Osmium Impregnation Research Articles

Whole-cell observation of ZIO-stained Golgi apparatus in rat hepatocytes with serial block-face scanning electron microscope, SBF-SEM.

The Golgi apparatus, which plays a role in various biosynthetic pathways, is usually identified in electron microscopy by the morphological criteria of lamellae. A 3-dimensional analyses with serial block-face scanning electron microscope (SBF-SEM), a volume-SEM proficient in obtaining large volumes of data at the whole-cell level, could be a promising technique for understanding the precise distribution and complex ultrastructure of Golgi apparatus, although optimal methods for such analyses remain unclear since the observation can be hampered with sample charging and low image contrast, and manual segmentation often requires significant manpower. The present study attempted the whole-cell observation and semi-automatic classification and segmentation of the Golgi apparatus in rat hepatocytes for the first time by SBF-SEM via ZIO staining, a classical osmium impregnation. The staining electron-densely visualized individual Golgi lamellae, and their ultrastructure could stably be observed without any noticeable charging. The simple thresholding of the serial images enabled the efficient reconstruction of the labeled Golgi apparatus, which revealed plural Golgi apparatus in one hepatocyte. The combination of the heavy metal-based histochemistry of zinc, iodine and osmium (ZIO) staining and SBF-SEM was useful in the 3-dimensional observation of the Golgi apparatus at the whole-cell level because of two technical advantages: (i) visualization of the Golgi apparatus without any heavy metal staining and efficient acquisition of the block-face images without additional conductive staining or any devices for eliminating charging; (ii) easy identification of the staining and hassle-free, semi-automatic classification and segmentation by simple thresholding of the images. This novel approach could elucidate the topographic characteristics of the Golgi apparatus in hepatocytes.

An optimized approach using cryofixation for high-resolution 3D analysis by FIB-SEM

Compared with conventional two-dimensional transmission electron microscopy (TEM), focused ion beam scanning electron microscopy (FIB-SEM) can provide more comprehensive 3D information on cell substructures at the nanometer scale. Biological samples prepared by cryofixation using high-pressure freezing demonstrate optimal preservation of the morphology of cellular structures, as these are arrested instantly in their near-native states. However, samples from cryofixation often show a weak back-scatter electron signal and bad image contrast in FIB-SEM imaging. In addition, it is impossible to do large amounts of heavy metal staining. This is commonly achieved via established osmium impregnation (OTO) en bloc staining protocols. Here, we compared the FIB-SEM image quality of brain tissues prepared using several common freeze-substitution media, and we developed an approach that overcomes these limitations through a combination of osmium tetroxide, uranyl acetate, tannic acid, and potassium permanganate at proper concentrations, respectively. Using this optimized sample preparation protocol for high-pressure freezing and freeze-substitution, perfect smooth membrane morphology, even of the lipid bilayers of the cell membrane, was readily obtained using FIB-SEM. In addition, our protocol is broadly applicable and we demonstrated successful application to brain tissues, plant tissues, Caenorhabditis elegans, Candida albicans, and chlorella. This approach combines the potential of cryofixation for 3D large volume analysis of subcellular structures with the high-resolution capabilities of FIB-SEM.

Establishing sample-preparation protocols for X-ray phase-contrast CT of rodent spinal cords: Aldehyde fixations and osmium impregnation

