Potency of microwave irradiation during fixation for electron microscopy

Liver, skeletal muscle, peripheral nerves, pancreas, thyroid and adrenal cortex were prepared for electron microscopy employing microwave energy either during prefixation with glutaraldehyde or instead of prefixation. Microwave irradiation in the presence of glutaraldehyde in Na/K-phosphate or Na-cacodylate containing CaCl2 and MgCl2 led to distinct appearance of membranes, mainly plasma membrane, and membranes of SER, Golgi complex and mitochondria in liver, pancreas and muscle. The area of high quality fixation, however, was limited to the periphery of samples. On the other hand, SER was dilated in cells of the adrenal cortex, and RER markedly vacuolated in thyroid follicular cells. Microwave irradiation in the presence of Na/K-phosphate and subsequent osmication resulted in preservation of the ultrastructure in similar quality as was obtained by osmication without previous immersion in glutaraldehyde. However, the preservation of SER and Golgi complex in liver and pancreas, and of mitochondria in muscle was greatly improved. Small myelin sheaths remained intact whereas large ones showed focal disintegration. We consider that enhancement of fixation by microwave energy may greatly improve preservation of membranes in some tissues. Successful fixation depends on the use of glutaraldehyde during microwave irradiation, the type of buffer, the addition of ions to increase stabilization, the exposure time to heat, and on postosmication.     

The influence of buffers during fixation on the appearance of smooth endoplasmic reticulum and glycogen in hepatocytes of normal and glycogen-depleted rats.

Liver tissue of normal and glycogen depleted rats was prepared for transmission electron microscopy by perfusion fixation and subsequent osmication in the presence of various buffers, dehydration in aethanol and embedding in epon. The use of Na/K-phosphate or Na-cacodylate to buffer glutaraldehyde led to similar appearance and distribution of SER. When Na-cacodylate was used during osmication, more SER membranes were retained but less accumulations of glycogen were found than after osmication in the presence of Na/K-phosphate. Fixation with s-collidine buffered osmium led to an easily recognisable network of SER comprising wide tubules whereas glycogen was hindered to be stained. Veronal acetate or Na-cacodylate supplemented with sucrose resulted in marked dilation and disintegration of SER. A similar effect was obtained when Na/K-phosphate or Na-cacodylate was used in hyposmolar concentration as buffer for glutaraldehyde. Liver of fasted rats or glucagon-treated rats after perfusion with Na/K-phosphate buffered glutaraldehyde and osmication in the presence of Na/K-phosphate or Na-cacodylate comprised glycogen-depleted hepatocytes which contained abundant SER membranes occupying the entire space between other organelles even in samples harvested 3 h after glucagon administration. The diversity in appearance and distribution of SER and glycogen granules, which depends to a large extend on the buffer used, suggests that SER membranes may not be sufficiently stabilized during aldehyde fixation and osmication. We thus consider it likely that large accumulations of glycogen granules are the consequence of disintegration of SER membranes during processing rather than they represent the morphologic substrate of physiological degradation of SER membranes in the course of glycogen synthesis and deposition.     

The influence of buffers during fixation on the appearance of smooth endoplasmic reticulum and glycogen in hepatocytes of normal and glycogen-depleted rats

Summary Liver tissue of normal and glycogen depleted rats was prepared for transmission electron microscopy by perfusion fixation and subsequent osmication in the presence of various buffers, dehydration in aethanol and embedding in epon. The use of Na/K-phosphate or Nacacodylate to buffer glutaraldehyde led to similar appearance and distribution of SER. When Na-cacodylate was used during osmication, more SER membranes were retained but less accumulations of glycogen were found than after osmication in the presence of Na/K-phosphate. Fixation with s-collidine buffered osmium led to an easily recognisable network of SER comprising wide tubules whereas glycogen was hindered to be stained. Veronal acetate or Na-cacodylate supplemented with sucrose resulted in marked dilation and disintegration of SER. A similar effect was obtained when Na/K-phosphate or Na-cacodylate was used in hyposmolar concentration as buffer for glutaraldehyde. Liver of fasted rats or glucagon-treated rats after perfusion with Na/K-phosphate buffered glutaraldehyde and osmication in the presence of Na/K-phosphate or Na-cacodylate comprised glycogen-depleted hepatocytes which contained abundant SER membranes occupying the entire space between other organelles even in samples harvested 3 h after glucagon administration. The diversity in appearance and distribution of SER and glycogen granules, which depends to a large extend on the buffer used, suggests that SER membranes may not be sufficiently stabilized during aldehyde fixation and osmication. We thus consider it likely that large accumulations of glycogen granules are the consequence of disintegration of SER membranes during processing rather than they represent the morphologic substrate of physiological degradation of SER membranes in the course of glycogen synthesis and deposition.     

