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Low-vacuum scanning electron microscopy (LV-SEM) is a powerful tool that allows to observe light microscopic specimens with periodic acid-silver methenamine (PAM) staining at a higher magnification, simply by removing the coverslip. However, it is not suitable for observation of immunohistochemistry (IHC) using 3,3′-diaminobenzidine (DAB) due to insufficient backscattered electron image. Traditional heavy metal enhancement techniques for DAB in IHC, (1) osmium tetroxide and iron, (2) cobalt, (3) methenamine silver (Ag), (4) gold chloride (Gold), and (5) both Ag and Gold (Ag + Gold), were examined by LV-SEM. Tissue specimens from Thy1.1 glomerulonephritis rat kidney stained with α-smooth muscle actin and visualized with DAB were enhanced by each of these enhancement methods. We found, in light microscopic and LV-SEM, that the enhancement with Ag, Gold, or Ag + Gold had better intensity and contrast than others. At a higher magnification, Ag + Gold enhancement showed high intensity and low background, although only Ag or Gold enhancement had nonspecific background. Even after observation by LV-SEM, the quality of specimens was maintained after remounting the coverslip. It was also confirmed that Ag + Gold enhancement could be useful for IHC using clinical human renal biopsy. These findings indicate that Ag + Gold provided an adequate enhancement in IHC for both LM and LV SEM observation.

Lobomycosis is a skin infection produced by the fungus Lacazia loboi, which mainly affects some indigenous and afro-descendant populations in Tropical America. We previously reported the comparative effect of osmium tetroxide (OsO4 ) and ruthenium tetroxide (RuO4 ) in the electron microscopy (EM) of other related microorganisms. The objective of this study is to compare the effect of postfixation with OsO4 and RuO4 in the ultrastructure of L. loboi yeasts. Skin biopsies on patients diagnosed with lobomycosis were fixed in glutaraldehyde at 3% and postfixed in the following solutions: (a) 1% OsO4 , (b) 0.2% RuO4 , and (c) OsO4 at 1% followed by RuO4 at 0.2%. They were then processed using the conventional method for EM. Unlike OsO4, the treatment with RuO4 revealed different shades of gray and electron dense bands in the cell wall and other cell components of L. loboi. The most notable finding was the presence of radial filamentous structures around the yeast, which made the image look like the sun. Postfixation with RuO4 revealed ultrastructural details that had not been previously reported for L loboi. The combined use of OsO4 and RuO4 in EM of microorganisms with cell walls can be useful to evaluate the effect of microbicide substances.

Chestnut rose, R. roxburghii Tratt. (Rosaceae) (RR) is an important crop in China due to its nutritional and medicinal values. RR frequently produces trichomes on the surfaces of a diverse range of organs, however a genetic component exists to the control of trichome development, with some cultivars having significantly fewer trichomes to others. Certain varieties have fruits that are thickly covered with macroscopic trichomes, which is an undesirable trait for fruit processing and consumption. However, smooth-fruit cultivars exist, such as R. roxburghii Tratt. f. esetosa Ku (RRE). Despite their economic importance, the anatomical features of trichomes have not been explored in detail for these two chestnut rose germplasms. Here, we investigate the ultrastructure of trichomes distributed on the stem, sepal, and fruit of RR and RRE using transmission electron microscopy (TEM). The internal structure of stem prickle trichomes in RR and RRE was oval in shape and did not contain nucleoli or other organelles. The cell walls of stem prickles in RR are thick and the intercellular spaces occupied with liquid, whereas the cells wall of stem prickles in RRE are thin and have air-filled intercellular spaces. The cells of sepal acicular trichomes in RR and glandular trichomes (GTs) of sepals in RRE had similar vacuole sizes, cytoplasm content, intercellular spaces, and arrangement of plastids within cells. However, there were osmiophilic granules present in the GTs of RRE. The flagelliform trichomes in the sepals of the two germplasms are composed of oval or rod-shaped cells. Although the flagelliform trichomes in the sepals of the two germplasms had a similar internal structure, and both contained starch grains and plastids with visible thylakoid membranes, the flagelliform trichomes in the sepals of RR had a thinner cell wall and a higher proportion of cytoplasm which was more evenly distributed across the cell. There were granules that stained heavily with osmium tetroxide which occurred infrequently in the flagelliform trichomes of sepals in RRE but were not observed in RR. On the acicular trichomes of fruit in RR, the flagelliform trichomes and the GTs of fruit in RRE shared similar cell morphology, arrangement and vacuole size as well as intercellular space. Both the fruit flagelliform trichomes and GTs in RRE contain granules which stain heavily with osmium tetroxide, and the GTs contain plastids and starch grains. These differences in trichome cell ultrastructure may be related to developmental processes or biological functions of the trichomes. These results also suggest that the two chestnut rose germplasms are good candidates for further study of trichome ontogeny in the genus and subsequent breeding of the smooth organ trait in this species.

Leafy liverwort is one of the most abundant and diverse plants in Indonesia. Their high variation and beneficial secondary metabolites contained in the oil bodies have attracted researchers' attention. The ultrastructural analysis of leafy liverworts is important as a means of species identification and also for further exploration of their oil bodies. However, the optimization of the preparation steps for observing leafy liverworts by SEM is necessary to avoid sample destruction. Fixation and drying play important roles in maintaining a sample's structure as close to its natural state as possible. Thus, in this study, we evaluated the effect of 4% Osmium tetroxide (OsO4 ) and drying on leafy liverworts ultrastructure. Microlejeunea, Acrolejeunea, and Frullania were fixed with 2.5% glutaraldehyde. Some samples were then post-fixed with 4% OsO4 , while the rest were directly dehydrated with an ethanol series and then subjected to different drying methods, i.e. air drying, freeze drying, and drying with hexamethyldisilazane (HMDS). According to the data obtained, post-fixation with 4% OsO4 could better maintain the integrity of the samples and enhance the contrast of leafy liverwort SEM images. In addition, samples dried with HMDS showed more detailed structures compared to those that were air dried. Different ultrastructure were found among the different leafy liverworts observed by SEM. Our data suggested the advantages of SEM in providing ultrastructure information on leafy liverworts as well as the optimum conditions to observe them with less deformation. OsO4 post-fixation could enhance the contrast of leafy liverwort SEM images and maintain the structure of the samples. Drying with HMDS provided a convenient way for rapid SEM preparation with less structural distortion.

