PATTERN OF OSMIUM DEPOSITION IN THE PARIETAL CELLS OF THE STOMACH

Parietal cells of the stomach of the hamster show extensive amounts of dense material in a variety of organelles after prolonged exposure to a solution of osmium tetroxide. Conspicuous amounts of reduced osmium compounds are evident within the granular endoplasmic reticulum, perinuclear cisterna, and vesicular elements of the Golgi complex. Dense material is also apparent within cristae of the mitochondria, the surface coat of the microvilli of the intracellular canaliculus, and vesicular elements of the multivesicular bodies. Multivesicular bodies, containing numerous small osmiophilic elements, are often seen surrounding and/or in close contact with mitochondria. The proximity of the multivesicular bodies to the mitochondria appears to be related to an autophagic process involving degradation of mitochondria. The distribution and intensity of the precipitates within the organelles of the parietal cells vary in different regions of the gastric glands. The findings of this study emphasize that cell structures other than the Golgi complex may contain large concentrations of reduced osmium compounds after prolonged exposure to a solution of osmium tetroxide.     

Ultrastructural localization of calcium in prothoracic gland cells of Galleria mellonella L. (Insecta,Lepidoptera) in relation to secretory activity

Summary Localization of intracellular calcium was demonstrated by precipitation with potassium hexahydroxoantimonate in the fixation medium containing osmium tetroxide or osmium tetroxide and glutaraldehyde. The presence of calcium in the precipitates was confirmed by X-ray microanalysis. Cells from active prothoracic glands contain more calcium deposits than inactive glands. The calcium precipitates are mainly localized in the nucleus, in the smooth endoplasmic reticulum, in the hyaloplasm and to a lesser degree in the mitochondria. These findings are consistent with the proposed role of calcium in the stimulation of steroidogenesis.     

The zinc iodide-osmium tetroxide staining-fixative of Maillet. Nature of the precipitate studied by x-ray microanalysis and detection of Ca2+-affinity subcellular sites in a tonic smooth muscle.

A mechanism of osmium reduction during zinc iodide-osmium tetroxide (ZIO) fixation is proposed. X-ray powder microanalyses of ZIO precipitates formed both in the presence or absence of tissues are identical with those of CuOsO4 and CuRuO4. Therefore, and based on indexation methods, ZnOsO4 was found to be the formula of the ZIO mixture reduction; this zinc osmate has an orthorhombic crystalline lattice. In smooth muscle preparations, ZIO electron dense deposits are localized in both cisternae of the sarcoplasmic reticulum and in mitochondria after a short fixation time. According to the microanalysis results, the zinc osmate has been associated to Ca2+ high affinity sites since Zn2+ is either replacing Ca2+ and/or displacing it by having a higher affinity for Ca2+ binding sites. Consequently, the ZIO mixture might be useful in revealing some Ca2+ storage sites in cells. This hypothesis was tested in ABRM preparations by selectively depleting sites which are known to bind Ca2+. In this case, the sarcoplasmic reticulum only retains the staining deposits after a short ZIO fixation. It is likely that OsO4 alone, used as fixative in cytology might be due to the formation of metallic osmates (e.g., divalent osmates like CaOsO4). In addition, of course, reduction of osmium during tissue fixation is accompanied by oxidation of double bonds of lipoproteic complexes or unsaturated lipids, and oxidation of sulfhydryl groups and amino groups.     

The zinc iodide-osmium tetroxide staining-fixative of Maillet. Nature of the precipitate studied by X-ray microanalysis and detection of Ca2+-affinity subcellular sites in a tonic smooth muscle

Summary A mechanism of osmium reduction during zinc iodide-osmium tetroxide (ZIO) fixation is proposed.X-ray powder microanalyses of ZIO precipitates formed both in the presence or absence of tissues are identical with those of CuOsO₄ and CuRuO₄. Therefore, and based on indexation methods, ZnOsO₄ was found to be the formula of the ZIO mixture reduction; this zinc osmate has an orthorhombic crystalline lattice.In smooth muscle preparations, ZIO electron dense deposits are localized in both cisternae of the sarcoplasmic reticulum and in mitochondria after a short fixation time.According to the microanalysis results, the zinc osmate has been associated to Ca²⁺ high affinity sites since Zn²⁺ is either replacing Ca²⁺ and/or displacing it by having a higher affinity for Ca²⁺ binding sites. Consequently, the ZIO mixture might be useful in revealing some Ca²⁺ storage sites in cells. This hypothesis was tested in ABRM preparations by selectively depleting sites which are known to bind Ca²⁺. In this case, the sarcoplasmic reticulum only retains the staining deposits after a short ZIO fixation.It is likely that OsO₄ alone, used as fixative in cytology might be due to the formation of metallic osmates (e.g., divalent osmates like CaOsO₄). In addition, of course, reduction of osmium during tissue fixation is accompanied by oxidation of double bonds of lipoproteic complexes or unsaturated lipids, and oxidation of sulfhydryl groups and amino groups.     

