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.     

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.     

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.     

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.     

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.     

Use of membrane filters and osmium tetroxide etching in the preparation of sperm for scanning electron microscopy

A new method was developed which is suitable for the preparation of mammalian sperm for scanning electron microscopy under either laboratory or field conditions. Samples of ejaculates from humans, two ferret species, and epididymal sperm from the African elephant were diluted in Millonig phosphate buffer and then fixed in glutaraldehyde solution. A small sample of the fixed sperm suspension was diluted in the same buffer, withdrawn with a syringe, and injected very slowly onto either a cellulose acetate or a polycarbonate membrane filter. This step was essential to concentrate the dilute sperm samples. During the various dilution steps most of the granular prostatic secretions were lost. However, a protein-like sheath, which remained attached to most sperm, obscured the surface features and had to be removed for SEM studies. It was removed by prolonged fixation/etching in 1% osmium tetroxide. Membrane filters containing sperm on their surfaces then were dehydrated, dried by the critical point drying method, and sputter coated with gold. Polycarbonate filters were superior to cellulose acetate filters in producing a flat and homogeneous background.     

Electron dense granules and the role of buffers: artefacts from fixation with glutaraldehyde and osmium tetroxide

Electron dense granules may appear in tissues after glutaraldehyde prefixation and osmium tetroxide postfixation. In order to determine the conditions under which the granules are formed various vehicles in pre- and post-fixatives were tested on lymph node, thymus and heart. If granules appeared they were found in all cell types investigated, particularly in the nuclei. There was no difference in the distribution of the granules in the different compartments of these organs. The granules probably consist of complexes of glutaraldehyde, osmium and phosphate. The ultimate phosphate concentration in the tissues during the postfixation was shown to determine whether or not the artefacts appeared. Local conditions in the tissues also contributed to the appearance of the granules. It is concluded that phosphate buffers can be used in the double fixation procedure, but to avoid the granules in lymph node, thymus and heart, a concentration of 0·1 mol/l or less phosphate should be used. For brain and kidney other conditions apply.     

Dimensional stability in tissues dehydrated with 2,2-dimethoxypropane for electron microscopy

Dimensions of tissues fixed in glutaraldehyde-osmium tetroxide mixture, osmium tetroxide and uranyl acetate and then dehydrated in 2,2-dimethoxypropane (DMP) were measured using transmission and scanning electron microscopy. Rat cardiac muscle, kidney and other tissues were examined in this study. The mean dimensions of characteristic ultrastructural features of material prepared by this method are similar or larger than those reported in the literature for conventionally processed samples. Critical point drying of specimens dehydrated with DMP does not produce abnormal shrinkage. Simultaneous primary fixation of lipids and proteins in a glutaraldehyde osmium tetroxide mixture and omission of the water wash after uranyl acetate appear to be important in stabilizing the tissue for rapid dehydration. The rapid reaction of DMP and water yielding the products acetone and methanol does not appear to denature tissue components to a greater extent than conventional solvent exchange dehydration.     

Methods for electron microscopy of the lamellated osmiophilic bodies of the lung

A study has been made of methods of fixing, staining, and embedding for electron microscopy the lamellated osmiophilic bodies (LOPBs) of the type II cells of the lung, which are probably connected with the lung surfactant. The preferred method for fixing and staining uses successively 2% glutaraldehyde, 1% osmium tetroxide, and a mixture of 10 vol M/40 lead nitrate with 10.5 vol M/60 potassium ferricyanide: all reagents are in cacodylate buffer. Unless the ferri-cyanide is in excess, the sections show blotchy precipitation. The bodies are damaged by ethanol and 1:2 epoxypropane. Dioxan or (with a quick time schedule) acetone may be used as a dehydrant and thinner prior to Araldite embedding. Durcupan water-soluble resin may also be used for dehydration and embedding; the preferred embedding mixture is: 5 ml resin A, 11.7 ml hardener B, 1.5 ml hardener C, and 0.4 ml plasticizer D, cured for 24–48 h at 60°C. Examination of LOPBs on a goniometer stage shows that they are composed of layers spaced at 4 nm, each lamella containing several such layers. Further evidence is adduced linking the LOPBs with the surfactant, and the application of the new methods to various problems involving the LOPBs is discussed.     

