A preparation method for observing intracellular structures by scanning electron microscopy

In order to observe intracellular structures by scanning electron microscopy, excess cytoplasmic matrix must be removed from the fractured surface of cells. Previously we reported an Osmium-DMSO-Osmium method devised for this purpose. This method is very effective in revealing intracellular structures, but requires osmium tetroxide for initial fixation with some consequent disadvantages. In the present study, a revised Osmium-DMSO-Osmium method is reported, in which an aldehyde mixture is used as the initial fixative instead of osmium tetroxide. As fixation is carried out by perfusion in this revised method, better preservation of fine structures is achieved than by the original method, especially in the central nervous tissue which tends to suffer from post-mortem degeneration. Moreover this method can be applied to cytochemical studies of intracellular structures with a scanning electron microscope (SEM). In this study, acid phosphatase of lysosomes is demonstrated in a coloured SEM micrograph.     

Comparison of different preparation methods of biological samples for FIB milling and SEM investigation

When a new approach in microscopy is introduced, broad interest is attracted only when the sample preparation procedure is elaborated and the results compared with the outcome of the existing methods. In the work presented here we tested different preparation procedures for focused ion beam (FIB) milling and scanning electron microscopy (SEM) of biological samples. The digestive gland epithelium of a terrestrial crustacean was prepared in a parallel for FIB/SEM and transmission electron microscope (TEM). All samples were aldehyde-fixed but followed by different further preparation steps. The results demonstrate that the FIB/SEM samples prepared for conventional scanning electron microscopy (dried) is suited for characterization of those intracellular morphological features, which have membranous/lamellar appearance and structures with composition of different density as the rest of the cell. The FIB/SEM of dried samples did not allow unambiguous recognition of cellular organelles. However, cellular organelles can be recognized by FIB/SEM when samples are embedded in plastic as for TEM and imaged by backscattered electrons. The best results in terms of topographical contrast on FIB milled dried samples were obtained when samples were aldehyde-fixed and conductively stained with the OTOTO method (osmium tetroxide/thiocarbohydrazide/osmium tetroxide/thiocarbohydrazide/osmium tetroxide). In the work presented here we provide evidence that FIB/SEM enables both, detailed recognition of cell ultrastructure, when samples are plastic embedded as for TEM or investigation of sample surface morphology and subcellular composition, when samples are dried as for conventional SEM.     

Comparing different methods to fix and to dehydrate cells on alginate hydrogel scaffolds using scanning electron microscopy

Scanning electron microscopy (SEM) is commonly used in the analysis of scaffolds morphology, as well as cell attachment, morphology and spreading on to the scaffolds. However, so far a specific methodology to prepare the alginate hydrogel (AH) scaffolds for SEM analysis has not been evaluated. This study compared different methods to fix/dehydrate cells in AH scaffolds for SEM analysis. AH scaffolds were prepared and seeded with NIH/3T3 cell line; fixed with glutaraldehyde, osmium tetroxide, or the freeze drying method and analyzed by SEM. Results demonstrated that the freeze dried method interferes less with cell morphology and density, and preserves the scaffolds structure. The fixation with glutaraldehyde did not affect cells morphology and density; however, the scaffolds morphology was affected in some level. The fixation with osmium tetroxide interfered in the natural structure of cells and scaffold. In conclusion the freeze drying and glutaraldehyde are suitable methods for cell fixation in AH scaffold for SEM, although scaffolds structure seems to be affected by glutaraldehyde. Microsc. Res. Tech. 78:553–561, 2015. © 2015 Wiley Periodicals, Inc.     

Dilute aldehyde treatment on aldehyde-fixed cells for observing chromosomes in situ with the scanning electron microscope

A new preparation method is introduced to reveal intracellular structures in the scanning electron microscope and its application to mitotic cells in root meristems of Vicia faba is demonstrated. The root tips are fixed with a mixture of formaldehyde and glutaraldehyde and the fixed tissues are frozen and fractured in liquid nitrogen. They are then incubated successively in dilute solutions of aldehyde (formaldehyde or glutaraldehyde) and osmium tetroxide. By this treatment, the excess cell-matrix is removed from the fractured surface of the cell, and a deep view into the cell-interior can be obtained with the scanning electron microscope. Varied levels of substructure are observed on the surface of chromosomes.     

Ultrastructure of submandibular salivary glands of mouse: TEM and HRSEM observations

The fine structure of submandibular glands of mouse were analyzed using light microscopy (LM), high resolution scanning electron microscopy (HRSEM), and transmission electron microscopy (TEM) methods. For LM, the specimens were embedded in Spurr resin, stained by toluidin blue solutions. For TEM, the tissues of submandibular salivary glands were fixed with modified Karnovsky solution and postfixed with osmium tetroxide. For HRSEM, the tissues were fixed with 2% osmium tetroxide solution in 1/15M sodium phosphate buffer (pH 7.4). The samples were immersed successively in dymethylsulphoxide and freeze cracked. The maceration was made in diluted osmium tetroxide for 24-48 h. The samples were examined by high resolution scanning electron microscopy. The intracellular components of acinar and ductal cells revealed clearly the Golgi apparatus, rough endoplasmic reticulum, secretory granules, and mitochondria. The end bulbs of Golgi lamellae and flattened cisterns of rough endoplasmic reticulum showed the luminal surface. A few mitochondria were identified intermingling between the rough endoplasmic reticulum and the mitochondriales cristae in three-dimensional HRSEM images. Secretory granules were numerous and presented different sizes. Small granules of ribosomes were attached on cistern surface, measuring 20-25 nm in diameter. Numerous arranged microvilli were found on the luminal surface of secretory canaliculus. The contact surfaces of acinar cells revealed complicated interdigitations by cytoplasmic processes. The mitochondria of duct cells were disposed vertically and surrounded by basal infoldings of plasma membranes. Basement membrane showed a spongy-like structure having an irregular surface with various strands and meshes of fine collagen fibrils.     

