Field-emission scanning electron microscopy of the internal cellular organization of fungi

Internal viewing of the cellular organization of hyphae by scanning electron microscopy is an alternative to observing sectioned fungal material with a transmission electron microscope. To study cytoplasmic organelles in the hyphal cells of fungi by SEM, colonies were chemically fixed with glutaraldehyde and osmium tetroxide and then immersed in dimethyl sulfoxide. Following this procedure, the colonies were frozen and fractured on a liquid nitrogen-precooled metal block. Next, the fractured samples were macerated in diluted osmium tetroxide to remove the cytoplasmic matrix and subsequently dehydrated by freeze substitution in methanol. After critical point drying, mounting, and sputter coating, fractured cells of several basidiomycetes were imaged with field-emission SEM. This procedure produced clear images of elongated and spherical mitochondria, the nucleus, intravacuolar structures, tubular- and plate-like endoplasmic reticulum, and different types of septal pore caps. This method is a powerful approach for studying the intracellular ultrastructure of fungi by SEM.     

Membrane blisters: A fixation artifact a study in fixation for scanning electron microscopy

It is been shown by scanning electron microscopy that fixation in glutaraldehyde followed by fixation in osmium tetroxide results in the presence of membrane blisters on the surface of a variety of cells. Fixation in glutaraldehyde alone or osmium tetroxide alone does not result in such extensive artifacts. The blisters, usually 0.2–0.6 μm in diameter, are seen by transmission electron microscopy to be membrane-bound, virtually empty vesicles. It is concluded that the optimum preservation of the cell surface for scanning electron microscopy is provided by fixation in glutaraldehyde alone.     

Candida albicans biofilms: comparative analysis of room‐temperature and cryofixation for scanning electron microscopy

Biofilms are frequently related to invasive fungal infections and are reported to be more resistant to antifungal drugs than planktonic cells. The structural complexity of the biofilm as well as the presence of a polymeric extracellular matrix (ECM) is thought to be associated with this resistant behavior. Scanning electron microscopy (SEM) after room temperature glutaraldehyde‐based fixation, have been used to study fungal biofilm structure and drug susceptibility but they usually fail to preserve the ECM and, therefore, are not an optimised methodology to understand the complexity of the fungal biofilm. Thus, in this work, we propose a comparative analysis of room‐temperature and cryofixation/freeze substitution of Candida albicans biofilms for SEM observation. Our experiments showed that room‐temperature fixative protocols using glutaraldehyde and osmium tetroxide prior to alcohol dehydration led to a complete extraction of the polymeric ECM of biofilms. ECM from fixative and alcohol solutions were recovered after all processing steps and these structures were characterised by biochemistry assays, transmission electron microscopy and mass spectrometry. Cryofixation techniques followed by freeze‐substitution lead to a great preservation of both ECM structure and C. albicans biofilm cells, allowing the visualisation of a more reliable biofilm structure. These findings reinforce that cryofixation should be the indicated method for SEM sample preparation to study fungal biofilms as it allows the visualisation of the EMC and the exploration of the biofilm structure to its fullest, as its structural/functional role in interaction with host cells, other pathogens and for drug resistance assays.     

Identification and study of the same cell in monolayer culture by different methods of light microscopy,scanning, and transmission electron microscopy. Application for cryodamaged cells examination

Cells were cultivated on transparent conductive substrates, glass slides coated with indium oxide; individual cells were marked with a diamond indentor. Cell cultures were frozen (–15°C), thawed, and then stained with fluorescent dyes to determine cell damage. The marked cells were examined by phase contrast, fluorescence, and Nomarski DIC microscopy. After aldehyde and osmium tetroxide fixation, the cell preparations were sequentially treated with tannic acid, uranyl acetate, and lead citrate. The same marked cell could be sequentially studied by light microscopy (LM; in water immersion conditions), scanning electron microscopy (SEM; after dehydration and critical point drying), and transmission electron microscopy (TEM; after embedding of cell samples in epoxy resin and laser marking of the cell previously marked with a diamond indentor). The method used ensures good preservation of cell morphology, cell surface relief, and intracellular structures. The treatment used renders the cells conductive and permitted SEM of uncoated culture cells on conductive substrates.     

Anhydrous fixation of dry plant tissue using non-aqueous fixatives

Non-aqueous preparation techniques such as freeze fracture of the dry tissue, fixation using dimethylsulphoxide as a vehicle for glutaraldehyde, and osmium vapour as a fixative have been used to investigate the fine structure of plant material in the dry state. Fixation using anhydrous glutaraldehyde dissolved in dimethylsulphoxide preserves existing cell structure and cell wall characteristics whereas conventional aqueous fixation methods hydrate the tissue during fixation and this gives a distorted impression of true cell structure.     