BackgroundDense and unbiased cellular-resolution representations of extended volumetric central nervous system soft-tissue anatomy are difficult to obtain, even in experimental post-mortem settings. Interestingly, X-ray phase-contrast computed tomography (X-PCI-CT), an emerging soft-tissue-sensitive volumetric imaging technique, can provide multiscale organ- to cellular-level morphological visualizations of neuroanatomical structure. New MethodHere, we tested different nervous-tissue fixation procedures, conventionally used for transmission electron microscopy, to better establish X-PCI-CT-specific sample-preparation protocols. Extracted rat spinal medullas were alternatively fixed with a standard paraformaldehyde-only aldehyde-based protocol, or in combination with glutaraldehyde. Some specimens were additionally post-fixed with osmium tetroxide. Multiscale X-PCI-CT datasets were collected at several synchrotron radiation facilities, using state-of-the-art setups with effective image voxel sizes of 3.03 to 0.33 μm3, and compared to high-field magnetic resonance imaging, histology and vascular fluorescence microscopy data. ResultsMultiscale X-PCI-CT of aldehyde-fixed spinal cord specimens resulted in dense histology-like volumetric representations and quantifications of extended deep spinal micro-vascular networks and of intra-medullary cell populations. Osmium post-fixation increased intra-medullary contrast between white and gray-matter tissues, and enhanced delineation of intra-medullary cellular structure, e.g. axon fibers and motor neuron perikarya. Comparison with Existing MethodsVolumetric X-PCI-CT provides complementary contrast and higher spatial resolution compared to 9.4 T MRI. X-PCI-CT’s advantage over planar histology is the volumetric nature of the cellular-level data obtained, using samples much larger than those fit for volumetric vascular fluorescence microscopy. ConclusionsDeliberately choosing (post-)fixation protocols tailored for optimal nervous-tissue structural preservation is of paramount importance in achieving effective and targeted neuroimaging via the X-PCI-CT technique.

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

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

Osmium impregnation detection of pulmonary intravascular fat in sudden death: A study of 65 cases

Pulmonary fat embolism is widely recognised in forensic pathology. Pulmonary fat embolism requires mobilisation of free fat, entry of free fat into the circulation and lodging of fat globules in fine venous capillaries. This paradigm of fat embolisation has been used to support the evidence of antemortem fat depot disruption when the presence of intravascular fat is confirmed at autopsy. However, sporadic reports of intravascular fat in various medical conditions, which contradict the above mechanism, have opened questions about the alternative pathogenesis. In this study, 65 cases of sudden deaths were examined for the presence of pulmonary intravascular fat (PIF) by osmium impregnation. Cases were selected based on the criteria that were designed to eliminate the possible confounding effect from medical intervention or postmortem changes. Slides were graded based on their ease of search and only the fat droplets confined by the blood vessel or capillary wall were considered as a positive finding. The results show surprisingly high PIF incidences of varying degrees in all the categories of sudden deaths. Further study is needed to devise criteria for diagnosis of fatal fat embolism since the histological appearance of the high-grade PIF in natural sudden death may not be easily distinguishable from the traumatic fat embolism.

High-contrast en bloc staining of neuronal tissue for field emission scanning electron microscopy

Conventional heavy metal poststaining methods on thin sections lend contrast but often cause contamination. To avoid this problem, we tested several en bloc staining techniques to contrast tissue in serial sections mounted on solid substrates for examination by field emission scanning electron microscopy (FESEM). Because FESEM section imaging requires that specimens have higher contrast and greater electrical conductivity than transmission electron microscopy (TEM) samples, our technique uses osmium impregnation (OTO) to make the samples conductive while heavily staining membranes for segmentation studies. Combining this step with other classic heavy metal en bloc stains, including uranyl acetate (UA), lead aspartate, copper sulfate and lead citrate, produced clean, highly contrasted TEM and scanning electron microscopy (SEM) samples of insect, fish and mammalian nervous systems. This protocol takes 7-15 d to prepare resin-embedded tissue, cut sections and produce serial section images.

Tannic acid-mediated osmium impregnation after freeze-substitution: A strategy to enhance membrane contrast for electron tomography

In this technical note we report a tannic acid-mediated osmium impregnation method that, applied after freeze-substitution, increases membrane contrast in cells for transmission electron microscopy and tomography studies. The general staining that is achieved allows visualization of organelles, plasma membrane and associated specializations (e.g. caveolae) in non-post-stained plastic sections by conventional transmission electron microscopy. In combination with electron tomography it results in membranes with a proper contrast and equal staining pattern through the depth of the tomograms. The protocol that we contribute can serve as starting point for those willing to improve the membrane contrast of their specimens or to make 3D studies on the architecture of membranous compartments by electron tomography.