Tissue fixation and staining with osmium tetroxide: the role of phenolic compounds.

It has been postulated that phenol-containing areas of plant and animal tissues were osmiophilic, but proof of direct interaction between osmium tetroxide and phenolic materials, or the nature of such reactions, has been lacking. We find that, under conditions similar to those of normal tissue fixation, osmium tetroxide reacts rapidly with those phenols containing o-dihydroxy groups (including such species found in plant tissues) to give very stable chelate complexes. We conclude that these complexes are responsible for the observed electron-density in phenol-containing areas of tissue treated with osmium tetroxide, so that such phenols are indeed osmiophilic.     

ULTRASTRUCTURAL DEMONSTRATION OF CYTOCHROME OXIDASE ACTIVITY BY THE NADI REACTION WITH OSMIOPHILIC REAGENTS

A new method for the subcellular and cytochemical demonstration of cytochrome oxidase has been developed with the introduction of N-benzyl-p-phenylenediamine (BPDA) and the discovery that indoanilines are osmiophilic. These indoanilines produced upon oxidation of BPDA in the presence of naphthols are highly colored compounds that yield electron-opaque coordination polymers of osmium (osmium black) that are amorphous, insoluble in water, and in organic solvents. The best methods for preparing rat tissue were in decreasing order: fixation in formaldehyde solution, fresh tissue slices, and frozen sections of fresh or fixed tissue. Ultrathin sections were counterstained by bridging with the thiocarbohydrazide-osmium tetroxide (T-O) procedure for enhancing underlying membranous structures. Cytochrome oxidase activity was noted primarily in mitochondria and occasionally in sarcotubules of heart, in mitochondria and occasionally in infoldings of the plasma membrane of renal tubular cells, and in mitochondria and, to a great extent, in endoplasmic reticulum of hepatic cells. Cytochrome oxidase activity produced deposits in droplet form, whereas dehydrogenase activity resulted in uniform staining of mitochondrial cristae, as recently demonstrated with an osmiophilic tetrazolium salt. Even more recently we have succeeded in demonstrating cytochrome oxidase activity in nondroplet staining on mitochondrial cristae with an osmiophilic benzidine-type reagent that apparently polymerizes upon oxidation (to be published later).     

A morphologic and cytochemical study on the great alveolar cell.

Lungs from marsupials, bats and rodents were studied by light and electron microscopy. In all three groups, the great alveolar cells exhibit similar morphologic and cytochemical characteristics. Cytoplasmic vacuoles seen in these cells by light microscopy correspond to cytosomes that are demonstrable in them by electron microscopy. Such cytosomes are osmiophilic, periodic acid-Schiff-positive and stainable with Sudan black after acetone extraction. After fixation in a mixture of aldehydes, followed by extraction in chloroform-methanol and postfixation in osmium tetroxide, cytosomes lose their osmiophilia. The cytoplasm of the great alveolar cell is notable for a loosely ordered granular endoplasmic reticulum, an extensive Golgi apparatus and numerous multivesicular bodies. Many forms transitional in appearance between multivesicular bodies and cytosomes are present. In these, osmiophilic matter occupies the intervesicular space. It is proposed that these bodies are the precursors of cytosomes. The cytosomes are interpreted to be products of the "lysosomal" system in this cell. Ultimately they are secreted onto the alveolar surface.     

The alveolar-lining layer in the lung of the axolotl,Ambystoma mexicanum

Summary Lungs of neotenic larvae of Ambystoma mexicanum were prepared for maintaining the air-tissue boundary during aldehyde fixation. Four methods of postfixation were applied: 1) osmium tetroxide followed by en-bloc staining with uranyl acetate and phosphotungstic acid, 2) ruthenium redosmium tetroxide, 3) osmium tetroxide-ferrocyanide, and 4) tannic acidosmium tetroxide.Three types of cells line the inner surface of the axolotl lung: 1) pneumocytes, covering the capillaries with flat cellular extensions and containing two types of granules: the osmiophilic lamellar bodies, precursors of extracellular membranous material, and apical granules of unknown significance; 2) ciliated cells, also containing osmiophilic lamellar bodies; and 3) goblet cells filled with secretory granules as well as osmiophilic bodies.The extracellular material forms membranous whorls as well as tubular myelin figures, consisting of membranous backbones combined with an intensely stained substance. This material strikingly resembles the surfactant of amphibian lungs.     