Parathyroid ultrastructure after aldehyde fixation, high-pressure freezing, or microwave irradiation

Parathyroid cell variants, commonly observed in parathyroid glands fixed by immersion in glutaraldehyde, are believed to be the result of cyclic changes in the course of parathyroid hormone secretion. Immersion of bovine parathyroid glands in a mixture consisting of 1% glutaraldehyde, 1.5% formaldehyde, and 2.5% acrolein, followed by post-fixation in 1% osmium tetroxide, resulted in high uniformity with only one cell variant, whereas the same fixation procedure led to disruption of cell membranes and formation of cell variants in rat parathyroids. Parathyroid glands of both cattle and rats prepared by high-pressure quick-freezing and subsequent freeze-substitution contained only one cell variant. Excellent preservation of the ultrastructure of bovine and rat parathyroids, also exhibiting only one cell variant, was achieved by microwave irradiation in the presence of 2.5% glutaraldehyde in Na-cacodylate followed by post-fixation with OsO4 in Na-cacodylate or s-collidine, both containing Ca2+ and Mg2+. Use of the appropriate buffer, as well as osmication, is essential for successful fixation utilizing microwave energy. The main effects are considered to be heating specimens within sufficient short periods and enhancement of subsequent osmium fixation. The results support the idea, arising after examination of perfusion-fixed parathyroid tissue, that parathyroid cell variants occur during improper aldehyde fixation rather than that they express functional diversity.     

Microwave-stimulated glutaraldehyde and osmium tetroxide fixation of plant tissue: ultrastructural preservation in seconds.

Microwave-enhanced fixation of animal tissues for electron microscopy has gained in interest in recent years. Attempts to use microwave irradiation for the preparation of plant tissues are rare. In this study; I report on microwave conditions which allow a high quality preservation of plant cell structure. Tissues used were: internodes of Chara vulgaris, leaves of Hordeum vulgare, root tips of Lepidium sativum. Microwave irradiation was done with a commercial microwave oven (Sharp R-5975). Fixatives used were: 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.2 and 1% osmium tetroxide in veronal/acetate buffer, pH 7.2. Conventional fixations with glutaraldehyde/osmium were compared with microwave fixations. Examinations of thin sections showed that microwave fixation (glutaraldehyde or sequential aldehyde/osmium) is an attractive and rapid alternative method for processing plant tissues for electron microscopy. The optimal conditions found were: microwave oven at power level 50 W, 6.5 ml of fixative solution, irradiation times between 32-34 s, final temperature between 40 degrees C and 47 degrees C.     

Quantitative assessment of cellular changes provoked by microwave enhanced fixation of parathyroids

Summary Rat parathyroids fixed by microwave enhancement, i.e. microwave irradiation in the presence of glutaraldehyde for 8 s and postfixation with OsO₄ after a delay of 5 min, were compared with parathyroids fixed by perfusion with glutaraldehyde followed by immersion in glutaraldehyde and finally in OsO₄. Morphometric analysis revealed that microwave enhanced fixation led to larger mean cell volume, to larger cell surface area, and to larger suface area in membranes of RER and secretory granules. Though it is not known by which method parathyroid cells are conserved closer to the living state it is obvious that microwave enhanced fixation retains more membranes but provokes centrifugal dislocation of membranes mimiking exocytosis.     

