation and processing techniques used in our laboratory to ensure reproducible specimen preparation and accurate interpretation. The methods include perfusion fixation, secondary fixation and dehydration, preparation for the scanning electron microscope and analysis. Finally, we provide a step by step method for standardized image analysis which will benefit all researchers in the field.     

Morphological studies on the nutlet of Turkish Cyclotrichium Manden. & Scheng. (Lamiaceae)

Morphological characters of the nutlets of Cyclotrichium Manden. & Scheng. in Turkey were examined using scanning electron microscopy (SEM). The genus Cyclotrichium is represented by six species in Turkey, of which two are endemic for Turkey. Nutlet morphology of the species was studied using stereoscopic microscopy and SEM, and the features were described and illustrated. Nutlet morphology of the examined specimens exhibits some variation in size, shape and sculpture. Nutlets range from 1.1 to 2.0 mm in length and 0.5 to 1.0 mm in width and are elliptic, oblong to ovoid and trigonous in shape. Nutlet sculpture can be divided into two main types: distinctly or weakly reticulate pattern in C. niveum, C. origanifolium, C. longiflorum and rounded cell arrangement in the remaining species. Micromorphological characters could be useful in solving taxonomic problems of Cyclotrichium.     

Use of ionic liquid in fungal taxonomic study of ultrastructure of basidiospore ornamentation

An ionic liquid (IL) is a kind of salt that stays in a molten state even at room temperature. Since ILs do not vaporize even under vacuum conditions and show high ionic conductivity, they can be used in scanning electron microscopy (SEM) studies. The ultrastructural features of basidiospore ornamentation are considered to be important in the delimitation of taxa for fungi. In the present study, we carried out SEM observations on basidiospores that were subjected to an IL treatment, and evaluated the usefulness of this method in comparison with a conventional preparation method in which dehydrating, drying and platinum (Pt) coating were used. Using the conventional method, a considerable number of basidiospores was lost from the gill tissues; however, using the IL method, the decrease in basidiospores was extremely small. No significant differences in ultrastructural morphology or basidiospore size were found between Pt-coated basidiospores and IL-treated ones. SEM images of Pt-coated basidiospores tended to have higher contrast than those of IL-treated ones. Charging effects were observed with Pt-coated basidiospores, especially at the tips of the ornaments, whereas no such effects occurred for the IL-treated ones. In addition, small crinkles were observed in the Pt-coated basidiospores, but not in the IL-treated ones. These results suggest that the IL method is useful for fungal taxonomic studies.     

A preliminary study of human pineal gland concretions: structural and chemical analysis

Acervuli and fragments of pineal gland obtained from 33 subjects of both sexes and age ranging from 1 to 87 years, (30 autopsy and 3 biopsy specimens) were analyzed by light microscopy, transmission and scanning electron microscopy, X-ray diffraction and X-ray energy dispersive microanalysis. It was found that primary mineralization occurs in an organic matrix formed by pinealocytes and that hydroxyapatite also takes place in mineral deposition. From our analysis, the formation of acervuli appears to be age and sex independent and can be possibly related to the secretory activity of the gland.     

Transmission and scanning electron microscope preparations of the same cell culture.

A technique has been developed which allows transmission electron microscopy and scanning electron microscopy to be performed on the same cell culture sample. The technique uses the Costar 3,393 Leighton Tube containing a plastic insert, which does not stick to epoxy, for transmission electron microscopy. A cut piece of the plastic insert can be critical point dried, sputter coated and viewed under high vacuum with the scanning electron microscope.     

A Study of Criteria Permitting the Use of Plastinated Specimens for Light and Electron Microscopy

Plastination permits the preservation of anatomical specimens in a physical state approaching that of the living condition. We studied the possibility of using silicone plastinated fragments of spleen and pancreas for optical and electron microscopy, and found that with an adequate fixation protocol, plastinated specimens can be used for both light microscopy and ultra-structural studies. Deplastination with sodium methoxide permitted production of clean sections. Artifacts produced by plastination/deplastination could be nearly eliminated by glutaraldehyde/formaldehyde fixation. The (Biodur) silicone S10 polymer is transparent and stable in an electron beam, and plastinated tissues can be contrasted or colored similar to tissues embedded in Epon 812. In addition to being very life-like, plastinated tissues are stable and easy to handle. They can also be used for electron and light microscopic studies. This technique may also allow retrospective epidemiological studies of archived pathology specimens.     