Unbuffered osmium staining of cell organelles: alterations induced by cell injury

We have studied the localization of osmium reduction products to investigate the functional state of organelles as well as organelle interrelationships during cell injury. In normal hepatocytes osmium deposits of variable intensity are seen in nuclear envelope, endoplasmic reticulum. Golgi cisternae and vesicles and lysosomes. Buffering of osmium with s- collidine (pH 7.4) prevents the deposition of osmium. Reversible (30 min) and irreversible (60 min) ischemia without reflow causes no change in the pattern of osmium deposition. Irreversible ischemia followed by reflow causes decreased staining of endoplasmic reticulum (ER) and redistribution of the osmium deposits through the cytoplasm. Reversibly injured pancreatic acinar cells in cultured explants manifest a similar loss of osmium staining in the endoplasmic reticulum cisternae. The administration of antimicrotubule drugs induces an accentuation of osmium staining in localized cisternal elements of hepatocytes. These heavily stained cisternae appear to give rise to the bounding membranes of drug-induced autophagic vacuoles. Cytoplasmic organelles sequestered inside the autophagic vacuoles acquire intense staining when they begin to undergo degradation. In homogenized liver tissue all the subcellular organelles show osmium deposits. The deposits are preferentially localized along the organelle membranes. In particular the dense deposits in the ER lumen are not seen in the subcellular fractions. Phospholipase A2 (3 units/mg protein) enhances the deposition of osmium in the lumen of microsomal vesicles, whereas the presence of detergent has no such effect. Addition of EDTA to the homogenizing medium enhances the ultrastructural preservation of the subcellular fractions but has little effect on the deposition of osmium. OsO4 deposition occurs at acid pH and the intensity and pattern of the stain can be modified in vivo and in vitro. Osmium tetroxide deposition is induced at sites of membrane transformation (autophagic vacuoles) and degradation (lysosomes). Calcium influx and phospholipase activation (ischemia, tissue homogenization, phospholipase addition) enhance osmium deposition and/or influence the localization of the staining pattern.     

The release of membrane-associated calcium from rabbit neutrophils by fixatives. Implications for the use of antimonate staining to localize calcium

Summary The addition of oxalate to a suspension of rabbit peritoneal neutrophils before fixation with glutaraldehyde and postfixation with osmium tetroxide-antimonate greatly enhanced the amount of calcium antimonate precipitate subsequently detectable with the electron microscope. Using chlortetracycline as a fluorescent probe for membrane-associated calcium, it was found that both glutaraldehyde and osmium tetroxide release calcium from membrane-associated stores in suspensions of living neutrophils. These findings suggest that some of the calcium released from cellular stores during fixation with glutaraldehyde is trapped within the neutrophil by oxalate which then reacts with potassium antimonate. This produces a more copious precipitate of calcium antimonate than fixation without oxalate. It is suggested, therefore, that the histochemical localization of calcium by antimonate techniques may not always represent thein vivo situation. The use of oxalate during fixation, however, may give a better indication of the amount of calcium stored within a cell.     

EFFECTS OF FIXATIVES ON THE ULTRASTRUCTURE OF PHYSODES IN VEGETATIVE CELLS OF SCYTOSIPHON LOMENTARIA (SCYTOSIPHONACEAE,PHAEOPHYTA)1

The effects of different glutaraldehyde-osmium fixation schedules on the ultrastructure of the vegetative cells from the meristematic regions of Scytosiphon lomentaria (Lyngbye) Link fronds are described. The best overall preservation of cell structure was obtained with a 2 h fixation in 2.5–3.5% glutaraldehyde in 0.1 M cacodylate buffered seawater (pH 7.0), followed after washing by 1 h post fixation in 1% osmium tetroxide. The addition of 1% caffeine to the glutaraldehyde fixative resulted in better retention and spatial localization of the electron dense phenolic deposits within the cells. Particular attention was paid to the effects of the various fixation schedules on the electron-dense material within the cells and the images obtained were compared with previous accounts of brown algal cells. It is proposed that the term physode should be restricted to the discrete electron dense spherical bodies within the vacuoles and not applied to electron dense material in general. Although the organization of Scytosiphon cells was similar to that previously reported in the Scytosiphonaceae, the organization of the plasmodesmata into pit fields is at variance with previous accounts.     