Simultaneous fixation using glutaraldehyde and osmium tetroxide or potassium ferricyanide-reduced osmium for the preservation of monogenean flatworms: an assessment for Merizocotyle icopae

Simultaneous fixation was investigated for a marine organism: the monogenean flatworm ectoparasite Merizocotyle icopae. Four protocols for primary fixation were compared: 3% glutaraldehyde alone in 0.1M cacodylate buffer for a minimum of 2 hours; 1% glutaraldehyde in combination with 1% osmium tetroxide, both in 0.1M cacodylate buffer, until tissues darkened (5-20 minutes); 1% glutaraldehyde in 0.1M cacodylate buffer in combination with 0.5% potassium ferricyanide-reduced osmium until tissues darkened (5-20 minutes); 1% glutaraldehyde in combination with 1% osmium tetroxide, both in 0.1M cacodylate buffer, for 30 minutes. The study confirms that the standard method for transmission electron microscopic fixation (first listed protocol) routinely applied to platyhelminths is optimal for ultrastructural preservation, but some simultaneous fixation methods (second and third listed protocols) are acceptable when rapid immobilization is needed. Scanning electron microscopic preparations may be improved using simultaneous primary fixation.     

Metabolic state dependent preservation of cells by fixatives for electron microscopy

The preservation of mitochondria, cytoplasmic vacuoles and cytoplasm by various fixatives after various pretreatments of ethothelial heart cells from Xenopus laevis tadpoles in tissue culture was investigated. The study was based on phase contrast cinemicrographic recordings and on qualitative and quantitative observations with the electron microscope. Three fixatives were used: 3% glutaraldehyde in phosphate buffer, followed by 1% osmium tetroxide postfixation, fixation only with 1% osmium tetroxide in phosphate buffer and the fixing medium according to Dalton. Cells were either not treated or pretreated for 20 min: 10 microM FCCP (Carbonylcyanide-p-trifluoromethoxy-phenylhydrazone) or 4 mM KCN. The superiority of glutaraldehyde was exemplified by its very rapid action, good preservation of cytoplasm, vacuoles, and mitochondria. It was the only medium which maintained an electron density of the mithochondria matrix. In both of the other fixatives swelling of mitochondria and coagulated appearance of cytoplasm (in phase contrast) was more pronounced in cells pretreated with metabolic inhibitors than in controls. Observations with the light microscope have been confirmed by morphometry of electron micrographs of mitochondria. The relation of matrix space to intracristal space is changed in opposite directions after glutaraldehyde and after the Dalton-type fixation. The results indicate a higher sensitivity against fixation artifacts in cells under pathological conditions than normal cells.     

An improved method for the scanning electron microscopy of spermatozoa

The authors carried out this research on an SEM technique and obtained a good preservation of spermatozoa. Among the many fixatires tested, potassium di-chromate mixed with osmium tetroxide and NaCl gave the best results. Generally, a rapid freeze drying of the fixed material, after a treatment with dimethylsul-phoxide, isopentane and liquid nitrogen improves the general standard of preservation.     

The effects of osmium tetroxide post‐fixation and drying steps on leafy liverwort ultrastructure study by scanning electron microscopy

Leafy liverwort is one of the most abundant and diverse plants in Indonesia. Their high variation and beneficial secondary metabolites contained in the oil bodies have attracted researchers' attention. The ultrastructural analysis of leafy liverworts is important as a means of species identification and also for further exploration of their oil bodies. However, the optimization of the preparation steps for observing leafy liverworts by SEM is necessary to avoid sample destruction. Fixation and drying play important roles in maintaining a sample's structure as close to its natural state as possible. Thus, in this study, we evaluated the effect of 4% Osmium tetroxide (OsO₄) and drying on leafy liverworts ultrastructure. Microlejeunea, Acrolejeunea, and Frullania were fixed with 2.5% glutaraldehyde. Some samples were then post‐fixed with 4% OsO₄, while the rest were directly dehydrated with an ethanol series and then subjected to different drying methods, i.e. air drying, freeze drying, and drying with hexamethyldisilazane (HMDS). According to the data obtained, post‐fixation with 4% OsO₄ could better maintain the integrity of the samples and enhance the contrast of leafy liverwort SEM images. In addition, samples dried with HMDS showed more detailed structures compared to those that were air dried. Different ultrastructure were found among the different leafy liverworts observed by SEM. Our data suggested the advantages of SEM in providing ultrastructure information on leafy liverworts as well as the optimum conditions to observe them with less deformation. OsO₄ post‐fixation could enhance the contrast of leafy liverwort SEM images and maintain the structure of the samples. Drying with HMDS provided a convenient way for rapid SEM preparation with less structural distortion.     