A New Drying Method of Plant Specimens for Scanning Electron Microscopy the t-Butyl Alcohol Freeze-Drying Method

The t-butyl alcohol freeze-drying method (lnoue and Osatake 1988) is a simple drying method of biological materials for scanning electron microscopy Fixed specimens were immersed in t-butyl alcohol after dehydration throgh a graded series of ethanol. Specimens in the alcohol were then forzen in a refrigerator. They were placed in the bell jar of a vacuum evaporator and simply evacuated with a rotary pump. The samples were completely dried within 40–60 min after the frozen alcohol was sublimated in the vacuum, when the specimen was examined by scanning electron microscopy (SEM), both surface and intracellular structures were demonstrated in three-dimension without any significant drying artifacts. Careful comparison of the results indicated that the SEM imayes obtained by this method were either superior or equal to those obtained by the critical point drying method.     

Fluid transport and dimensions of epithelial cells and intercellular spaces in frog gallbladder

Summary Morphologic findings of widely dilated intercellular spaces in fluid transporting epithelia have been claimed as evidence for the existence of an epithelial compartment in which the coupling between solute and water fluxes takes place. The validity of using epithelial geometry in sectioned material as an argument can be questioned. The present report describes the morphological appearance of frog gallbladder epithelium — normal and ouabain-treated — in the living state in vitro and after fixation, dehydration and embedding. Gallbladder segments were photographed in the living state and at the end of each step of the preparative procedure. Direct observations of whole-mounted gallbladder segments were carried out, taking advantage of the possibility of optical sectioning and high resolution by Nomarski-microscopy. The same specimens were then sectioned and examined by conventional light and electron microscopy. The observations were quantitated and showed that the epithelial cells of normal and ouabain-treated gallbladders experienced an average linear shrinkage down to 70% of their length in Ringer's solution, which corresponds to a volume shrinkage down to 35%. Moreover, dilated lateral intercellular spaces appeared during the dehydration and embedding procedure in normal but only very moderately or not at all in ouabain-treated gallbladder specimens.     

Transmission And Scanning Electron Microscope Preparations Of The Same Cell Culture

A technique has been developed which allows transmission electron microscopy and scanning electron microscopy to be performed on the same cell culture sample. The technique uses the Costar 3393 Leighton Tube containing a plastic insert, which does not stick to epoxy, for transmission electron microscopy. A cut piece of the plastic insert can be critical point dried, sputter coated and viewed under high vacuum with the scanning electron microscope.     

Correlative Light and Scanning Electron Microscopy for Observing the Three-Dimensional Ultrastructure of Membranous Cell Organelles in Relation to Their Molecular Components

Although the osmium maceration method has been used to observe three-dimensional (3D) structures of membranous cell organelles with scanning electron microscopy (SEM), the use of osmium tetroxide for membrane fixation and the removal of cytosolic soluble proteins largely impairs the antigenicity of molecules in the specimens. In the present study, we developed a novel method to combine cryosectioning with the maceration method for correlative immunocytochemical analysis. We first immunocytochemically stained a semi-thin cryosection cut from a pituitary tissue block with a cryo-ultramicrotome, according to the Tokuyasu method, before preparing an osmium-macerated specimen from the remaining tissue block. Correlative microscopy was performed by observing the same area between the immunostained section and the adjacent face of the tissue block. Using this correlative method, we could accurately identify the gonadotropes of pituitary glands in various experimental conditions with SEM. At 4 weeks after castration, dilated cisternae of rough endoplasmic reticulum (RER) were distributed throughout the cytoplasm. On the other hand, an extremely dilated cisterna of the RER occupied the large region of the cytoplasm at 12 weeks after castration. This novel method has the potential to analyze the relationship between the distribution of functional molecules and the 3D ultrastructure in different composite tissues.     