THE FINE STRUCTURE OF THE PURKINJE CELL

This paper describes the fine structure of the Purkinje cell of the rat cerebellum after fixation by perfusion with 1 per cent buffered osmium tetroxide. Structures described include a large Golgi apparatus, abundant Nissl substance, mitochondria, multivesicular bodies, osmiophilic granules, axodendritic and axosomatic synapses, the nucleus, the nucleolus, and the nucleolar body. A new and possibly unique relationship between mitochondria and subsurface cisterns is described. Possible functional correlations are discussed.     

Ultrastructural cytochemistry of the secretory granules of the hamster submandibular gland

Summary The ultrastructure and cytochemistry of the secretory granules of the male hamster submandibular salivary gland were studied. After fixation in glutaraldehyde followed by osmium tetroxide the granules exhibit a characteristic bipartite substructure, with an electron lucid crescenteric rim and a more dense central core. A differentiation into two regions of the granules could also be visualized in specimens primarily fixed in Millonig's osmium tetroxide or in potassium permanganate. The electron lucid peripheral portion of the membrane bounded secretory granules further displays a strong positive reaction after staining of ultrathin sections with the periodic acid-chromic acid-(PA-CrA)-silver technique. The strong periodate reactivity of the rim relative to the core, suggests a difference in mucin composition of the two granule regions. With the PA-CrA-silver staining technique a positive reaction was also observed within the Golgi apparatus of the acinar cells.     

Studies of Osmium Deposits in the Carotid Body of the Cat

Double aldehyde fixed carotid bodies and small pieces of vagus nerve of cats were incubated in 3 mM copper sulfate and 0.5 mM potassium ferricyanide in 0.05 M acetate buffer (pH 5.6) for 30 minutes at room temperature. Several modifications of this procedure were also attempted. Tissues were then postosmicated with 2% unbuffered osmium tetroxide and heated to 50-55 C for ten minutes. Under the electron microscope carotid body cells exhibited fine osmium deposits within cisternae of endoplasmic reticulum, saccule and vesicles of Golgi complex, and cristae of mitochondria. Intense osmium precipitation was also noted in the mitochondria of nerve endings. In addition, much more intense, more conspicuous and more localized reaction wan noted in the intraperiod lines of the myelin sheath of nerves. Deposits here were rod-shaped, displaying considerable variation in length. These results are discussed in the light of previous findings on osmium deposits in various tissues. It was concluded that the osmium reaction is unspecific, and that histochemical methods employing hot osmium tetroxide to amplify enzymatic activities may therefore not be reliable.     

OBSERVATIONS ON THE FINE STRUCTURE OF PHARYNGEAL MUSCLE IN THE PLANARIAN DUGESIA TIGRINA

Pharyngeal muscle of the planarian Dugesia tigrina was studied by electron microscopy after osmium tetroxide fixation. The muscle cell was observed to contain one myofibril or bundle of myofilaments parallel to its longitudinal axis. The myofilaments were of two types, different in size and distribution. No Z lines or myofilament organization into cross or helical striations were seen. Dense bodies were seen as projections from an invagination of the plasma membrane and as dense lines parallel to the myofilaments. The muscle cells are surrounded by a plasma membrane which is structurally associated with dense body projections, with vesicles and cisternae of sarcoplasmic reticulum, and with synaptic nerve endings. The cell has sarcoplasmic projections perpendicular to its long axis; these projections are seen to contain the nucleus or mitochondria and granules. Mitochondria and granules are also seen in a sarcoplasm rim around the fibril. The dense bodies may serve as attachment for thin myofilaments and function in transmission of stimuli from plasma membrane to the interior of the fibril.     

The relationship between synthesis of secretory products and reducing capacity in pancreas and parotid acinar cells

The secretory products in exocrine pancreas acinar cells in utero were found to reduce osmium tetroxide. This reducing capacity was also exhibited by adult pancreas and parotid glands in different phases of synchronized secretion, and after single or chronic administration of a secretagogue, pilocarpine or isoprenaline. In utero, the reducing capacity appeared in the pancreas concomitantly with the synthesis of secretory products, and was limited to the transitional vesicles on the cis Golgi side. After birth, osmium staining occurred in the cis Golgi vesicles and cisternae of both glands. In the chronically-treated parotid gland, where the occupational programme for secretory proteins had been altered, the reducing capacity was diminished, resembling that in embryonic exocrine pancreas.     

