Chemical analysis of osmium tetroxide staining in adipose tissue using imaging ToF-SIMS

Osmium tetroxide (OsO₄) is a commonly used stain for unsaturated lipids in electron and optical microscopy of cells and tissues. In this work, the localization of osmium oxide and specific lipids was independently monitored in mouse adipose tissue by using time-of-flight secondary ion mass spectrometry with Bi cluster primary ions. Substance-specific ion images recorded after OsO₄ staining showed that unsaturated C18 fatty acids were colocalized with osmium oxide, corroborating the view that osmium tetroxide binds to unsaturated lipids. In contrast, saturated fatty acids (C14, C16 and C18) and also unsaturated C16 fatty acids show largely complementary localizations to osmium oxide. Furthermore, the distributions of saturated and unsaturated diglycerides are consistent with the specific binding of osmium oxide to unsaturated C18 fatty acids. The abundance of ions, characteristic of phospholipids and proteins, is strongly decreased as a result of the osmium staining, suggesting that a large fraction of these compounds are removed from the tissue during this step, while ions related to fatty acids, di- and triglycerides remain strong after osmium staining. Ethanol dehydration after osmium staining results in more homogeneous distributions of osmium oxide and unsaturated lipids. This work provides detailed insight into the specific binding of osmium oxide to different lipids.     

Lipid staining for the electron microscope: a new method.

Tissues fixed in osmium tetroxide or in combined osmium and glutaraldehyde (Hinde), embedded in Spurr's medium, cut at 0-5-I mum and mounted in Farrants' gum medium containing ethyl gallate, show good staining of lipid-contaning structures (droplets of triglyceride, membranes, mitochondria, etc.) in the light microscope. Such preparations show moderate contrast in the electron microscope without further staining. But a specific increase in contrast in lipid-rich structures is obtained by partition of the tissues, before embedding, in 70% ethanol saturated with the monoterpene hydrocarbon myrcene, with or without the addition of 0-I % ethyl gallate, followed by osmium tetroxide. This method will visualize both saturated and unsaturated lipids, including waxes.     

A comparison of standard lipid staining techniques used in electron microscopic studies of mammalian tissues

Comparisons of several standard techniques for staining lipids in ultrastructural studies have been undertaken using the rat uterine epithelium as the experimental tissue. The best technique for clarity, retention of stain, and acceptability of cellular ultrastructure utilized p-phenylenediamine after primary fixation in glutaraldehyde and postfixation in osmium tetroxide. While osmium by itself stained only unsaturated lipids and p-phenylene-diamine stained no lipids in spot tests, when acting together, the staining of unsaturated lipids was enhanced and some staining of saturated lipids was seen. Further, the marked extraction of stained lipids normally found during dehydration did not then occur.     

The distribution of lipid in the cell structure: An improved method for the electron microscope

An improved partition method for visualizing lipid consists in fixing tissues in paraformaldehyde-glutaraldehyde followed by osmium tetroxide. Three progressive grades of lipid staining are then obtained: (i) by renewed osmium tetroxide alone, (ii) by partition in myrcene or farnesol solutions followed by renewed osmium, (iii) by saturated thymol in sucrose followed by partition and renewed osmium. No additional metallic stains are used. The thymol treatment in (iii) renders ‘masked’ lipid accessible to partition—the effect being regulated as required by time and temperature. Thymol used before the first osmium facilitates lipid extraction which provides a complementary test for lipid. The possibilities of the method have been demonstrated on sections of familiar tissues of insect (mainly Rhodnius, Hemiptera) and mammal (mouse). By and large the results support what is known already about the distribution of lipid in cells, but observations on lipid in muscle fibres, in the nucleolus and chromatin, in the cells of the adrenal cortex, in the lung and intestine suggest that the method might prove a source of new information.     