Microwave-stimulated glutaraldehyde and osmium tetroxide fixation of plant tissue: ultrastructural preservation in seconds

Summary Microwave-enhanced fixation of animal tissues for electron microscopy has gained in interest in recent years. Attempts to use microwave irradiation for the preparation of plant tissues are rare. In this study, I report on microwave conditions which allow a high quality preservation of plant cell structure. Tissues used were: internodes of Chara vulgaris, leaves of Hordeum vulgare, root tips of Lepidium sativum. Microwave irradiation was done with a commercial microwave oven (Sharp R-5975). Fixatives used were: 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.2 and 1% osmium tetroxide in veronal/acetate buffer, pH 7.2. Conventional fixations with glutaraldehyde/osmium were compared with microwave fixations. Examinations of thin sections showed that microwave fixation (glutaraldehyde or sequential aldehyde/osmium) is an attractive and rapid alternative method for processing plant tissues for electron microscopy. The optimal conditions found were: microwave oven at power level 50 W, 6.5 ml of fixative solution, irradiation times between 32–34 s, final temperature between 40° C and 47° C.     

Parathyroid cell variants may be provoked during immersion fixation

Summary Parathyroid glands of cattle, dogs, cats, mice and rats were immersed in glutaraldehyde or mixtures consisting of glutaraldehyde, formaldehyde and acrolein in either Na-phosphate, Na/K-phosphate or Na-cacodylate buffer, and postfixed with OsO₄ in the same buffers or, alternatively, in s-collidine.Excellent preservation of bovine, feline and murine parathyroid glands was achieved with fixation mixtures containing 1% glutaraldehyde, 1.5–2% formaldehyde and 2.5–5% acrolein in 0.1 M Na-cacodylate with or without Ca²⁺ and Mg²⁺, Na-phosphate or Na/K-phosphate at 4°C followed by postfixation with 1% OsO₄ in the same buffers or in s-collidine containing sucrose, Ca²⁺ and Mg²⁺. This procedure largely abolished the occurence of parathyroid cell variants. Bovine parathyroid glands were also satisfactorily preserved with 1% glutaraldehyde and 2% formaldehyde whereas 1% glutaraldehyde and 2.5 or 5% acrolein, lower or higher buffer osmolarity, or immersion at room temperature led to vacuolization of RER and to breakdown of membranes. In contrast, all fixation protocols led to the formation of dark and light cell variants and to multinucleated syncytial cells in dog and rat parathyroids. The results thus show that parathyroid cell variants arise during immersion fixation and that aldehydes, buffers and temperature are important factors for provoking parathyroid cell variants.     

Parathyroid cell variants may be provoked during immersion fixation

Parathyroid glands of cattle, dogs, cats, mice and rats were immersed in glutaraldehyde or mixtures consisting of glutaraldehyde, formaldehyde and acrolein in either Na-phosphate, Na/K-phosphate or Na-cacodylate buffer, and postfixed with OsO4 in the same buffers or, alternatively, in s-collidine. Excellent preservation of bovine, feline and murine parathyroid glands was achieved with fixation mixtures containing 1% glutaraldehyde, 1.5-2% formaldehyde and 2.5-5% acrolein in 0.1 M Na-cacodylate with or without Ca2+ and Mg2+, Na-phosphate or Na/K-phosphate at 4 degrees C followed by postfixation with 1% OsO4 in the same buffers or in s-collidine containing sucrose, Ca2+ and Mg2+. This procedure largely abolished the occurrence of parathyroid cell variants. Bovine parathyroid glands were also satisfactorily preserved with 1% glutaraldehyde and 2% formaldehyde whereas 1% glutaraldehyde and 2.5 or 5% acrolein, lower or higher buffer osmolarity, or immersion at room temperature led to vacuolization of RER and to breakdown of membranes. In contrast, all fixation protocols led to the formation of dark and light cell variants and to multinucleated syncytial cells in dog and rat parathyroids. The results thus show that parathyroid cell variants arise during immersion fixation and that aldehydes, buffers and temperature are important factors for provoking parathyroid cell variants.     

A cytochemical study of the calcium-activated adenosinetriphosphatase in hamster adrenal medulla: its occurrence in the golgi region of chromaffin cells

Summary Several different fixation procedures and incubation media were used in order to demonstrate the ultrastructural localisation of Ca²⁺-activated adenosinetriphosphatase (ATPase) in the hamster adrenal medulla. Fixation by perfusion with 2.5% glutaraldehyde gave the best preservation of fine structure without markedly inhibiting the enzymic activity. The localisation of Ca²⁺-activated ATPase was different from that of Mg²⁺-activated ATPase: the Mg²⁺-dependent enzyme was confined to plasma membranes. Ca²⁺-dependent ATPase also occurred on the plasma membranes of neurons and of some chromaffin cells, but the most prominent site of this enzyme was in the Golgi apparatus of chromaffin cells. Most of the reaction product was localised between Golgi lamellae, but some was found in Golgi vesicles and in prosecretory granules. The nucleus, mature chromaffin granules, roughsurfaced endoplasmic reticulum and mitochondria were usually free of reaction product. Rarely, some precipitate was found in the matrix of mitochondria and in lysosomes.Wellcome Research Fellow.J. H. Burn Research Scholar.This work was supported by a grant from the Medical Research Council.     