Lipid fixation during preparation of chloroplasts for electron microscopy

Reaction of osmium tetroxide with isolated spinach chloroplasts fixed completely the glycolipids, phosphatidyl glycerol, and phosphatidyl choline. Under the same reaction conditions only 30% of the chlorophyll was fixed. Reaction of potassium permanganate with isolated spinach chloroplasts fixed more than 90% of the glycolipids, phosphatidyl glycerol, and phosphatidyl choline, provided the reaction period was long enough. Potassium permanganate also fixed the chlorophyll. Reaction of osmium tetroxide and potassium permanganate with isolated (14)C-lipids from Chlorella pyrenoidosa fixed 59% and 66% of the radioactivity, respectively. The lipids that were not fixed included sterols and pigments. Electron micrographs show that chloroplasts extracted with chloroform-methanol after fixation in osmium tetroxide or potassium permanganate differ from those dehydrated with acetone mainly in that in the former, osmiophilic globules have been removed and there seems to be some fusion of the boundary membranes and grana membranes. These effects may be due to the extraction of unfixed, neutral lipids such as sterols and quinones.     

The effects of 6-hydroxydopamine on the appearance of granulated vesicles in glomus cells of the rat carotid body

The glomus cells of the rat carotid body reveal an intense fluorescence after exposure to paraformaldehyde vapor and contain catecholamines. After initial fixation in glutaraldehyde, many granulated vesicles are seen in the glomus cells. After initial fixation in osmium tetroxide, most of the vesicles are depleted of their dense interiors and granulated vesicles occur infrequently. Administration of 6-hydroxydopamine followed by initial fixation in osmium tetroxide leads to the reappearance of dense interiors in virtually all vesicles. 6-Hydroxydopamine apparently is taken up by the membrane pump of the glomus cell and is incorporated into the amine storage granules, thereby displacing the endogenous monoamines. Osmium tetroxide does not dissolve the 6-hydroxydopamine from the vesicles, as it apparently does for the normal vesicular contents. The 6-hydroxydopamine does not fluoresce, hence 6-hydroxydopamine administration results in a decreased intensity of formaldehyde induced fluorescence in the glomus cells. Administration of reserpine after 6-hydroxydopamine treatment (and subsequent initial fixation in osmium tetroxide) depletes the previously restored dense material from the vesicles of the glomus cells. 6-Hydroxydopamine acts like a monoamine in that it is taken up by the glomus cell, incorporated into the vesicles, and can be depleted from the vesicles by reserpine.     

Ultrastructural Distribution of Calcium in Cutaneous Electroreceptor Organs of Teleost Fish

The calcium distribution in the ampullary electroreceptor and the type B electrore-ceptor organ (gymnarchomast) of Gymnarchus niloticus (Gymnarchidae) and in the tuberous organ of Apteronotus leptorhynchus (gymnotidae) was studied. Endogenous calcium appeared as electron-dense precipitates when the cutaneous organs were pre-fixed with phosphate-buffered glutaraldehyde and postfixed with osmium tetroxide plus potassium bichromate. Calcium precipitates were localized in both intracellular compartments of sensory cells and afferent nerve fibers. In contrast to sensory cells, small amounts of calcium precipitates were found in the cytoplasm of accessory cells. In sensory cells, electron-dense deposits were apparent mainly in synaptic vesicles near synaptic ribbons, inside vacuoles of the endoplasmic reticulum, and between the layers of the nuclear membrane. Very few deposits were found in mitochondria. Precipitates were also observed within the axons of afferent nerves and between the layers of the myelin sheath. The synaptic cleft was devoid of calcium. Calcium deposits have a specific cellular distribution in electro-receptor organs of teleost fish.     

Subcellular distribution of calcium in zona fasciculata cells of the rat adrenal cortex

Zona fasciculata cells from the adrenal cortex of female Sprague-Dawley rats were fixed by immersion in potassium pyroantimonate-osmium tetroxide and potassium pyroantimonate-glutaraldehyde to study the distribution of calcium. Potassium pyroantimonate-osmium tetroxide treatment gave reproducible patterns of electron-opaque precipitate, whereas inconsistent deposits of reaction product were seen after potassium pyroantimonate-glutaraldehyde fixation. Nuclei showed sparse precipitate over heterochromatin and dense aggregates over areas of nucleoli surrounded by portions of the nucleolar-dense component. Two major cytoplasmic sites of precipitate were identified: mitochondria and vesicles of smooth endoplasmic reticulum. Most of the intramitochondrial precipitate was localized to the intracristal space. Precipitate was also seen in vesicles of Golgi apparatus. The extracellular space was filled with closely packed electron-opaque particles. Observation of tissues treated with control fixative saturated with EGTA showed little if any reaction, confirming that calcium was the primary cation precipitated by potassium pyroantimonate. Our results provide a method suitable for accurate localization of calcium in adrenocortical cells.