Rat Myocellular and Perimysial Intramuscular Triacylglycerol: A Histological Approach

There is controversy as to the use of intramuscular triacylglycerol (IMTAG) during exercise and to whether endurance training increases its utilization, despite the various methodologies used to address these questions. We used a histological-morphometrical approach to study the relative contribution of the two compartments of IMTAG storage, intramyocellular, and perimysial adipocytes, during exercise in sedentary and endurance-trained rats. After osmium impregnation, the soleus (SOL) and gastrocnemius (GAS) were studied under light and electron microscopy. IMTAG content (after Triton WR1339 treatment or not) and 14C-oleate incorporation into the muscles were studied. In GAS, training, but not exercise alone, decreased extramyocellular lipid (P < 0.001 vs sedentary), an effect not found for SOL. Both muscles presented reduced lipid inclusion number (P < 0.001) and area (P < 0.05), immediately after exercise in sedentary and trained rats. For SOL, a greater number (P < 0.001 vs sedentary) of inclusions was found 24 h after exercise in trained rats. Triton WR1339 treatment decreased IMTAG content 12 h after exercise in SOL (but not in GAS), in sedentary (33%), and trained rats (52%). The multi-analytical approach adopted allowed the discernment between the IMTAG compartments and provided evidence for an effect of training upon storage of lipid in perimysial adipocytes in rat gastrocnemius, as well as clearly showed that the IMTAG mobilized during submaximal exercise in sedentary and trained rats derives from intramyocellular lipid, both in SOL and GAS. Moreover, the reposition of these stores 12 h after exercise was shown to be different in GAS and SOL, as plasma triacylglycerol clearly contributed to the process only in the latter, possibly reflecting the differences in lipoprotein lipase activity in the muscles reported by others.

Buds of the Golgi apparatus in parotid acinar cells.

The Golgi apparatus (GA) is a membranous organelle composed of stacked cisterns with associated vesicles. This study was undertaken to determine its origin in rat parotid acinar cells. The morphogenesis of the GA could be recognized in the developmental process as well as in mitotic division of cells. EM studies depicted an aggregation of small vesicles in the early stage of postnatal development or mitosis, that appeared to be the rudimental element of GA. Brefeldin A induced rapid degradation of the cisternal structure to vesicular aggregates. Reconstruction of the GA structure based on these remnant vesicles was observed upon removal of the drug. Similar membranous assembly could be observed after destruction of microtubules. These membranous aggregates presumably corresponded to 'buds of the GA' in parotid acinar cells. However, conventional cytochemical markers for GA were not detected on such immature form of GA. We found that the GA matrix protein GM130 and osmium reductivity (a classical marker for cis-Golgi elements) were consistently localized in the GA elements. Therefore, immunohistochemical distribution of GM130 and osmium impregnation of parotid acinar cells were studied under various dynamic conditions that produced structural modification of the GA.

The subglandular tissue of Hibiscus rosa-sinensis nectaries

Summary The nectary of Hibiscus rosa–sinensis is subtended by subglandular parenchymatous tissue extending from the bases of the nectary hairs to the depth of about 30 cells. Several of these cells contain large crystals. The subglandular tissue shows a well developed vascularization. In principal two cell types were found. When Sudan Black B or the selective osmium impregnation method were applied, many cells, especially those around the parenchymatous tissue and under the vascular bundles, were stained brown to black. They can be distinguished from neighboring cells by their large size and intense absorption of stains or high electron density. They deposit in their large vacuoles osmiophilic substances, presumably oil and have been named oil cells. Groups of oil droplets, in contact with the nucleus, were also present in the parenchymatous and oil cells. Tissue fixation in glutaraldehyde and osmium tetroxide vapors revealed granular electron dense remnants within oil cells. Another cell type, the mucilage cells, was found in the subglandular tissue. These cells became evident after the application of histochemical methods for polysaccharides. Autoradiographical study showed that, externally applied labelled glucose incorporated into the mucilage and cell walls. Mucilage plays an important role in the regulation of water economy, which is decisive in nectar secretion. The oil cells mainly protect the sugar containing tissues (phloem, parenchyma) against herbivores.