Quantitative assessment of cellular changes provoked by microwave enhanced fixation of parathyroids

Rat parathyroids fixed by microwave enhancement, i.e. microwave irradiation in the presence of glutaraldehyde for 8 s and postfixation with OsO4 after a delay of 5 min, were compared with parathyroids fixed by perfusion with glutaraldehyde followed by immersion in glutaraldehyde and finally in OsO4. Morphometric analysis revealed that microwave enhanced fixation led to a larger mean cell volume, to larger cell surface area, and to larger surface area in membranes of RER and secretory granules. Though it is not known by which method parathyroid cells are conserved closer to the living state it is obvious that microwave enhanced fixation retains more membranes but provokes centrifugal dislocation of membranes mimicking exocytosis.     

Improved structural preservation in freeze-substituted sporidia ofUstilago avenae—a comparison with low-temperature embedding

Summary In the present study the cellular fine structure of freeze-substituted sporidia of the phytopathogenic fungusUstilago avenae is investigated by means of thin-section electron microscopy. A conventional embedding method using Spurr's low viscosity resin is compared with the recently developed methacrylate mixtures Lowicryl^® K 4 M and HM 20 resin. Generally, freeze-substitution yields improved preservation of fine structural details of the fungus compared to previously applied conventional fixation methods. Using double fixation during freeze-substitution prior to conventional embedding the fungal membrane system (plasmalemma, endoplasmic reticulum), organelles (mitochondria, nucleus etc.) and other cytoplasmic features (ribosomes, cytoskeleton) appear well resolved and smoothly contoured. Aldehyde fixed and Lowicryl embedded sporidia ofU. avenae resemble these double fixed fungal specimens fairly closely. The complete low-temperature preparation produces visualization of distinct cellular details although contrast reversal of cellular membranes (er, mitochondria etc.) is sometimes observed. In particular, fine structure resolution is enhanced in Lowicryl HM 20 embedded fungal cells. This is due also to significant improvement in staining of the cellular membranes, cytoskeleton (microfilaments and microtubules) and Golgi apparatus-like areas, using tannic acid. In case of the fungusU. avenae, freeze-substitution in combination with mild glutaraldehyde fixation and final low-temperature embedding in HM 20 resin prove suitable for improved preservation of cellular ultrastructure.Abbreviations cw cell wall - cy cytoplasm - FS freeze-substitution - FS-A GA/OsO₄ freeze-substitution and Spurr's LV-embedding - FS-B GA freeze-substitution and Lowicryl K 4 M LT-embedding - FS-C GA freeze-substitution and Lowicryl HM 20 LT-embedding - go Golgi apparatus-like body - GA glutaraldehyde - g glycogen deposit - l lipid droplet - LT low temperature - Lowicryl LT-embedding Lowicryl low-temperature embedding - Lowicryl LT-resin Lowicryl low-temperature resin - MeOH methanol - mf microfilament - mt microtubule - m mitochondrion - mvb multivesicular body - ne nuclear envelope - np nuclear pore - npl nucleoplasm - nu nucleolus - n nucleus - OsO₄ osmium tetroxide - pl plasmalemma - pr polyribosomes - Pb-citrate Reynolds' lead citrate - r ribosome - RT room temperature - rer rough endoplasmic reticulum - Spurr's LV-embedding Spurr's low viscosity embedding - Spurr's LV-resin Spurr's low viscosity resin - t tonoplast - Uac uranyl acetate - v vacuole     

STUDIES ON SEEDS : I. Fixation of Seeds

Several fixation procedures were studied to determine those most suitable for preservation of seeds during late stages of development and early stages of germination. These are the periods when the tissues are partially dehydrated and are most difficult to fix for electron microscopy. It was found that a prefixation with a mixture of glutaraldehyde, reconstituted formaldehyde (i.e. paraformaldehyde), and acrolein, followed by a postfixation in OsO₄ or KMnO₄, gives very acceptable images. The results also indicate that glutaraldehyde is necessary for preservation of cell shape, paraformaldehyde for stabilization of reserve proteins, and acrolein for rapid penetration of tissues. Phosphate, cacodylate, and collidine are all acceptable buffers, although collidine gives the most consistent results.     