Electron microscope studies of the human epidermis: the clear cell of Masson (dendritic cell or melanocyte)

The human epidermis has been studied by electron microscopy following osmium tetroxide and potassium permanganate fixation. An anatomically distinct cell in the human epidermis has been demonstrated with features similar to the melanocyte of the hair bulb described by Barnicot, Birbeck and Cuckow (3). It is dendritic in form and does not contain tonofilaments. "Intercellular bridges" are not formed. The mitochondria are larger and more numerous than those of other epidermal cells and the endoplasmic reticulum is more complex. Some of these cells contain melanin but others are melanin-free. The cell has been interpreted as being identical with the dopa-positive, clear cell of Masson (dendritic cell of Bloch or melanocyte). We have found that many membranous structures in the human epidermis are better preserved by permanganate fixation than by osmium tetroxide fixation.     

Ultrastructural studies of neutral lipid localisation inStreptomyces

Triacylglycerol is accumulated by Streptomyces spp. when grown in submerged culture. Ultrastructural studies using transmission electron microscopy (TEM), staining and freeze-fracture/freeze-etch procedures, and light microscopy confirmed the accumulation of neutral lipid by S. lividans and S. coelicolor during the stationary phase and its storage within membrane-bound globular structures within the cytoplasm. These structures were of various sizes and occupied up to approximately 80% of the total cell volume at that time. There was no evidence of such material within cells examined during the early exponential phase of growth. The globules visualised by TEM were electron-transparent since they comprised lipids containing saturated fatty acids that did not react with osmium tetroxide. The globules appeared to be bounded by a single membrane. Received: 6 June 1995 / Accepted: 4 September 1995     

Imidazole-buffered osmium tetroxide: an excellent stain for visualization of lipids in transmission electron microscopy

Summary The usefulness of imidazole-buffered osmium tetroxide as a stain for lipids in transmission electron microscopy has been investigated. Rat liver and other tissues were fixed by perfusion with glutaraldehyde and post-fixed with osmium-imidazole and the appearance of lipid droplets was compared with that after post-fixation in unbuffered aqueous osmium tetroxide or an osmium solution buffered otherwise. Prominent electron-opaque staining of lipid droplets and of lipoprotein particles was noted after post-fixation with 2% osmium-imidazole, pH 7.5, for 30 min. The lipid droplets appeared well circumscribed with no evidence of diffusion. In contrast, the intensity of staining was much less and there was some diffusion around lipid droplets in material post-fixed in aqueous or cacodylate-buffered osmium tetroxide. Spot tests on filter paper revealed that unsaturated fatty acids, especially linolenic and linoleic acids reacted more intensely with osmium-imidazole than with aqueous osmium tetroxide. These findings demonstrate that osmium-imidazole provides an excellent stain for lipids in transmission electron microscopy and that most probably it stains lipids with unsaturated fatty acids.     

Osmium dependent argentaffin staining of lysosomes

Summary Lysosomes stain with the argentaffin reaction after fixation with glutaraldehyde followed by osmium tetroxide. The reaction works well both at the level of the light and electron microscope. Control experiments show that this argentaffinity is caused by reduced osmium tetroxide. No staining could be observed in freeze-dried material, in tissues fixed only with glutaraldehyde, or after bleaching of the sections with hydrogen peroxide solutions. In the electron microscope, the population of lysosomes appears heterogeneous as related to the density of silver deposits over the organelles. No correlation is found between size and argentaffinity of lysosomes. X-ray microanalysis of sections from glutaraldehyde/osmium tetroxide fixed material reveals significantly higher amounts of osmium in lysosomes, as compared to other cell organelles (e.g. peroxisomes or mitochondria). A significant peak for silver is observed in lysosomes after treatment of the sections with ammoniacal silver solution, whereas the signal for osmium is reduced. Amounts of sulphur are too low to be detected in lysosomes. It is concluded that argentaffin staining of lysosomes is an osmium dependent reaction.Parts of these results have been presented as a poster during the 20th Congress of Electron Microscopy, joint session of the Austrian Society of Electron Microscopy and the German Society of Electron Microscopy, August 23–28, 1981, Innsbruck, Austria     