Ultrastructure of the echinoderm cuticle after fast-freezing/freeze substitution and conventional chemical fixations

The cuticles of the pedicellaria primordia in the sea urchin Paracentrotus lividus and of the tube foot disk in the sea star Asterias rubens were preserved by different methods, viz., glutaraldehyde fixation followed by osmium tetroxide postfixation, glutaraldehyde-ruthenium red fixation followed by osmium tetroxide-ruthenium red postfixation, and two fast freezing / freeze substitution methods (FF/FS). The gross ultrastructure of the cuticle as well as the influence of the preservation method on this ultrastructure were identical for the two tissues studied. The cuticle ultrastructure was poorly preserved after glutaraldehyde fixation / osmium tetroxide postfixation. Its preservation was improved after ruthenium red was added in the fixative and postfixative, but the best preservation was consistently achieved using FF/FS. Both low-pressure freezing (plunge freezing) and high-pressure freezing were tested, the latter giving seemingly better results. With these methods, the cuticle appeared to be composed of a proximal lower cuticle, an intermediate upper cuticle, and a distal fuzzy coat. In particular, cryoimmobilization methods emphasized or revealed the occurrence of a well-developed fibrillar lower cuticle in the pedicellaria, the complexity of the upper cuticle which consisted of several zones, and the importance of the usually poorly preserved fuzzy coat that is actually the thickest layer of the cuticle. These observations bring new insights on the functions of the cuticle, and particularly of the fuzzy coat. According to its preservation characteristics, the fuzzy coat presumably consists mostly of proteoglycans. This composition could give it shock absorption and antifouling properties. Furthermore, its important thickness also implies that molecules detected by the short sensory cilia must diffuse through and could be selected by the fuzzy coat.     

Air-drying of human leucocytes for scanning electron microscopy using the GTGO procedure

The utilization of tannic acid and guanidine hydrochloride as mordants for better osmium binding has been shown to serve as an excellent alternative to metal coating of organ tissue specimens for scanning electron microscopy (SEM). The present report describes the GTGO procedure, a modification of the TAO technique introduced by Murakami et al. (1977, 1978), which we have found successful for the preparation of air dried peripheral blood leucocytes for SEM studies. Air dried, GTGO-treated leucocytes show excellent preservation of surface features with minimal cell shrinkage. When critical point dried, GTGO-treated cells are examined, they also show less shrinkage than cells prepared with standard glutaraldehyde fixation and critical point drying. The potential application of this air drying procedure (GTGO-AD) to other soft biological specimens is currently under investigation. This technique is recommended as a new and effective air drying procedure for the successful preparation of cells for SEM.     

Effects of glutaraldehyde or osmium tetroxide fixation on the osmotic properties of lung cells

The osmotic properties of lung cells have been tested before and after perfusion fixation of isolated, perfused lungs with either glutaraldehyde or osmium tetroxide. The testing procedure was to add hypertonic sucrose to the perfusate for several minutes and monitor the lung weight response (an ‘osmotic transient’). Each lung was perfused with one or the other fixative solutions for 10 min, then the perfusate was changed back to Ringer-lactate before the post-fixation test was conducted. The results indicate that osmium tetroxide makes the cell membranes as permeable to sucrose as to water, and that sucrose thus causes no osmotic volume change. Glutaraldehyde, on the other hand, apparently preserves the impermeability of the cell membranes to sucrose, but the osmotic volume response is attenuated, indicating that significant changes in the cells have occurred.     

A technique for processing mucous coated marine invertebrate spermatozoa for scanning electron microscopy

A technique for preparing heavily mucous coated marine invertebrate spermatozoa for scanning electron microscopy (SEM) is described. This technique involves washing in 1500 NF units/ml hyaluronidase in millipored sea water to remove mucus, followed by fixation in glutaraldehyde and osmium tetroxide. Following primary fixation, spermatozoa are enclosed in Nuclepore membrane bags positioned within Teflon specimen capsules allowing them to be processed and critical point dried without excessive mechanical damage or loss.     