Fixation of platelets for scanning and transmission electron microscopy.

A double fixation method of preparing platelet suspensions for both scanning and transmission electron microscopy is outlined. Prefixation in 0.1% glutaraldehyde allows for immediate preservation of morphologic characteristics induced by experimental procedures, but does not completely destroy platelet surface stickiness. Preservation of surface stickiness allows subsequent production of a platelet pellet for processing for transmission electron microscopy. This pelleting cannot be achieved when higher initial concentrations of glutaraldehyde are used for prefixation. Prefixation in 0.1% glutaraldehyde is also an appropriate initial step for preservation of platelets in suspension for scanning electron microscopy.     

Fixation of Platelets for Scanning and Transmission Electron Microscopy

A double fixation method of preparing platelet suspensions for both scanning and transmission electron microscopy is outlined. Prefixation in 0.1% glutaraldehyde allows for immediate preservation of morphologic characteristics induced by experimental procedures, but does not completely destroy platelet surface stickiness. Preservation of surface stickiness allows subsequent production of a platelet pellet for processing for transmission electron microscopy. This pelleting cannot be achieved when higher initial concentrations of glutaraldehyde are used for prefixation. Prefixation in 0.1% glutaraldehyde is also an appropriate initial step for preservation of platelets in suspension for scanning electron microscopy.     

Microwave irradiation improvements in the silver staining of the nucleolar organizer (Ag-NOR) technique

The well-know technique of silver staining of the nucleolar organizer (Ag-NOR) is improved in contrast, selectivity and speed when performed with microwave irradiation. The Ag-NOR technique is a very useful tool for studies on the functional morphology and molecular architecture of the nucleolus, and is reputed to be one of the best techniques for diagnosis and prognosis of cancer lesions. To test the generality of the enhancing effects. our study has involved the use of both mammalian and plant cells. Two steps in the process are improved quantitatively by microwave irradiation: fixation and staining itself. Fixation with the ethanol-based reagent, Kryofix, for 3 min in the microwave oven, resulted in good structural preservation at the optical level, and enhanced the contrast and selectivity of silver staining. On the contrary, we found that neither glutaraldehyde fixation, nor a treatment of sections with Carnoy's solution, improved Ag-NOR staining. After an analysis of the effects of the different substances involved in sample preparation, we conclude that ethanol is an essential factor for fixation for nucleolar staining, particularly if aldehydes are eliminated from fixative solutions. The process of staining was performed with a drop of staining solution on a semithin section of plastic-embedded tissue intthe microwave oven for 1 min. Staining under these conditions always improved the visualization of nucleoli, regardless of the fixation procedure. Therefore, microwave irradiation at both steps is recommended for giving the best results. Microwave irradiation probably enhances fixation by controlled heat, whereas the increase in reactivity of the staining solution is a direct effect by the microwaves on the silver ions themselves. We used this method to study nucleolar materials during mitosis in proliferating plant cells. Current applications of Ag-NOR staining can be improved with this technical modification.     

Use of nitrogen mustard N-oxide for the fixation of electron microscopic tissues

Summary Nitrogen mustard N-oxide was tried for the fixation of tissue for electron microscopy. A fixative consisting of 1% nitrogen mustard N-oxide, 1% glutaraldehyde and 1% paraformaldehyde buffered at pH 7.4 followed by 1% O_sO₄ buffered at pH 7.4 was found useful for the tissues examined: thyroid, anterior pituitary, adrenal gland and oviduct of mice.If the tissues are fixed and the sections are stained with uranyl acetate and lead acetate doubly, the follicle colloid, colloid droplets, and secretory granules containing thyroglobulin in the thyroid become higher in electron density. The cisterna of the maturing face of the Golgi apparatus, secretory granules, ribosomes, nucleolus and chromatin in the cells examined are extremely electron dense. Tubular elements of smooth endoplasmic reticulum in the adrenal cortical cell and microtubules in all the cells examined are also well preserved. The fixative containing nitrogen mustard N-oxide is useful also for cytochemistry. Using tissue fixed by this method and stained en bloc by uranyl acetate, the noradrenaline and adrenaline cells in the adrenal medulla are clearly distinguished by light microscopy.This study was supported by a grant from the Japan Educational Ministry     