The use of high resolution, three-dimensional visualization has been receiving growing interest within life sciences, with non-invasive imaging tools becoming more readily accessible. Although initially useful for visualizing mineralized tissues, recent developments are promising for studying soft tissues as well. Especially for micro-CT scanning, several X-ray contrast enhancers are performant in sufficiently contrasting soft tissue organ systems by a different attenuation strength of X-rays. Overall visualization of soft tissue organs has proven to be possible, although the tissue-specific capacities of these enhancers remain unclear. In this study, we tested several contrast agents for their usefulness to discriminate between tissue types and organs, using three model organisms (mouse, zebrafish and Xenopus). Specimens were stained with osmium tetroxide (OsO4), phosphomolybdic acid (PMA) and phosphotungstic acid (PTA), and were scanned using high resolution microtomography. The contrasting potentials between tissue types and organs are described based on volume renderings and virtual sections. In general, PTA and PMA appeared to allow better discrimination. Especially epithelial structures, cell-dense brain regions, liver, lung and blood could be easily distinguished. The PMA yielded the best results, allowing discrimination even at the level of cell layers. Our results show that those staining techniques combined with micro-CT imaging have good potential for use in future research in life sciences.

OBJECTIVE A preliminary study to determine collagen fibril diameter (CF-ED) distribution on medial and lateral sides of cleft lip (CL). MATERIAL AND METHODS Tissue samples from medial and lateral sides of CL were fixed in 2.5% glutaraldehyde and 1% osmium tetroxide and embedded in Araldite CY212 resin for transmission electron microscopy. The analysis of CF-ED was performed using the ImageJ program. To characterize the packaging of collagen fibrils (CFs) in the two tissues, we estimated the collagen number density (CF-ND) and fibril-area-fraction (FAF). Differences in measurements across the two sides were calculated using Wilcoxon signed-rank test. RESULTS The CF-ED was statistically significantly (p

Knowledge about the three-dimensional fine structure of human heart, as a crucial vital organ of the body, is not only fascinating from the scientific or educational points of view, but has a very important clinical impact. Therefore, we decided to create a three-dimensional atlas of fine structure of the human heart. Tissue samples from ten human hearts were rinsed in phosphate-buffer solution, fixed by glutaraldehyde buffered solution, and post-fixed in osmium tetroxide solution. A gentle dehydration with ethanol in different concentration and drying at the critical point of CO2 were applied as next procedures. Non-conductive specimens were galvanized with thin gold layer and observed in scanning electron microscope. In this study, we present the three-dimensional ultrastructural architecture of the human heart from patients after myocardial infarction, end-stage failing heart as well as without apparent cardiac abnormalities at the time of autopsy. The results are presented as a histological atlas. Its images illustratively describe the fine structure of endocardium including the morphology of Purkyně (Purkinje) fibres, spatial arrangements of cardiac muscle cells inside myocardium or the arrangements of adipose tissue of epicardium. We present also figures of the ultrastructure of papillary muscles, intercalated discs as well as the connective tissue scars after myocardial infarction. The scanning electron microscopy could be a reliable technique to perform a more complete morphological data and to improve our knowledge of some pathological changes of tissues and cells, not always detected by conventional morphological examinations.

For nearly 50 years immunogold labeling on ultrathin sections has been successfully used for protein localization in laboratories worldwide. In theory and in practice, this method has undergone continual improvement over time. In this study, we carefully analyzed circulating protocols for postembedding labeling to find out if they are still valid under modern laboratory conditions, and in addition, we tested unconventional protocols. For this, we investigated immunolabeling of Epon-embedded cells, immunolabeling of cells treated with osmium, and the binding behavior of differently sized gold particles. Here we show that (in contrast to widespread belief) immunolabeling of Epon-embedded cells and of cells treated with osmium tetroxide is actually working. Furthermore, we established a "speed protocol" for immunolabeling by reducing antibody incubation times. Finally, we present our results on three-dimensional immunogold labeling.

Intramuscular fat (IMF) accumulates in muscles of the rotator cuff after tendon tear. The number and cross-sectional area of fat clumps and of adipocytes were quantified on osmium tetroxide stained sections of the proximal, middle and distal quarters of SSP muscles 4, 8 and 12 weeks after SSP tendon division in a rabbit model. Linear mixed-effects models were fitted to the data and statistical significance was evaluated by ANOVA. Both the number (P0.01). IMF accumulation was more important in the distal quarter of detached SSP muscle near tendon sectioning and characterized by increases of the number (P0.01). Interestingly, the number of adipocytes in the distal quarter increased (P

Intracellular accumulation of excessive glycogen or lipid in the liver occurs as a manifestation of metabolic dysfunction or drug-induced toxicity in humans or animals. The identification of individual components (lipids, glycogen, or water) involved is essential to the diagnosis of disease and pathogenesis. The aqueous fixatives and processing solvents used in paraffin embedding for routine hematoxylin and eosin stains remove both lipid and glycogen, leaving clear vacuoles of which the precise content is unknown in routine hematoxylin and eosin-stained sections. Traditional methods for detecting intracellular lipids and glycogen require two separate techniques, such as periodic acid-Schiff (PAS) reaction, with or without diastase digestion for glycogen, and osmium tetroxide (OsO4) post-fixation for lipid and utilization of different tissue sections. We describe here, for the first time, a novel OsO4/PAS dual-staining technique that permits a simple color-coded detection and direct visualization of both l...