ENHANCEMENT OF IMMUNOGOLD-LABELLED FOCAL ADHESION SITES IN FIBROBLASTS CULTURED ON METAL SUBSTRATES: PROBLEMS AND SOLUTIONS

Visualisation of cell adhesion patterns by scanning electron microscopy requires special preparation and labelling. The membranes and cytoplasm must be removed, without damaging the antigen, to facilitate antibody access to vinculin in the focal adhesions. Low beam energy imaging is used to visualise the cell undersurface (embedded in resin after staining with osmium tetroxide) and immunogold-labelled adhesion sites. The gold probe, must be large enough (>40 nm) for detection, while viewing the whole cell, but large gold markers increase steric hindrance and decrease labelling efficiency. This problem can be overcome by using small gold probes (1-5 nm) followed by enlargement with silver enhancement, but osmium tetroxide stain etches the silver. We demonstrated that metal substrates increased this etching. Reducing the concentration of osmium tetroxide and incubation time reduced the amount of etching. We have demonstrated that gold enhancement was not etched by osmium tetroxide, irrespective of the substrate. Therefore, comparative studies of cell adhesion to different biomaterial substrates can be performed using immunogold-labelling with gold enhancement.     

Histochemistry of glycogen in the inner ear

Synopsis The effect of fixation and processing upon the morphological appearance of glycogen within the outer hair cells of the guinea-pig was investigated using two methods. In each method, tissue was fixed for 12 h in cold phosphate-buffered 4% paraformaldehyde and eventually dehydrated in ethanol, embedded in Epon 812, and cut into 4 m sections. In procedure A, after complete processing, the sections were tained using the periodic acid-Schiff reaction (PAS) or the periodic acid-thiocarbo-hydrazide-osmium tetroxide (PATCO) reaction which resulted in the appearance of listinct, coarse granules in the cytoplasm of the outer hair cells. Diastase digestion on one of the two matched sections after Epon removal and prior to staining, confirmed the granules to be glycogen. In procedure B, after primary fixation, the tissue was post-fixed in 1% osmium tetroxide and then processed exactly as in procedure A. Here, unless the Epon and osmium was remoyed, there was no staining of the outer hair cell cytoplasm. However, after Epon removal there was diffuse, grainy appearance of the outer hair cell cytoplasm which we considered to be due to glycogen although diastase confirmation was not possible. We have concluded that osmium tetroxide (1) inhibits PAS or PATCO staining, (2) prevents diastase digestion, and (3) prevents the appearance by light microscopy of distinct granules of glycogen.     

Osmium tetroxide/p-phenylenediamine staining of nucleoli and Balbiani Rings in Chironomus salivary glands.

This paper deals with the application of the osmium tetroxide fixation followed by p-phenylenediamine treatment to salivary gland cells from Chironomus larvae. After this procedure, cytoplasm, nucleoli and Balbiani rings show a high degree of staining both in light and electron microscopy, while chromatin remains unstained. Ethanol fixation followed by osmium tetroxide/p-phenylenediamine does not modify the above mentioned staining pattern. Under these conditions, extractive procedures for lipids do not affect the osmiophilia of nucleoli and Balbiani rings, while RNase or trichloroacetic acid treatment decreaes the staining degree of these structures. In osmium tetroxide/p-phenylenediamine treated salivary glands, the highest contrast within nuclei is seen to occur in the pars granulosa from normal or segregated nucleoli, as well as in Balbiani ring granules, which appear either as hollow granules or with a bipartite or horseshoe-like structure.     

A Comparison of Standard Lipid Staining Techniques Used in Electron Microscopic Studies of Mammalian Tissues

Comparisons of several standard techniques for staining lipids in ultrastructural studies have been undertaken using the rat uterine epithelium as the experimental tissue. The best technique for clarity, retention of stain, and acceptability of cellular ultrastructure utilized p-phenylenediamine after primary fixation in glutaraldehyde and postfixation in osmium tetroxide. While osmium by itself stained only unsaturated lipids and p-phenylenediamine stained no lipids in spot tests, when acting together, the staining of unsaturated lipids was enhanced and some staining of saturated lipids was seen. Further, the marked extraction of stained lipids normally found during dehydration did not then occur.     