A simple protocol for paraffin-embedded myelin sheath staining with osmium tetroxide for light microscope observation

Experimental investigation of peripheral nerve fiber regeneration is attracting more and more attention among both basic and clinical researchers. Assessment of myelinated nerve fiber morphology is a pillar of peripheral nerve regeneration research. The gold standard for light microscopic imaging of myelinated nerve fibers is toluidine blue staining of resin-embedded semithin sections. However, many researchers are unaware that the dark staining of myelin sheaths typically produced by this procedure is due to osmium tetroxide postfixation and not due to toluidine blue. In this article, we describe a simple pre-embedding protocol for staining myelin sheaths in paraffin-embedded nerve specimens using osmium tetroxide. The method involves immersing the specimen in 2% osmium tetroxide for 2 h after paraformaldehyde fixation, followed by routine dehydration and paraffin embedding. Sections can then be observed directly under the microscope or counterstained using routine histological methods. Particularly good results were obtained with Masson's trichrome counterstain, which permits the imaging of connective structures in nerves that are not detectable in toluidine blue-stained resin sections. Finally, we describe a simple protocol for osmium etching of sections, which makes further immunohistochemical analysis possible on the same specimens. Taken together, our results suggest that the protocol described in this article is a valid alternative to the conventional resin embedding-based protocol: it is much cheaper, can be adopted by any histological laboratory, and allows immunohistochemical analysis to be conducted.     

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.     

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.     

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.     

Preparation of fetal rat brains for light and electron microscopy

To study cellular shapes, growth patterns, and fine structure during early stages of CNS development in rat embryos, preparative procedures were evaluated and modified to meet two criteria: (1) Coronal semithin sections should reveal undeformed telencephalic hemispheres that were symmetrically expanded on both sides of midline structures and were surrounded by contiguous mesenchyme. (2) In electron micrographs, cells should have intact, undistorted surface membranes, evenly distributed nucleoplasm and well preserved cytoplasmic organelles. To meet these criteria, 378 fetuses with a gestational age of 11–20 days (E11–E20) were used to test and modify procedures for anesthesia, embryo removal and handling, dissection, fixation, dehydration, and embedding of the embryonic CNS. Most specimens were in an early stage of development (E11–E13), which, in case of the neopallial wall, is the preneural period. The tests produced methods that met the above criteria and identified the most common artifacts and their causes. Deformities of the cerebral hemispheres and separations between the brain and its coverings were usually caused by trauma during embryo removal and during handling before fixation. Changes in cellular volumes, especially swelling during fixation and dehydration, were the most important causes of histological artifacts. The procedures and methods that consistently produced the best light and electron microscopic preservation of the E11–E13 rat CNS are described. Fixation was best when the brains were treated with glutaraldehyde and s-collidine buffer, followed by osmium tetroxide in s-collidine buffer. A surprisingly beneficial effect of sodium chloride in the dehydrating alcohol was noted.     

The fixation,dehydration, drying and coating of cultured cells for SEM

Cultivated cells form a valuable model system for studies on the effects of various preparative protocols for scanning electron microscopy (SEM). The various effects of each preparative step can be followed in detail in the light microscope and no diffusion gradients complicate the fixation and other procedures as in the case of solid tissues. Studies on cultivated cells indicate that the glutaraldehyde component of a glutaraldehyde-based fixative does not contribute to the effective osmotic pressure of the fixative and thus the osmolarity of the buffer, and other components, must be equalized to that of the medium in which the cells grow. Even small deviations from this ideal effective osmotic pressure will result in osmotically induced artefacts. Disturbances of pH and temperature of the cultures prior to and during fixation will result in changes in the appearance of many cellular structures such as microspikes and ruffles. We find that osmium fixation is advisable in most instances for best possible membrane preservation and that even long periods of glutaraldehyde fixation do not compensate for osmium fixation. Dehydration always results in shrinkage. Freeze drying (FD) and critical point drying (CPD) also give rise to shrinkage, the former to a lesser degree than the latter. A gold-palladium alloy gives a less granular coating that does gold alone. When cultured cells are studied, a metal thickness of between 5 and 15 nm is usually sufficient to give rise to an adequate secondary electron production and to avoid charging even at accelerating voltages of 30–40 kV. Without treatment with OsO₄ a thicker metal coating is required.     

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.     

Periodic acid-Schiff-phosphotungstic acid as an electron histochemical method for ultrathin sections

Structures which are regarded as ‘PAS positive’ may be strongly and selectively stained for electron microscopy in ultrathin sections by using the following procedure. Tissues were fixed by buffered osmium tetroxide or by glutaraldehyde followed by postfixation in osmium tetroxide. After dehydration the fixed tissues were embedded in butyl methacrylate-methyl methacrylate (10:1 v/v). Other embedding media do not give successful results. Ultrathin sections were cut and mounted on formvar-coated gold grids. The sections were treated with 1% w/v periodic acid in aqueous alcohol (water-ethanol 1:1 v/v) for 10 min, washed in water for 2–3 min, then treated with Schiff reagent for 20 min. The Schiff reagent was made according to the method of de Tomasi and allowed to age for 3–5 h. The sections were then washed in two changes of water for 3–5 min each, dried in air, stained in 1% w/v aqueous phosphotungstic acid for 1.5 h then rinsed in water for 2–3 min.     

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.     

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.     

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.