We have used X-ray phase contrast tomography to resolve the structure of uncut, entire myelinated optic, saphenous and sciatic mouse nerves. Intrinsic electron density contrast suffices to identify axonal structures. Specific myelin labeling by an osmium tetroxide stain enables distinction between axon and surrounding myelin sheath. Utilization of spherical wave illumination enables zooming capabilities which enable imaging of entire sciatic internodes as well as identification of sub-structures such as nodes of Ranvier and Schmidt-Lanterman incisures.

Although scanning electron microscopy (SEM) has been broadly used for the examination of fixed whole insects or their hard exoskeleton-derived structures, including model organisms such as Drosophila, the routine use of SEM to evaluate vulnerable soft internal organs and tissues was often hampered by their fragile nature and frequent surface contamination. Here, we describe a simple four-step protocol that allows for the reliable and reproducible preparation of the larval and prepupal salivary glands (SGs) of Drosophila for SEM devoid of any surface contamination. The steps are to: first, proteolytically digest the adhering fat body; second, use detergent washes to remove contaminating coarse tissue fragments, including sticky remnants of the fat body; third, use nonionic emulsifying polysorbate emulsifiers to remove fine contaminants from the SGs surface; and fourth, use aminopolycarboxylate-based chelating agents to detach sessile hemocytes. Short but repeated rinses in 100 μL of a saline-based buffer between steps ensure efficient removal of remnants removed by each treatment. After these steps, the SGs are fixed in glutaraldehyde, postfixed in osmium tetroxide, dehydrated, critically point-dried, mounted on aluminum stubs, sputter coated with gold-palladium alloy and examined in the SEM.

Flashing fireflies were permitted to breathe osmium tetroxide vapor, after which the lanterns were removed and the sites of absorption of the osmium into the tissues were detected in two ways: (1) by sonication to remove soft tissues, that is, those that had not been fixed by the osmium gas, and (2) by intensification with thiocarbohydrazide and silver nitrate, in a modification of the osmium-thiocarbohydrazide-osmium (OTO) stain technique. The results of both procedures indicate that the gas first enters into the tissues at the level of the tracheoles. These findings may be interpreted as underscoring the importance of the tracheolar cell and the tracheal end organ in the control of oxygen entry into the lantern tissues, and the implications of the results in the oxygen regulation theory of flash control are discussed.

Peripheral nerves extend throughout the body, innervating target tissues with motor or sensory axons. Due to widespread distribution, peripheral nerves are frequently damaged because of trauma or disease. As methods and strategies have been developed to assess peripheral nerve injury in animal models, function and regeneration, analyzing the morphometry of the peripheral nerve has become an essential terminal outcome measurement. Toluidine blue staining of nerve cross sections obtained from resin embedded nerve sections is a reproducible method for qualitative and quantitative assessments of peripheral nerves, enabling visualization of morphology number of axons and degree of myelination. This technique, as with many other histological methods, can be difficult to learn and master using standard written protocols. The intent of this publication is therefore to accentuate written protocols for toluidine blue staining of peripheral nerves with videography of the method, using sciatic nerves harvested from rats. In this protocol, we describe in vivo peripheral nerve fixation and collection of the tissue, and post-fixation with 2% osmium tetroxide, embedding of nerves in epoxy resin, and ultramicrotome sectioning of nerves to 1-2μm thickness. Nerve sections then transferred to a glass slide and stained with toluidine blue, after which they are quantitatively and qualitatively assessed. Examples of the most common problems are shown, as well as steps for mitigating these issues.

Introduction: Knowledge of the neuro-anatomical architecture of the inner ear contributes to the improvement and development of cochlear and vestibular implants. The present knowledge ismainly based on two-dimensional images (histology) or derived models that simplify the complexity of this architecture. This study investigated the feasibility of visualizing relevant neuro-anatomical structures of the inner ear in a dynamic three-dimensional reproduction, using a combination of staining, micro-CT imaging and an image processing algorithm. Methods: Four fresh cadaveric temporal bones were postfixed with osmium tetroxide (OsO4) and decalcified with EDTA. Micro-CT was used for scanning at 10 mu m (4 scans) and 5.5 mu m (1 scan) voxel resolution. A new image processing algorithm was developed and the scans were visualized in open source software. Results: OsO4 enhanced the contrast in all scans and the visualization was substantially improved by the image processing algorithm. The three-dimensional renderings provided detailed visualization of the whole inner ear. Details were visible up to the size of individual neurons, nerve crossings and the specific neuro-anatomical structures such as the tunnel of Corti. Conclusion: The combination of OsO4, micro-CT and the proposed image processing algorithm provides an accurate and detailed visualization of the three-dimensional micro-anatomy of the human inner ear.

International audience; Bone homeostasis is influenced by the bone marrow adipose tissue (BMAT). BMAT distribution varies from one anatomical location in the skeleton to another. We developed an advanced microfocus computed tomography imaging and analysis protocol that allows accurate alignment of both the BMAT distribution and bone micro-architecture as well as calculation of the distance of the BMAT adipocytes from the bone surface. Using this protocol, we detected a different spatial BMAT distribution between the rat tibia and mandible: in the proximal metaphysis of the tibia a large amount of BMAT (~ 20% of the total BMAT) was located close to the bone surface (

The aim of our study is to obtain, as accurately as possible, porcine ocular tissue visualization using microtomography (micro-CT) method. We propose image contrast enhancement by different staining procedures with combination of micro-CT scanning. Porcine eye globes were investigated with Bruker-SkyScan 1172 micro-CT. We used 4F1G and Bouin's as sample fixation solutions and tincture of iodine, 100% Lugol, phosphotungstic acid and 1% osmium tetroxide solutions for staining. Quantitative and qualitative analysis was performed based on micro-CT reconstruction images histograms and 3D volume rendering models of investigated samples. This investigation showed that staining methods improved micro-CT image quality in case of ocular anatomy visualization. Characteristic profiles of the grey level distributions and quality of the cross-section and 3D volume rendering images confirmed the staining effect. Most significant contrast enhancement was obtained after 96 h staining in osmium tetroxide and Lugol solutions. The images of eye anatomical structures were characterized: cornea, lens, iris, ciliary body, vitreous, retina, choroid and sclera, vasculature and optic nerve. Staining of porcine eye globes used in this work leads to quality improvement of the micro-CT imaging. The most contrast images were obtained for Lugol and osmium tetroxide solutions. Different affinity of staining solutions to eye anatomical structures has been observed in the obtained images. Osmium tetroxide provides sharper image of conjunctiva, sclera, choroid, retina, iris and ciliary body structure. Lugol staining leads to more accurate vessels, cornea and optic nerve imagining.