The optical near-field and cell biology

A new form of scanning light microscopy is described in which the lens is replaced by a point of light that is smaller than the wavelength. Resolution is obtained that is defined not by the wavelength but by the size of the spot of light. This is the case so long as the point of light is within the dimension of a wavelength from the surface that is to imaged or within the optical near-field. This new form of light microscopy is called near-field scanning optical microscopy (NSOM). Resolutions are being obtained with NSOM that are similar to scanning electron microscopy but without the destructive effects of a vacuum or of an electron beam. In addition such a microscope is readily interfaced with fluorescent and non-fluorescent contrast enhancing stains that are commonly used in cell biology. The possibility of a near-field/far-field microscope is discussed with overlapping resolutions from a few hundred of a conventional microscope to the tens of thousand that can be obtained with NSOM.     

Utilization of hydrogen,acetate, and “noncompetitive”; substrates by methanogenic bacteria in marine sediments

We have used a relatively new microscopical technique, environmental scanning electron microscopy (ESEM), along with transmission electron microscopy (TEM) and light microscopy, to investigate a unique microbial community from a temperate-climate, cold sulfide spring near Ancaster, Ontario, Canada. ESEM allows the viewing of fully hydrated specimens that have not undergone the structural or chemical alterations imposed by the extensive procedures necessary for viewing biological specimens in a vacuum. Besides allowing visualization of microorganisms in their natural form and as intact assemblages, ESEM also detects elements, especially those lighter than Si, which tend to be lost or masked by the processes used to prepare samples for conventional SEM and for TEM thin sections. In this study we report new information about the structure of bacteriogenic sulfur deposits and their relationship to the structural aspects of a natural microbial community from a cold sulfide spring.     

Insect morphology in the age of phylogenomics: innovative techniques and its future role in systematics

A brief account of the history of insect morphology is given. Different techniques and analytical methods used in current projects on insect morphology and phylogeny and their optimized combined application are described. These include fixation, dissection, maceration, histology (microtome sectioning), scanning electron microscopy (SEM), transmission electron microscopy (TEM), serial block‐face scanning electron microscopy (SBFSEM), focused ion beam scanning electron microscopy (FIB/SEM), confocal laser scanning microscopy (CLSM), bleaching, micro‐computed tomography (μCT), computer‐based three‐dimensional reconstruction, focus stacking of digital images, geometric morphometrics and the storage of morphological metadata. The role of insect morphology in the “age of phylogenomics” is discussed.     

Silver-enhanced colloidal gold probes as markers for scanning electron microscopy

Silver enlargement of small colloidal gold particles has been extensively used for the light microscopical visualization of gold probes. Very recently, a few investigators have employed physical developers in electron microscopy (both pre-embedding and on-grid staining methods). We now demonstrate that physical development of small colloidal gold particles advantageously can be exploited for labelling biological surfaces in scanning electron microscopy. This novel application of silver enhancement of colloidal gold particles is characterized by a high detection efficiency. Thus, specimens are labelled with small gold probes affording high immunocytochemical efficiency but being impossible to detect with the present scanning microscopes. These particles are subsequently scanning electronmicroscopically visualized by silver enhancement.     

Electrical performance of low cost cathodes prepared by plasma sputtering deposition in microbial fuel cells

Microbial fuel cells (MFCs) could potentially be utilized for a variety of applications in the future from biosensors to wastewater treatment. However, the amount of costly platinum (Pt) used as a catalyst should be minimized via innovative deposition methods such as sputtering. In addition, alternative and low-cost catalysts, such as cobalt (Co), should be sought. In this study, ultra low Pt or Co cathodes (0.1 mg cm(-2)) were manufactured by plasma sputtering deposition and scanning electron micrographs revealed nano-clusters of metal catalyst in a porous structure favorable to the three-phase heterogeneous catalytic reaction. When operated in single-chamber air-cathode MFCs, sputtered-Co cathodes generated on average the same power as sputtered-Pt cathodes (0.27 mW cell(-1)) and only 27% less than conventional Pt-ink cathodes with a catalyst load 5 times higher (0.5 mg cm(-2)). Finally, microscopy and molecular analyses showed evidence of biocatalysis activity on metal-free cathodes.