Extraction of membrane lipids during fixation,dehydration and embedding of Acholeplasma laidlawii-cells for electron microscopy

The aim of the present investigation was to study the extent to which lipids are extracted from biological membranes during dehydration and embedding procedures carried out at high or low temperatures. Cells of Acholeplasma laidlawii were used as experimental material, since the lipids of this bacterium easily can be radioactively labelled without labelling the rest of the cell, and the lipids are almost entirely located in the cytoplasmic membrane. The cells were fixed at 277 K with glutaraldehyde, sequentially with this reagent and osmium tetroxide, or with glutaraldehyde, osmium tetroxide and uranyl acetate in that order. Loss of lipid during these procedures was negligible. When cells fixed with glutaraldehyde and osmium tetroxide were dehydrated with ethanol at room temperature and embedded in Epon at 333 K, i.e. subjected to a conventional treatment, about 90% of the lipid content of the cells was extracted. The loss was reduced to c. 20% when treatment with uranyl acetate was included in the procedure and the non-polar methacrylate resin Lowicryl HM20 was substituted for Epon. When cells fixed with glutaraldehyde and osmium tetroxide were dehydrated with ethanol at 238 K and embedded in Lowicryl HM20 at room temperature, practically no lipid was extracted. Substitution of the polar methacrylate-acrylate resin Lowicryl K4M for Lowicryl HM20 resulted in the loss of about half of the lipid content of the cells. The use of ethanediol as dehydrating agent instead of ethanol did not diminish the extraction. Cells fixed solely with glutaraldehyde lost about half of their lipid content, even when both dehydration and embedding was performed at 238 K. The lipid material extracted from glutaraldehyde-fixed cells contained slightly more saturated fatty acids than that remaining in the cells. The reverse was true for osmium tetroxide-fixed cells. With respect to lipid species, the extractions were generally rather unspecific.     

Changes of particle frequency in freeze-etched erythrocyte membranes after fixation

The frequency of particles on the membrane fracture faces of freeze-etched human erythrocytes was measured, and the effect of fixation procedures on the particle frequencies was studied. Fresh blood, buffer washed cells and cells fixed in one of the following ways were examined: glutaraldehyde, glutaraldehyde followed by osmium tetroxide, osmium tetroxide alone. Quantitative analyses showed that some treatments produced a significant reduction in the number of particles on the fracture faces as compared with the fresh cells. After both osmium tetroxide fixations, the loss of particles was greater from the outer fracture face (OFF) than the inner fracture face (IFF), whilst after the other treatments approximately the same number of particles were lost from both fracture faces. The results are discussed with respect to some current concepts of the molecular architecture of the erythrocyte membrane and the action of fixatives. The reduction of particle frequencies is thought to be due to both leaching of membrane proteins, and deviations of the usual fracture plane within the membrane. Glutaraldehyde alone was shown to have less effect on particle frequency than the other fixatives and it is therefore a suitable fixative for the preparation of freeze-etch specimens.     

Transmission and scanning electron microscopic examination of intracellular organelles in freeze-substituted Kloeckera and Saccharomyces cerevisiae Yeast Cells

The application of the conventional double-fixation method (glutaraldehyde and osmium tetroxide) to whole yeast cells is difficult because the thick cell wall of the yeast prevents the penetration of osmium tetroxide. However, this problem was solved by using the freeze-substitution fixation method. Therefore, it was possible to examine the intracellular structures of the yeast cells without digestion of the cell wall. In the present method, specimens for transmission electron microscopy and for scanning electron microscopy were prepared simultaneously. By scanning electron microscopic observation, three-dimensional information about internal structures was obtained. In the cytological analysis of the yeasts, intracellular structures were well preserved by using the freeze-substitution fixation method. On the outer leaflet of the nuclear envelope, many ribosomes were attached. The rough endoplasmic reticulum and Golgi apparatus were clearly seen in the yeast cytoplasm. The Golgi stack appeared to consist of smooth membranes, and small vesicles were present beside it. The details of other structures such as the nuclear division apparatus, actinlike filaments, and viruslike particles were also revealed. The present technique can be applied to most species of yeast cells. With this new information, the previous model of a yeast cell was modified.     

Quantitative studies on the preservation of choline and ethanolamine phosphatides during tissue preparation for electron microscopy. I. Glutaraldehyde, osmium tetroxide, Araldite methods