Scanning electron microscopy (SEM) is commonly used in the analysis of scaffolds morphology, as well as cell attachment, morphology and spreading on to the scaffolds. However, so far a specific methodology to prepare the alginate hydrogel (AH) scaffolds for SEM analysis has not been evaluated. This study compared different methods to fix/dehydrate cells in AH scaffolds for SEM analysis. AH scaffolds were prepared and seeded with NIH/3T3 cell line; fixed with glutaraldehyde, osmium tetroxide, or the freeze drying method and analyzed by SEM. Results demonstrated that the freeze dried method interferes less with cell morphology and density, and preserves the scaffolds structure. The fixation with glutaraldehyde did not affect cells morphology and density; however, the scaffolds morphology was affected in some level. The fixation with osmium tetroxide interfered in the natural structure of cells and scaffold. In conclusion the freeze drying and glutaraldehyde are suitable methods for cell fixation in AH scaffold for SEM, although scaffolds structure seems to be affected by glutaraldehyde.

The El Plomo mummy was a pre-Columbian Incan child who was found mummified in the Andes Mountains above an altitude of 17,700 feet. In the environment, natural mummification occurred due to low temperatures and strong winds. Dating measurements (relative dating) by experts from the National Museum of Natural History of Chile established that the mummified body corresponds the Inca period (1,450 to 1,500 AD). In 2003, the body was transferred to the University of Chile Medical School for exhaustive medical examination. Tissue samples from the right quadriceps muscle were extracted and fixed in glutaraldehyde and postfixed in osmium tetroxide to obtain ultrathin sections to be observed by transmission electron microscope. Images were recorded on photographic paper, digitalized and analyzed by experts on morphology. Results showed a preservation of cell boundaries in striated muscle cells, but specific subcellular organelles or contractile sarcomeric units (actin and myosin) were unable to be recognized. However, the classical ultrastructural morphology of the polypeptide collagen type I was preserved intact both in primary and secondary organization. Therefore, we concluded that the process of natural mummification by freezing and strong winds is capable of damaging the ultrastructure of muscle cells and preserving collagen type I intact.

The retina is a part of the central nervous system in terms of histological and embryological aspect. It is a thin layer of neural tissue lining the inner eye. This study assessed the histomorphometric changes in the thicknesses of the retina and its layers using light microscope (LM) and retinal photoreceptor cells using transmission electron microscope (TEM) as a consequence of diabetes and effect of aloe vera gel extract in male rats. Thirty Sprague-Dawley matured male rats (175 ± 25 g) in two age groups (12 and 16 weeks) were divided into three main groups as control, diabetic, and diabetic receiving 400 mg/kg aloe vera extract. There were two subgroups in each group. For inducing diabetes, a single dose of streptozotocin (STZ) (50 mg/kg) was injected through IP route. Their blood sugar was measured by a glucometer before STZ administration and 24 h thereafter. At the end, animals were anesthetized with sodium thiopental (40 mg/kg) via IP injection. The retina was collected for tissue processing following dissection of eyes, fixed in 4 % glutaraldehyde, postfixed in osmium tetroxide 1 %, dehydrated, and then embedded in TAAB resin. Semithin sections (1 μm) were stained with toluidine blue stain and viewed under light microscope. Ten slides were prepared from each animal. Ultrathin sections were prepared, stained, and viewed under TEM. The blood glucose levels and body weight in treated rats were reduced in comparison to diabetic groups. The neural retina thickness and its layers were also different. In the aloe vera-treated group, the thickness of the retina and its layers retained their normal histological structures. In diabetic groups, the vesicular mitochondria and degenerated cristae and dense nucleus were observed in photoreceptor cells. After treatment with aloe vera extract, cristae gained partially their arrangement and chromatin spread. It is concluded that the degenerated retina and the thickness of different layers of diabetic retina could be affected with long-term treatment of aloe vera gel extract.

We studied the effectiveness of heat-induced antigen retrieval (HIAR) in conventionally processed, epon-embedded specimens and the mechanisms of HIAR in the specimens. Frozen sections were first immunostained to examine the possibility of using HIAR for 18 antigens to avoid the effects of epoxy resin embedment. The antigenicity of 7 out of 18 antigens was retrieved with glutaraldehyde fixation followed by osmium tetroxide treatment whereas none were retrieved with glutaraldehyde fixation without post-osmication. Six antigens also exhibited positive immunostaining in semi-thin epon sections when the sections were deplasticized with sodium ethoxide followed by autoclaving. In the immunoelectron microscopy with the post-embedding method, positive reactions with fine ultrastructures were obtained using HIAR without deplasticization. These results suggested that osmium tetroxide binds to ethylene double bonds (which are introduced into protein crosslinks by glutaraldehyde) and forms an extremely stable resonance interaction with the Schiff bases, thus destabilizing the protein crosslinks. Heating also further degrades these crosslinks. The present study demonstrated that archival epon blocks can be useful resources for immunohistochemical studies for both light and electron microscopy.