Energy Filtering Imaging Of Thick Biological Specimens With In-Column "Omega"-Filter Microscopes.

Abstract Preliminary results were obtained from thick sections of biological specimens using 200 keV electron microscopes with an in-column “Omega” energy filter. Images of selectively stained specimens prepared by osmium impregnation of frog spinal ganglia, with thickness ranging from 0.5 μm to 2.5 μm, show drastic improvement in image quality with the use of the energy filter. Two instruments with Omega filters were used. One was a non-commercial research prototype at JEOL Ltd. equipped with a LaB6 gun and the other was a commercial instrument (JEM-2010FEF) with a field-emission gun, installed at Kyushu University. We found that, although the field-emission gun offered a brightness 2 to 3 orders of magnitude higher, the LaB6 gun was more suitable for studying thick specimens, primarily due to the higher beam current of the LaB6 gun.

Evidence of a tubular system for transendothelial transport in arterial capillaries of the rete mirabile.

The arterial endothelial cells of the rete capillaries of the eel were examined by transmission electron microscopy on thin sections, on freeze-fracture replicas, by scanning electron microscopy, after cytochemical osmium impregnation and perfusion with peroxidase. The study revealed the existence of membrane-bound tubules and vesicles that open at both the luminal and abluminal poles of the cell and at the level of the intercellular space. The tubules are straight or present successive dilations and constrictions. They branch in various directions and intrude deeply into the cell cytoplasm, forming a complex tubular network within the cell. Immunocytochemical techniques were applied on immersion-fixed tissues and on perfusion of the capillaries with albumin and insulin. These demonstrated that the tubular-vesicular system is involved in the transport of circulating proteins. Furthermore, protein A-gold immunocytochemistry has revealed the association of actin with the membranes of this system. On the basis of these results, we suggest that the transendothelial transport of serum proteins takes place by a transcytotic process through a membrane-bound tubular-vesicular system and is equivalent to the large pore system presumed from functional studies.

Globular and fibrous structure in barley chromosomes revealed by high-resolution scanning electron microscopy.

Barley chromosomes were prepared for high-resolution scanning electron microscopy using a combination of enzyme maceration, treatment in acetic acid and osmium impregnation using thiocarbohydrazide. Using this technique, the three-dimensional ultrastructure of interphase nuclei and mitotic chromosomes was examined. In Interphase, different levels of chromatin condensation were observed, consisting of fibrils 10 nm in diameter, 20- to 40-nm fibres and a higher order complex. In prophase, globular and strand-like structures composed of 20- to 40-nm fibres were dominant. As the cells progressed through the cell cycle and the chromatin condensed, globular and strand-like structures (chromomeres) were coiled and packed to form chromosomes. Chromomeres were observed as globular protuberances on the surface of metaphase chromosomes. These findings indicate that the chromomere is a fundamental substructure of the higher order architecture of the chromosome. In the centromeric region, there were no globular protuberances, but 20- to 40-nm fibres were folded compactly to form a higher level organization surrounding the chromosomal axia.

Ultrastructural and cytochemical study of the Golgi complex of molluscan (Mytilus galloprovincialis) digestive cells

The present study has been performed to investigate the ultrastructure and subcellular distribution of several marker enzymes within the Golgi complex of digestive cells of the mussel, Mytilus galloprovincialis. This cell type is involved in the intracellular digestion of food and contains a well-developed lysosomal-vacuolar system and a characteristic cup-shaped Golgi complex. The number and degree of compaction of the tubulo-filamentous structures extending across the distended regions of the cisternae, the number of Golgi stacks per cell and the number of cisternae per stack vary with season and nutritional status of the animals. The degree of compaction of the tubulo-filamentous structures defines the cis- and trans-portions of the Golgi apparatus, the less compacted structures being associated with the cis-portion. Acid phosphatase and arylsulphatase activities are located within some tubules and vesicles in the trans-Golgi network and in some membrane-bounded organelles comprising the lysosomal-vacuolar system. The tubulo-filamentous structures of the dilated rims are reactive for arylsulphatase activity only when located in the trans-portion. Osmium impregnation techniques label all the cisternae of the Golgi complex. No nicotinamide adenine dinucleotide phosphatase activity is present in Golgi bodies or other associated structures in digestive cells. Thiamine pyrophosphatase activity only rarely occurs in some elongate tubules that are interpreted as belonging to trans-cisternae.