Immunocytochemistry with osmium-fixed tissue. I. Light microscopic localization of growth hormone and prolactin with the unlabeled antibody-enzyme method.

Growth hormone and prolactin were localized on thin plastic sections of rat anterior pituitary gland and mammosomatotropic tumor MtTW15 that were fixed with osmium tetroxide (alone,mixed with aldehydes, or after aldehydes). Intense immunocytochemical staining for both antigens was obtained after plastic was removed from sections with an alcoholic solution of sodium hydroxide. The results indicated that antigenic determinants of rat prolactin and growth hormone were not completely destroyed or inactivated by fixation with osmium and embedment in epoxy resin, and that removal of the polymerized epoxy resin was necessary to obtain light microscopic postembedding immunocytochemical staining of these antigens. The results also demonstrated that tissues which have been conventionally processed for morphological evaluation by electron microscopy may be suitable for postembedding immunocytochemical staining of some antigens for light microscopy.     

On the stabilization by fixation of configurational states in beef heart mitochondria

The energized configuration of the cristal membrane of beef heart mitochondria can be maintained only as long as oxygen is available for electron transfer. When the oxygen supply is exhausted, the membrane undergoes a transition to the nonenergized configuration. Since the exhaustion of the available oxygen supply is complete in 5–20 sec, it is impossible to apply the method of sedimenting the mitochondria prior to fixation for studying the energized configurational states of mitochondria. The direct addition of glutaraldehyde followed by osmium tetroxide to the mitochondrial suspension is the most effective way of freezing the configurational state of the cristal membrane. Fixation with glutaraldehyde appears to be complete within 1–2 sec even at 0°. Osmium tetroxide alone can also freeze the energized configuration by fixation but the concentration of the fixative is critical. The problem of capturing the configurational state applies not only to energized transitions (nonenergized to energized) but also to nonenergized transitions (orthodox to aggregated). The freezing by fixation of the cristal membrane in the aggregated configuration is best accomplished by the sequential use of glutaraldehyde and osmium tetroxide. When the levels of glutaraldehyde and osmium tetroxide are respectively too low or too high, the mitochondrion will undergo a transition from the aggregated to the orthodox configuration before fixation is complete. Light-scattering studies provide an independent method for monitoring configurational changes in mitochondria; these light-scattering measurements confirm that the conditions for fixation which lead to stabilization of the energized state as judged by electron microscopy, also show maintenance of configuration as judged by absence of light-scattering changes after the fixatives are introduced. Reagents used in negative staining will induce the geometrical form of the energized configuration of the mitochondrion even under nonenergizing conditions. These reagents are thus unsuitable for use in studies of configurational transitions in mitochondria.     

Localization of plant lipids for light microscopy using p-phenylenediamine in tissues of Arachis hypogaea L.

p-Phenylenediamine (pPD) can be used en bloc to preserve and differentiate cell lipids in aldehyde-fixed peanut plant tissues treated with osmium tetroxide during dehydration in 70% ethanol. Semithin plastic sections for light microscopy need no further staining and can be mounted in Histoclad after drying on a slide. Brown staining above background differentiates lipid-containing structures. Nonspecific staining can be distinguished in control preparations extracted en bloc with lipid solvents.     

Osmium tetroxide/p-phenylenediamine staining of nucleoli and balbiani rings in Chironomus salivary glands

Summary This paper deals with the application of the osmium tetroxide fixation followed by p-phenylenediamine treatment to salivary gland cells from Chironomus larvae. After this procedure, cytoplasm, nucleoli and Balbiani rings show a high degree of staining both in light and electron microscopy, while chromatin remains unstained. Ethanol fixation followed by osmium tetroxide/p-phenylenediamine does not modify the above mentioned staining pattern. Under these conditions, extractive procedures for lipids do not affect the osmiophilia of nucleoli and Balbiani rings, while RNase or trichloroacetic acid treatment decreaes the staining degree of these structures. In osmium tetroxide/p-phenylenediamine treated salivary glands, the highest contrast within nuclei is seen to occur in the pars granulosa from normal or segregated nucleoli, as well as in Balbiani ring granules, which appear either as hollow granules or with a bipartite or horseshoe-like structure.     