Lamellar bone is the most common bone type in humans. The predominant components of individual lamellae are plywood-like arrays of mineralized collagen fibrils aligned in different directions. Using a dual-beam electron microscope and the Serial Surface View (SSV) method we previously identified a small, but significantly different layer in rat lamellar bone, namely a disordered layer with collagen fibrils showing little or no preferred orientation. Here we present a 3D structural analysis of 12 SSV volumes (25 complete lamellae) from femora of 3 differently aged human individuals. We identify the ordered and disordered motifs in human bone as in the rat, with several significant differences. The ordered motif shows two major preferred orientations, perpendicular to the long axis of the bone, and aligned within 10-20° of the long axis, as well as fanning arrays. At a higher organizational level, arrays of ordered collagen fibrils are organized into 'rods' around 2 to 3μm in diameter, and the long axes of these 'rods' are parallel to the lamellar boundaries. Human bone also contains a disordered component that envelopes the rods and fills in the spaces between them. The disordered motif is especially well-defined between adjacent layers of rods. The disordered motif and its interfibrillar substance stain heavily with osmium tetroxide and Alcian blue indicating the presence of another organic component in addition to collagen. The canalicular network is confined to the disordered material, along with voids and individual collagen fibrils, some of which are also aligned more or less perpendicular to the lamellar boundaries. The organization of the ordered fibril arrays into rods enveloped in the continuous disordered structure was not observed in rat lamellar bone. We thus conclude that human lamellar bone is comprised of two distinct materials, an ordered material and a disordered material, and contains an additional hierarchical level of organization composed of arrays of ordered collagen fibrils, referred to as rods. This new structural information on human lamellar bone will improve our understanding of structure-mechanical function relations, mechanisms of mechano-sensing and the characterizations of bone pathologies.

The palatine mucosa and filiform papillae of the dorsal tongue mucosae of rodents were examined using transmission electron microscopy (TEM) and high resolution scanning electron microscopy (HRSEM). In the HRSEM method, the samples were fixed in 2% osmium tetroxide, dehydrated in alcohol, critical point-dried, and coated with gold-palladium. In addition, the HRSEM technique was used for morphometric analysis (length, width, and length/width ratio of cocci and bacilli). For the TEM method, the tissues were fixed in modified Karnovsky solution (2.5% glutaraldehyde, 2% formalin in 0.1M sodium phosphate buffer, pH 7.4) and embedded in Spurr resin. The results demonstrated that there are thick polygonal keratinized epithelial cells where groups of bacteria are revealed in three-dimensional images on the surface of filiform papillae in these animals. The bacterial membranes are randomly attached to the microplicae surface of epithelial cells. Morphometrics showed higher values of length and width of cocci in newborn (0 day) as compared to newborn (7 days) and adults animals, the bacilli showed no differences in these measurements. At high magnification, the TEM images revealed the presence of glycocalyx microfilaments that constitute a fine adhesion area between bacterial membranes and the membranes of epithelial microplicae cells. In conclusion, the present data revealed the fine fibrillar structures of bacteria that facilitate adhesion to the epithelial cell membranes of the oral cavity and morphometric changes in newborn (0 day) rats as compared with other periods. Microsc. Res. Tech. 76:1226–1233, 2013. © 2013 Wiley Periodicals, Inc.

The aim of this study is to obtain information about the mouse tongue muscle rendered using micro-computed tomography (μCT) at low, middle, and high magnifications. Three-dimensional (3D) μCT is used in various fields. Most μCT observations are restricted to hard tissue in biomaterial samples. Recently, with the use of osmium tetroxide, μCT has been effectively employed to observe soft tissue; it is now believed that μCT observation of soft tissue is feasible. On the other hand, the structure of the tongue muscle has been well studied, but cross-sectional imaging enhanced by 3D rendering is lacking. We chose the mouse tongue as a soft tissue case study for μCT and generated cross-sectional images of the tongue enhanced by 3-D image rendering with histological resolution. During this observation, we developed new methods of low-magnification observation to show the relation between the tongue muscles and surrounding tissues. We also applied high-resolution μCT in high-magnification observation of muscle fiber fascicles. Our methodological techniques give the following results: (1) For low-magnification observation (field of view: 12,000 μm), pretreatment with decalcification and freeze drying is suitable for observing the area between the muscle of the tongue and the bone around the tongue using μCT. (2) For middle-magnification observation (Field of view: 3,500 μm), the use of osmium tetroxide to observe the muscle arrangement of the tongue by μCT is suitable. (3) For high-magnification observation (Field of view: 450 μm), high-resolution μCT is suitable for observation of the transversus muscle fiber fascicles.

Procamallanus (Spirocamallanus) halitrophus (Fusco and Overstreet, 1978) is an intestinal parasite of the flounders Syacium papillosum and Citharichthys macrops, both of which are native to waters off the coast of the state of Rio de Janeiro, Brazil. With transmission electron microscopy, we observed the body wall which is composed of the cuticle, hypodermis, and somatic musculature. The cuticle of P. (S.) halitrophus is composed of 5 layers: the epicuticle, cortical, median, fibrous, and basal layers. Underlying the cuticle is the hypodermis, a syncytium that contains mitochondria, glycogen granules, vesicles, inclusion bodies, and an endoplasmic reticulum. The region of hypodermal chords contains a nucleus in addition to the other organelles and there is a basal lamina surrounding each muscle cell. The use of imidazole-buffered osmium tetroxide solution revealed the presence of lipids in the epicuticle, the membrane that surrounds each muscle cell, the inclusion bodies, and the endoplasmic reti...