Maleate modifies apical endocytosis and permeability of endoplasmic reticulum membranes in kidney tubular cells.

Previous studies have shown that histochemical modifications of the endoplasmic reticulum in epithelial cells might be related to their transport function. We have examined the effect of sodium maleate, which produces generalized transport derangement reminiscent of Fanconi syndrome, on the organization, morphology and enzyme activities of endoplasmic reticulum in rat kidney cells. The osmium impregnation technique has revealed that apical vacuoles increase in volume and in number in most proximal tubule cells, and contain osmium deposits. Osmium impregnation of the endoplasmic reticulum is much reduced. In vitro studies, performed with isolated microsomes, show NADPH cytochrome c reductase activity in both normal and maleate-treated rats. As revealed by vanadate, Ca+-ATPase activity in isolated microsomes is unnaffected by maleate but the vanadate-insensitive or passive component of calcium uptake increases particularly later in the response. Therefore, the remaining calcium uptake in the presence of vanadate is indeed passive; in vivo maleate administration also appears to increase the passive entry of calcium into the microsomal compartment. The morphological and histochemical alterations of the endoplasmic reticulum cisternae occur rapidly and with a similar time course to the transport defects, suggesting that this organelle plays a role in transcellular transport. Maleate may directly affect the endoplasmic reticulum membranes whereby passive permeability to calcium is increased. The endocytotic apparatus and possibly exocytosis phenomena are modified by maleate as shown by the increased vacuolization and the presence of black osmium deposits in vacuoles.

Scanning Tunneling Microscopic Study of Osmium-Impregnated Collagen

The vapor-phase osmium impregnation method was applied to increase the conductivity of the collagen specimens used in scanning tunneling microscopy (STM), since the application of STM to biological materials has been limited by the poor conductivity, irregular sample geometry, and instability of these materials. The impregnation method was applied to collagen fibrils attached to highly oriented pyrolytic graphite, and the STM images obtained were compared with those of routinely processed uncoated specimens. The osmium-impregnated collagen fibrils showed a periodicity of about 67 nm, which was also observed on the negative-stained specimens by transmission electron microscopy. In addition, the periodicity of 3.1 nm, which corresponded to the helical cycle of the 3 alpha-chains within collagen, was clearly demonstrated by STM. Moreover, the comparative observations of collagen specimens without osmium impregnation disclosed the left-handed helical structure with a periodicity of 3.1 nm in a single collagen filament.

Organization of the endoplasmic reticulum in renal cell lines MDCK and LLC-PK1

The spatial organization of the endoplasmic reticulum has been studied in two renal cell lines, MDCK and LLC-PK1, which originate from the distal and proximal portions of the mammalian nephron, respectively, and which form a polarized epithelium when they reach confluence in tissue culture. The two renal cell lines, grown to confluence on either solid or permeable supports, were investigated by fluorescence microscopy, confocal microscopy, and transmission electron microscopy. Fluorescence labeling of the endoplasmic reticulum was achieved using the cationic fluorescent dye DIOC6 (3). In order to differentiate fluorescent labeling of the endoplasmic reticulum from that of the mitochondria, cells were also labeled with rhodamine 123. For electron microscopy, the spatial organization of the endoplasmic reticulum was examined in thick sections using the long-duration osmium impregnation technique or the ferrocyanide/osmium technique. In both cell lines, the endoplasmic reticulum formed an abundant tubular network of canaliculi that frequently abutted the basolateral domain of the plasma membrane and occasionally the apical membrane. Elements of the endoplasmic reticulum were also found in close proximity to mitochondria that, as in the nephron, formed branched structures. Canaliculi appeared circular or flattened and had an inner diameter of 10-70 nm for MDCK cells and 20-90 nm for LLC-PK1 cells. Such a three-dimensional organization might facilitate the translocation of defined lipid species between the endoplasmic reticulum and the plasma membrane, and between the endoplasmic reticulum and mitochondria.