Influence of calcium and magnesium containing fixatives of the ultrastructure of parathyroids

The effect of Ca²⁺ and Mg²⁺ on feline parathyroid cells during perfusion fixation with glutaraldehyde and subsequent immersion in OsO₄ was investigated. Both Ca²⁺ and Mg²⁺ may exert a stabilizing or destabilizing effect on cell membranes and on elements of the cytoskeleton. The effect depends (1) on the ion concentration, (2) on the buffer concentration and (3) on the fixative. Stabilization due to Ca²⁺ or Mg²⁺ during glutaraldehyde fixation is not altered during subsequent osmication but both cations may cause destabilization during osmication in tissue prefixed without cations. Ca²⁺ and Mg²⁺ also reduce cell volume in combination with low osmolar buffer but they prevent cells from excessive shrinkage due to high osmolar buffers. Ca²⁺ and Mg²⁺ alone or in combination reduce swelling of RER, extraction of cellular material and loss of subcellular compartments, such as secretory granules, under optimal conditions. Ca²⁺, however, provokes formation of dark (shrunken) and light (swollen) cells accompanied by loss of subcellular components when used in low concentration during osmication. Low concentrations of Mg²⁺ added to glutaraldehyde exert similar effects. Stabilization of membranes is assumed to be due to the binding capacity of Ca²⁺ and Mg²⁺ to both phospholipids and proteins. The influence of Ca²⁺ and Mg²⁺ to changes in cell volume is considered likely to be the result of ionic interaction in the cytoplasmic gel, the maintenance of cell volume being a matter of equilibrium between the swelling pressure of the cytoplasmic gel and osmotic pressure of the fixative solution.     

THE ULTRASTRUCTURE OF LIPID-DEPLETED ROD PHOTORECEPTOR MEMBRANES

The structure of lipid-depleted retinal rod photoreceptor membranes was studied by means of electron microscopy. Aldehyde-fixed retinas were exhaustively extracted with acetone, chloroform-methanol, and acidified chloroform-methanol. The effect of prefixation on the extractability of lipids was evaluated by means of thin-layer chromatography and fatty acid analysis. Prefixation with glutaraldehyde rendered 38% of the phospholipids unextractable, while only 7% were unextractable after formaldehyde fixation. Embedding the retina in a lipid-retaining, polymerizable glutaraldehyde-urea mixture allows a comparison of the interaction of OsO₄ with lipid-depleted membranes and rod disk membranes which contain all their lipids. A decrease in electron density and a deterioration of membrane fine structure in lipid-depleted tissue are correlated with the extent of lipid extraction. These observations are indicative of the role of the lipid bilayer in the ultrastructural visualization of membrane structure with OsO₄. Negatively stained thin sections of extracted tissue reveal substructures in the lipid-depleted rod membranes. These substructures are probably the opsin molecules which are the major protein component of retinal rod photoreceptor membranes.     

The effect of fixation conditions on the ultrastructural appearance of gastrin cell granules in the rat gastric pyloric antrum

The ultrastructural appearance of gastrin cell (G cell) granules was studied after different fixation procedures. When the pH of prefixation was varied there was greater preservation of the electron density of granule cores after acidic (pH 5.0 and 6.0) than after neutral or alkaline (pH 7.0 and 8.0) prefixation. Increasing duration of prefixation at pH 7.3 resulted in progressive loss of electron density of the granule core with swelling and occasional rupture of the limiting membrane. In tissues where most granules had been rendered electron lucent by fixation, those granules remaining dense cored were preferentially located close to the Golgi zone. These findings indicate that the electron density of G cell granules is profoundly affected by conditions of fixation, and that immature granules are more resistant to loss of core density than mature granules. They also suggest that the gastrin granule in vivo, like other polypeptide granules, may have a "solid", osmotically inactive core.