Niemann-Pick disease (types A and B), or acid sphingomyelinase deficiency, is an inherited deficiency of acid sphingomyelinase, resulting in intralysosomal accumulation of sphingomyelin in cells throughout the body, particularly within those of the reticuloendothelial system. These cellular changes result in hepatosplenomegaly and pulmonary infiltrates in humans. A knockout mouse model mimics many elements of human ASMD and is useful for studying disease histopathology. However, traditional formalin-fixation and paraffin embedding of ASMD tissues dissolves sphingomyelin, resulting in tissues with a foamy cell appearance, making quantitative analysis of the substrate difficult. To optimize substrate fixation and staining, a modified osmium tetroxide and potassium dichromate postfixation method was developed to preserve sphingomyelin in epon-araldite embedded tissue and pulmonary cytology specimens. After processing, semi-thin sections were incubated with tannic acid solution followed by staining with toluidine blue/borax. This modified method provides excellent preservation and staining contrast of sphingomyelin with other cell structures. The resulting high-resolution light microscopy sections permit digital quantification of sphingomyelin in light microscopic fields. A lysenin affinity stain for sphingomyelin was also developed for use on these semi-thin epon sections. Finally, ultrathin serial sections can be cut from these same tissue blocks and stained for ultrastructural examination by electron microscopy.

The high spatial resolution of micro-computed tomography (micro-CT) is ideal for 3D imaging of coronary arteries in intact mouse heart specimens. Previously, micro-CT of mouse heart specimens utilized intravascular contrast agents that hardened within the vessel lumen and allowed a vascular cast to be made. However, for mouse coronary artery disease models, it is highly desirable to image coronary artery walls and highlight plaques. For this purpose, we describe an ex vivo contrast-enhanced micro-CT imaging technique based on tissue staining with osmium tetroxide (OsO(4) ) solution. As a tissue-staining contrast agent, OsO(4) is retained in the vessel wall and surrounding tissue during the fixation process and cleared from the vessel lumens. Its high X-ray attenuation makes the artery wall visible in CT. Additionally, since OsO(4) preferentially binds to lipids, it highlights lipid deposition in the artery wall. We performed micro-CT of heart specimens of 5- to 25-week-old C57BL/6 wild-type mice and 5- to 13-week-old apolipoprotein E knockout (apoE(-/-) ) mice at 10 μm resolution. The results show that walls of coronary arteries as small as 45 μm in diameter are visible using a table-top micro-CT scanner. Similar image clarity was achieved with 1/2000th the scan time using a synchrotron CT scanner. In 13-week-old apoE mice, lipid-rich plaques are visible in the aorta. Our study shows that the combination of OsO(4) and micro-CT permits the visualization of the coronary artery wall in intact mouse hearts.

This study analyzed the ultrastructural characteristics of the myotendinous junction (MTJ) between anterior belly of digastrics muscle and the intermediate tendon in adult rats. Six male Wistar rats were used and were anesthetized with an overdose of urethane and sacrificed by intracardiac perfusion with modified Karnovsky solution, postfixed in 1% osmium tetroxide, dehydrated in increasing series of alcohols and embedded in Spurr resin for transmission electron microscopic analysis. Ultrastructural analysis showed conical shape of the fiber extremity in MTJ region, highlighting the presence of numerous mitochondria arranged in groups in the subsarcolemmal and intermyofibrillary regions. Atypical MTJ characteristics were seen interspersed with bundles of collagen fibers. Classic characteristics such as finger-like processes by means of sarcoplasmic projections were observed among interdigitations. Terminals and periphericals bundles of myofibrils showed close relationship with the adjacent muscle fiber's endomysium through lateral junctions. In the distal portion, it was observed that the communication region of microtendons forming the intermediate tendon of digastric muscle, and it can highlight the columns disposition of tenocytes. In conclusion, the MTJ ultrastructure between the anterior belly of digastric muscle and intermediate tendon of adult rats showed classical morphologic descriptions and presented an atypical region revealed by the subspecialization between the myofibrils bundles and collagen fibers in the MTJ region.

Conclusion: The use of both gadolinium chloride (GdCl3) and osmium tetroxide (OsO4) allowed for the visualization of the membranous labyrinth and other intralabyrinthine structures, at different intensities, as compared with the control sample. This initial comparison shows the advantages of GdCl3 in radiological assessments and OsO4 in more detailed anatomical studies and pathways of labyrinthine pathogenesis using X-ray microtomography (microCT). Objective: To assess an improved OsO4 staining protocol and compare the staining affinities against GdCl3. Methods: Guinea pig temporal bones were stained with either GdCl3 (2% w/v) for 7 days or OsO4 (2% w/v) for 3 days, and scanned in a microCT system. The post-scanned datasets were then assessed in a 3D rendering program. Results: The enhanced soft tissue contrast as presented in the temporal bones stained with either GdCl3 or OsO4 allowed for the membranous labyrinth to be visualized throughout the whole specimen. GdCl3-stained specimens presented more defined contours of the bone profile in the radiographs, while OsO4-stained specimens provided more anatomical detail of individual intralabyrinthine structures, hence allowing spatial relationships to be visualized with ease in a 3D rendering context and 2D axial slice images. © 2013 Informa Healthcare.

As a surface for respiratory gas diffusion, ion regulation and waste excretion, gill epithelium of Alosa Caspio Caspio has been investigated. Problem statement: Surely, morphological study of gill cells is the main route for giving the basic data which can be inductive for physiological and pathological investigations. Approach: Gills were removed from Alosa Caspio which is from euryhaline species and fixed in freshly prepared glutaraldehyde and osmium tetroxide. Thin sections were stained in Uranyl acetate-lead citrate and examined under LEO transmission electron microscope. Results: Flattened respiratory lamellae extend in two rows from the sides of the filaments. The pavement cell is the major cell covering the thin epithelia of lamellae, but mucous cell and chloride cell also can be seen on lamellar epithelia, however, most of them are observed in filamental epithelia. The other cell forms the wall of capillaries in lamellae is Pillar cell. Chloride cells which are as single cells without complex junction, are surrounded by the pavement cells, have high number of large mitochondria and extensive membranous tubular system, with a free and smooth surface. There is one type of chloride cell with a light cytoplasm. Conclusion: The main ultrastructure aspects of the major cells are similar in teleosts, although some features such as the localization of chloride cell on lamellae, one type of cell, small crypts and the smooth surface are special features of this cell in Alosa Caspio that resemble this cell to freshwater species.