The Golgi apparatus of the ciliated protozoon Ichthyophthirius multifiliis: A cytochemical study

Summary The trophozoites of the ciliated protozoon Ichthyophthirius multifiliis , a fish parasite, possess many small dictyosomes. Each one is associated with a modified cisterna of the endoplasmic reticulum. AcPase, TPPase and NDPase activities were located at the trans face of these dictyosomes. In the cell cortex, thick-membrane cisternae with coated dilated rims are abundant and possess AcPase, TPPase and NDPase activities. Our results suggest that the thick membrane cisternae of ciliated protozoa may be a terminal compartment of the Golgi apparatus, equivalent to the trans-Golgi network. After osmium impregnation all dictyosomal cisternae are filled with electron-dense deposits that are also found in thick-membrane cisternae and in some alveolar sacs. The presence of TPPase and NDPase activities in the alveolar sacs may be related to glycosyltransferase activities, as proved in other cells. Our new results suggest that the previous detection of AcPase activity in the alveolar sacs was an artefact.

Changes of cytochemical properties in the Golgi apparatus during in vivo differentiation of the ameloblast in developing rat molar tooth germs.

The cytochemical changes of the Golgi stacks occurring concomitantly with cell differentiation were examined in ameloblasts of developing rat molar tooth germs using osmium impregnation and cytochemistry with nicotinamide adenine dinucleotide phosphatase (NADPase), thiamine pyrophosphatase (TPPase), and acid phosphatase (Acpase). NADPase, TPPase, and Acpase activities were already present in the Golgi stacks of the inner enamel epithelial cells, the undifferentiated form of the ameloblast: NADPase activity existed in the medial Golgi cisternae, TPPase activity in the trans Golgi cisternae, and Acpase activity in almost all cisternae and strongly in the trans-most cisterna of the Golgi stack. At this stage, however, osmium deposits after impregnation were not observed in the cisterna of Golgi stacks but were present in some small vesicles. These vesicles were located throughout the cytoplasm. Osmiophilic cisternae in the Golgi stacks were apparent for the first time at the stage when the Golgi apparatus developed and migrated to the region distal to the nucleus with the progression of cell differentiation. These findings indicate that the cis subcompartment of the Golgi apparatus was incomplete in the inner enamel epithelial cells with regard to appearance of its cytochemical property, as compared with the medial and trans subcompartments. It is suggested that the cis compartment of the Golgi stack may be completed only in the last stage of the compartmentalized Golgi organization during differentiation of the ameloblast.

Changes in cell polarity during mitosis in rat parotid acinar cells.

We studied the ultrastructure and cytochemistry of mitotic parotid acinar cells in vivo after induction of mitosis by isoproterenol injection. With entrance of the cells into the division cycle, the Golgi apparatus lost its characteristic stacked structure and internal polarity among the cisternae, appearing as fragments distributed throughout the cytoplasm. These fragments consisted of electron-lucent vesiculotubular structures and electron-dense 70-nm vesicles; neither component showed thiamine pyrophosphatase activity, a marker for trans cisternae of the Golgi apparatus, but the 70-nm vesicles showed a positive reaction for osmium impregnation, indicating retention of the cis nature. The rough endoplasmic reticulum was dilated and fragmented. Recovery of the structure of Golgi apparatus and rearrangement of rough endoplasmic reticulum occurred in daughter cells during telophase. These changes were the same as those observed after drug-induced inhibition of protein transport. The secretory granules were not dispersed but were divided into two groups with which centrioles were closely associated. Both groups migrated with the centrioles as far as the next interphase. The distribution of 5'-nucleotidase on the luminal plasma membrane showed no change during the process of division, thus demonstrating that surface polarity was maintained during mitosis. These changes in organelle structure and distribution may be due to the conversion of cell function from a secretory to a mitotic action.