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.     

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.     

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.     

A balanced technique for preparation of specimens from pathogenicity studies for scanning electron microscopy

This paper reports our experiences with preparing delicate biological specimens for scanning electron microscopy. Three different washing methods were evaluated: One method allowed the analysis of the location of the bacterium Mycoplasma mobile on piscine gill epithelium and the optimal evaluation of histopathologic changes caused by this microbe. These results were achieved when specimens were washed three times in a cacodylic acid buffer after completion of the in vitro infection experiment in gill explant cultures. We also found that of three different concentrations of glutaraldehyde, a fixation with a 1.5% solution was sufficient to achieve excellent structural preservation, even without using post fixation in osmium tetroxide. Furthermore, this study showed that the use of acetone-carbon dioxide in the critical point drying procedure resulted in well-preserved piscine gill epithelium and mycoplasmas. Finally, long-term storage of tissue specimens in 0.1 M cacodylic acid buffer is possible if the buffer is changed on a monthly basis to avoid growth of unwanted microorganisms, such as fungi.     

Backscattered electron image of osmium‐impregnated/macerated tissues as a novel technique for identifying the cis‐face of the Golgi apparatus by high‐resolution scanning electron microscopy

The osmium maceration method with scanning electron microscopy (SEM) enabled to demonstrate directly the three‐dimensional (3D) structure of membranous cell organelles. However, the polarity of the Golgi apparatus (that is, the cis–trans axis) can hardly be determined by SEM alone, because there is no appropriate immunocytochemical method for specific labelling of its cis‐ or trans‐faces. In the present study, we used the osmium impregnation method, which forms deposits of reduced osmium exclusively in the cis‐Golgi elements, for preparation of specimens for SEM. The newly developed procedure combining osmium impregnation with subsequent osmium maceration specifically visualised the cis‐elements of the Golgi apparatus, with osmium deposits that were clearly detected by backscattered electron‐mode SEM. Prolonged osmication by osmium impregnation (2% OsO₄ solution at 40°C for 40 h) and osmium maceration (0.1% OsO₄ solution at 20°C for 24 h) did not significantly impair the 3D ultrastructure of the membranous cell organelles, including the Golgi apparatus. This novel preparation method enabled us to determine the polarity of the Golgi apparatus with enough information about the surrounding 3D ultrastructure by SEM, and will contribute to our understanding of the global organisation of the entire Golgi apparatus in various differentiated cells.     

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.     

Crazes in osmium tetroxide stained styrene polymers

Crazes in polystyrene and rubber modified polystyrene became opaque in the electron microscope after films of the polymers are exposed to osmium tetroxide. The osmium tetroxide with the polystyrene in the crazes is shown to form a flexible insoluble material that is easily folded and twisted without breaking.     

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.     

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.     

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.     

Cryo-sectioning and chemical-fixing ultramicrotomy techniques for imaging rubber latex particle morphology

Two methods adapted from biological microscopy are described for a new application in imaging the morphology of rubbery latex particles. In the first method, a drop of latex is frozen in liquid nitrogen, sectioned with a diamond knife and vapour-stained with osmium tetroxide, then viewed by transmission electron microscopy. When applied to latexes made by emulsion polymerization of methyl methacrylate in a natural rubber latex seed, inclusions are clearly visible. A chemical fixation method is then described for imaging the morphology of such rubbery latex particles. Glutaraldehyde is added to the latex, followed by osmium tetroxide. The sample is then dehydrated in ethanol, epoxy resin added, and the sample cured, ultramicrotomed, and imaged with transmission electron microscopy. An inclusion morphology is again clearly seen.     

High-resolution scanning electron microscopy of immunogold-labelled cells by the use of thin plasma coating of osmium

The feasibility of plasma coating of a thin osmium layer for high‐resolution immuno‐scanning electron microscopy of cell surfaces was tested, using Drosophila embryonic motor neurones as a model system. The neuro‐muscular preparations were fixed with formaldehyde and labelled with a neurone‐specific antibody and 10 or 5 nm colloidal gold‐conjugated secondary antibodies. The specimens were post‐fixed with osmium tetroxide and freeze‐dried. Then they were coated with a 1–2 nm thick layer of osmium using a hollow cathode plasma coater. The thin and continuous coating of amorphous osmium gave good signals of gold particles and fine surface structures of neurites in backscattered electron images simultaneously. This method makes it possible to visualize the antigen distribution and the three‐dimensionally complex surface structures of cellular processes with a resolution of several nanometres.     

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.     

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.     

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.