Aims: To assess methods for both measurement of and comparison between fibril spacing arrangement in transmission electron micrograph (TEM) images of collagen fibrils. Methods: Rabbit corneas were glutaraldhyde fixed, treated with osmium tetroxide and then thin sections stained with aqueous phosphotungstic acid. TEM images, at 28,000 magnification, of collagen fibrils in cross section were projected at a final magnification close to 250,000 to obtain overlays. Fibril centers were marked and the relative position of all fibrils measured either from a cluster of fibrils within a fixed region of interest (ROI) or by using a variable sequential ROI across the micrograph. The 2-D organization of the interfibril distance (IFD) data were analyzed using a radial distribution analysis and the difference in outcome statistically compared. Results: Repeated measures of IFDs provides data on the nearest neighbor distance within a fixed or sequential ROI of close to 48 nm and provides a numerical descriptor of the radial positions of all other fibril centers. From the set of radial positions (IFD) data, simple statistical comparisons can be made to both assess location-to-location and sample-to-sample variability. The sequential use of an ROI of fibril arrays however provide a more complete and accurate numerical descriptor. Conclusions: Methods are presented that will allow for assessment of the nearest neighbors distances and the overall 2-D pattern of collagen fibrils in electron micrographs. The sets of IFDs and their pattern can be compared using simple statistical analyses. Microsc. Res. Tech., 2010. © 2010 Wiley-Liss, Inc.

Few ultrastructural studies have been performed on members of the Dendrobatidae, although such investigations can be useful for the understanding of reproductive patterns, as a diagnostic method for males in breeding programs for endangered amphibians and for phylogenetic analysis. The sperm ultrastructure of the Green Poison Frog, Dendrobates auratus, from Panama is described following induced spermiation in living animals. To date only testicular spermatozoa in other dendrobatid frogs have been analysed. Moreover, an electron microscopic preparation method (transmission and scanning electron microscopy) for dendrobatid sperm cells in low concentration is presented. Sperm cells from stimulated frogs (100 IU human chorionic gonadotropin, hCG, twice at an interval of 1h) were recovered via cloaca lavage using 600 microl isotonic phosphate-free amphibian saline (IPS). Centrifuged flushings (5 min, 173 x g) were deposited on microscopic slides. Adherent spermatozoa were treated with Karnovsky fixative (overnight, 4 degrees C). After postfixation (2h, 1% osmium tetroxide), samples were dehydrated in series of ascending acetones (30-100%). For transmission electron microscopy sperm cells were encapsulated using Epon and 1.5% 2,4,6-tris(dimethylaminomethyl)phenol (DMP 30). Ultrathin sections (70 nm) were cut and stained with uranyl acetate (30 min) and lead citrate (5 min). Sperm cells are filiform with a 21.1+/-2.7 microm long and arcuated head and a single tail (35.0+/-4.2 microm length). Their acrosomal complex is located at the anterior portion of the head and consists of the acrosomal vesicle which has low electron density, and the subjacent electron-dense subacrosomal cone. In transverse section, the nucleus is circular (1.9+/-0.2 microm diameter) and conical in longitudinal section. It is surrounded by several groups of mitochondria. The chromatin is highly condensed and electron-dense but shows numerous electron-lucent inclusions. A short midpiece has a mitochondrial collar with a proximal and a distal centriole. The latter gives rise to the axoneme which alone forms the flagellum. The sperm ultrastructure of D. auratus differs from that of other Dendrobatidae because of the absence of a nuclear space and the absence of the undulating membrane associated with an axial fibre. This tail conformation is found in the Ranoidea but not in the Bufonoidea. These results show that the spermatozoa of D. auratus are the first within the Dendrobatidae without accessory tail structures. Methods of using sperm samples from hormonal treated frogs for ultrastructural studies is not only reasonable to examine e.g. amphibian phylogeny without killing frogs threatened with extinction but allows investigations in the field of assisted reproduction and male fertility for example in conservation programs for endangered amphibians.

The nerve terminals of intrinsic muscular fibers of the tongue of adult wistar rats was studied by using silver impregnation techniques, transmission electron microscopy (TEM), and high resolution scanning electron microscopy (HRSEM) to observe the nerve fibers and their terminals. Silver impregnation was done according to Winkelman and Schmit, 1957. For TEM, small blocks were fixed in modified Karnovsky solution, postfixed in 1% buffered osmium tetroxide solution, and embedded in Spurr resin. For HRSEM, the parts were fixed in 2% osmium tetroxide solution with 1/15 M sodium phosphate buffer (pH 7.4) at 4°C for 2 h, according to the technique described by Tanaka, 1989. Thick myelinated nerve bundles were histologically observed among the muscular fibers. The intrafusal nerve fiber presented a tortuous pathway with punctiform terminal axons in clusters contacting the surface of sarcolemma. Several myelinated nerve fibers involved by collagen fibers of the endoneurium were observed in HRSEM in three-dimensional aspects. The concentric lamellae of the myelin sheath and the axoplasm containing neurofilaments interspersed among the mitochondria were also noted. In TEM, myofibrils, mitochondria, rough endoplasmic reticulum, Golgi's apparatus, and glycogen granules were observed in sarcoplasm. It is also noted that the sarcomeres constituted by myofilaments with their A, I, and H bands and the electron dense Z lines. In areas adjacent to muscular fibers, there were myelinated and unmyelinated nerve fibers involved by endoneurium and perineurium. In the region of the neuromuscular junction, the contact with the sarcolemma of the muscular cell occurs forming several terminal buttons and showing numerous evaginations of the cell membrane. In the terminal button, mitochondria and numerous synaptic vesicles were observed. Microsc. Res. Tech., 2009. © 2009 Wiley-Liss, Inc.