The loss of ¹⁴C ethanolamine- and ³H choline-labelled phospholipids from rat liver during tissue preparation for electron microscopy has been examined. Column and thin-layer chromatography combined with double-label scintillation spectrometry were used to analyse the radioactive phospholipid content of the livers of rats injected simultaneously with ¹⁴C aminoethanol and ³H choline chloride. After 4 h (in vivo) the ¹⁴C and ³H labels were mainly incorporated into phosphatidyl ethanolamine and phosphatidyl choline respectively but some ¹⁴C label had been incorporated into phosphatidyl choline. Chopped rat liver was fixed in glutaraldehyde or osmium tetroxide or both sequentially and tissues were dehydrated in ethanol and embedded in Araldite. In each procedure examined the choline label proved more labile than the ethanolamine. After glutaraldehyde fixation alone complete loss of phosphatidyl choline occurred and half of the phosphatidyl ethanolamine was also lost. Following osmium tetroxide fixation negligible loss of either phosphatide occurred. In terms of phospholipid retention, no advantage was gained by glutaraldehyde fixation prior to osmium tetroxide fixation. The results show that both ethanols and embedding monomers are potent phospholipid solvents. The data also suggests that EM autoradiography of these two phosphatides may be carried out with reasonable confidence although it must be pointed out that a high degree of retention does not necessarily imply retention in situ.     

Ice crystal damage in frozen thin sections: freezing effects and their restoration

Thin sections of unfixed kidney, fast frozen without cryoprotectants, were fixed in osmium tetroxide vapour directly after freeze drying or after 30 min in a moist atmosphere. Dry sections fixed in vapour showed ice crystal damage characteristic for the freezing procedure. This was demonstrated with freeze fracture replicas from the same preparation. Ice crystal holes were obscured in serial sections which were freeze dried and allowed to rehydrate in a moist atmosphere. The same ultrastructural appearance was observed in frozen sections brought to room temperature immediately after cutting. Frozen thin sections from unfixed tissue, if freeze dried, are very sensitive to atmospheric conditions and need some form of stabilization (e.g. osmium vapour fixation, sealing with an evaporated carbon film) before electron microscope images can be interpreted as representative for the frozen state. Restoration of ice crystal damage can occur by melting frozen sections or by rehydration of freeze dried frozen sections. Restoration phenomena will impair studies aimed at the localization of diffusible substances by autoradiography or X-ray microanalysis.     

Critical-point drying versus freeze drying for scanning electron microscopy: a quantitative and qualitative study on isolated hepatocytes

Critical-point drying and freeze drying were compared both quantitatively and qualitatively as preparative procedures for scanning electron microscopy. Isolated hepatocytes were used as model cells. Nomarski differential interference contrast microscopy was used for light microscopic measurements of the hepatocytes in the unfixed, the glutaraldehyde fixed, the glutaraldehyde + OsO₄ fixed, the critical-point dried and the freeze dried states. Critical-point dried hepatocytes were found to shrink to 38% of glutaraldehyde + OsO₄ fixed volume, whereas optimal freeze dried hepatocytes (frozen in water saturated with chloroform and freeze dried at 183 K for 84 h) were found to shrink to 51% of glutaraldehyde + OsO₄ fixed volume. Transmission and scanning electron micrographs of the critical-point dried cells showed well-preserved ultrastructure and surface structure. Micrographs of the freeze dried cells showed ultrastructure destroyed by internal ice crystals and surface structure destroyed by external ice crystals. Double-fixed isolated hepatocytes were shown to swell during storage in buffer and to shrink during storage after critical-point drying. For low magnification scanning electron microscopy (up to about 3000 times) both critical-point drying and freeze drying can be used. However, for high magnification scanning electron microscopy, critical-point drying is superior to freeze drying.     

Freeze-substitution and low-temperature embedding of dairy products for transmission electron microscopy

Dairy products are comprised largely of fat, air and water, which makes it difficult to preserve their ultrastructure for electron microscopy. Keeping the samples frozen throughout fixation and embedding protects the structure and distribution of the components of emulsions and foams. Therefore, dairy products were freeze‐substituted and embedded at low temperature (?20 °C) to prepare them for transmission electron microscopy. Whipped cream, ice cream mix and dairy/non‐dairy mixed systems were frozen by plunging in propane, at its boiling point (?187 °C). Ice cream, because it is already frozen, was fractured into 1‐mm³ pieces in liquid nitrogen and then added to frozen fixative (?196 °C). Fixative solution consisted of glutaraldehyde, osmium tetroxide and uranyl acetate dissolved in either methanol or acetone. When material was to be stained after sectioning the fixative was limited to glutaraldehyde in methanol. The temperature was increased step‐wise from ?80 to ?20 °C. Solvent was replaced with resin; the polar resin Lowicryl HM4, the non‐polar resin Lowicryl HM20, LR White and LR Gold were tested. Samples were embedded and polymerized at ?20 °C using ultraviolet light to cross‐link the resin. Methanol proved to be the most effective solvent for substituting the ice; the hydrophobic resin Lowicryl HM20 was the most effective resin for retaining fat structure following osmium fixation.