Immunoelectron microscopic studies of the ultrastructure of myosin filaments in Dictyostelium discoideum

We have examined the characteristics of myosin in situ in Dictyostelium amoebae. By an improved immunofluorescence method, we previously found rod-like structures that contain myosin, which we call "myosin rods", in amoebae (Yumura. S., and Fukui, Y. (1985) Nature, 314: 194-196). Although we prepared samples for electron microscopy using conventional chemical fixation to clarify the ultrastructure of the myosin rods, we could not find any filamentous structures similar to myosin thick filaments. Therefore, we examined the effects of chemical fixatives on the myosin rods in situ by immunofluorescence staining. When cells were fixed in more than 0.05% glutaraldehyde or more than 1% osmium tetroxide at 4 degrees C, the myosin rods disappeared. These effects did not result from loss of the antigenicity, because a monoclonal myosin-specific antibody was able to react with synthetic myosin filaments treated with 0.5% glutaraldehyde or 2% osmium tetroxide. Cells fixed by the procedure used for immunofluorescence staining were post-fixed with permissible concentrations of chemical fixatives and prepared for examination by transmission electron microscopy. We found discrete filaments of about 12 nm thickness between the microfilaments. These filaments were shown to contain myosin by immunoelectron microscopy with an immunogold probe. These filaments were thinner than synthetic myosin thick filaments formed in vitro in the presence of 10 mM MgCl2, but they were similar to those formed in the presence of 2 mM MgCl2, or under nearly physiological ionic conditions. The images after immunofluorescence and immunogold labeling both suggested that these 12-nm-thick filaments in Dictyostelium amoebae were myosin filaments in situ.     

Localization of Plant Lipids for Light Microscopy Using P-Phenylenediamine in Tissues of Arachis Hypogaea L.

p-Phenylenediamine (pPD) can be used en bloc to preserve and differentiate cell lipids in aldehyde-fixed peanut plant tissues treated with osmium tetroxide during dehydration in 70% ethanol. Semithin plastic sections for light microscopy need o further staining and can be mounted in Histoclad after drying on a slide. Brown staining above background differentiates lipid-containing structures. Nonspecific staining can be distinguished in control preparations extracted en bloc with lipid solvents.     

Ruthenium red as a stain for electron microscopy. Some new aspects of its application and mode of action.

Commercial ruthenium red has been tested for its purity by spectrophotometry. Impurities detected by this method could be abolished by nitric acid-precipitation of ruthenium brown. This substance has no effect on cell surface staining and converts almost completely to ruthenium red under the conditions used in electron microscopy. It was found, by photometric analysis, that in the ruthenium red-osmium tetroxide-cacodylate combination, generally used for cell surface staining, chemical reactions between ruthenium red and osmium tetroxide occur. As aerial oxidation of hexammineruthenium2+ leads to a product with some surface staining capability, it is suggested that an oxidized product of ruthenium red is responsible for binding to cellular components, and that a reduced product of osmium tetroxide gives an additional contrast enhancement. In ruthenium red-osmium dioxide combinations ruthenium red seems to bind to cell surfaces without any molecular alteration, and contrast is gained by the model proposed by Blanquet (1976b). The latter method could open a way for investigating the binding of ruthenium red to certain natural compounds involved in calcium transport, as postulated by a number of authors. Both ruthenium-osmium combinations differ in their cell surface staining ability. The ruthenium red-osmium dioxide combination tends to form distinct subunits, whereas the osmium tetroxide variety stains homogeneously. In combination with osmium dioxide, the surface staining is affected by EDTA, and, in contrast to osmium tetroxide, a successive application of ruthenium red and osmium dioxide as possible.