Scanning electron microscopy of human cadaver corneas revealed a selective binding of ruthenium red-osmium tetroxide to some substance coating the posterior endothelial surface. A coating material was not found on autolyzed cells, on denuded areas of the membrane of Descemet, or on the anterior surface of endothelial cells. Partial digestion of the coating material by urokinase and trypsin suggests the presence of at least three different structural or chemical elements.

The combination of osmium tetroxide staining and high-resolution tomographic imaging using monochromatic X rays allows visualizing cellular structures of the human inner ear, that is, the organ of Corti, the stria vascularis and further soft tissues of the membranous labyrinth, in three-dimensional space with isotropic micrometre resolution. This approach permits to follow the course of nerve fibre bundles in a major part of the specimen and reveals the detailed three-dimensional arrangement of individual ganglion cells with distinct nuclei by means of X-ray tomography for the first time. The non-destructive neuron cell counting in a selected volume of 125 microm x 800 microm x 600 microm = 0.06 mm(3) gives rise to the estimate that 2000 ganglion cells are present along 1 mm organ of Corti.

Sympathetic chain ganglia of newborn rats were cultured in Rose chambers with and without guanethidine. After one week, the cultures were examined by light microscopy for formaldehyde-induced catecholamine fluorescence and by electron microscopy after fixation in 5% glutaraldehyde and 1% osmium tetroxide. Guanethidine sulphate (2 mg/l) caused an increase in the number of the small, intensely fluorescent (SIF) cells in the ganglion explants. Electron microscopic examination of guanethidine-containing cultures revealed an increased number of small, granule-containing (SGC) cells, which corresponded in size and shape to the SIF cells. Round vesicles (about 100 nm in diameter) and elongated vesicles (about 80 nm in cross section and about 200 nm in length) containing an electron-dense core were observed in the cytoplasm of the SGC cells both in control and guanethidine-containing cultures. The granular vesicles were most frequent in the periphery of the cytoplasm. In ganglia cultured with guanethidine, most SGC cells observed contained a greatly reduced number of granular vesicles as compared to SGC cells of the control cultures.

Objective: Microscopic computed tomography (microCT) has been recently applied to morphological evaluation of mouse embryos with or without congenital malformations, and 3-dimensional (3D) digital images of the whole embryo can be obtained. In the present study, the authors report a modified, rapid technique of 3D embryonic microCT without processing with osmium tetroxide. Methods: Normal embryonic days 10.5 to 11 mouse embryos, as well as those with craniofacial anomalies treated with teratogens, were examined. After fixation, we processed the embryo samples with hexamethyldisilazane, instead of highly toxic osmium tetroxide in the original method. Results: Our protocol enabled clear 3D craniofacial imaging of the normal and anomalous mouse embryos within a short period of 20 minutes or 1 hour. In addition, some anatomical landmarks were clearly detected in the reconstituted craniofacial section images. Conclusion: Our present data suggest a possible role of microCT for high-throughput morphological screening of the mouse embryos with craniofacial anomalies.

The present investigation concerns the true morphology of the attachment of the two otolith receptor organs the utricular and the saccular maculae in two and three dimensions. By applying a new visualization method, which utilized the application of X-ray microtomography and a method of contrast enhancement based on en-bloc staining in osmium tetroxide, we were able to overcome problems of artefact production such as tissue distortion and loss of valuable information that was present in previous studies. A series of more than 1000 axial sections were obtained for each of the specimens, which subsequently formed the basis for detailed 2D and 3D visualizations. Our interpretations of these data reveal that the saccular maculae are closely attached to the curved bony surface of the temporal bone as traditionally believed, but the utricular macula is attached to the temporal bone only at the anterior region of the macula.

This review summarizes the function, structure, and chemistry of the myelin sheaths of axons: discusses the mechanisms of several staining techniques using inorganic compounds, dyes, and antibodies; and provides technical instructions for 13 staining methods for normal and degenerating myelin. Myelin has alternating hydrophobic and hydrophilic layers. The former contain the hydrocarbon tails of lipid molecules and hydrophobic segments of transmembrane proteins; the latter contain the anionic heads of polar lipids and also basic proteins. Aqueous fixatives allow some myelin lipids to be retained in paraffin sections. Stains for normal myelin include histochemical reactions that detect choline-containing phospholipids (Baker's chromation-acid hematein) or double bonds (unsaturation) in the fatty acid components of lipids (osmium tetroxide or palladium chloride reduction; the pseudoplamal reaction and DAB fluorescence, for oxidized double bonds). Traditional fat stains (Sudan dyes) stain normal myeli...

Although the osmium maceration method has been used to observe three-dimensional (3D) structures of membranous cell organelles with scanning electron microscopy (SEM), the use of osmium tetroxide for membrane fixation and the removal of cytosolic soluble proteins largely impairs the antigenicity of molecules in the specimens. In the present study, we developed a novel method to combine cryosectioning with the maceration method for correlative immunocytochemical analysis. We first immunocytochemically stained a semi-thin cryosection cut from a pituitary tissue block with a cryo-ultramicrotome, according to the Tokuyasu method, before preparing an osmium-macerated specimen from the remaining tissue block. Correlative microscopy was performed by observing the same area between the immunostained section and the adjacent face of the tissue block. Using this correlative method, we could accurately identify the gonadotropes of pituitary glands in various experimental conditions with SEM. At 4 weeks after castration, dilated cisternae of rough endoplasmic reticulum (RER) were distributed throughout the cytoplasm. On the other hand, an extremely dilated cisterna of the RER occupied the large region of the cytoplasm at 12 weeks after castration. This novel method has the potential to analyze the relationship between the distribution of functional molecules and the 3D ultrastructure in different composite tissues.

Objectives - The vagus nerve has gained a role in the treatment of certain diseases by the use of vagus nerve stimulation (VNS). This study provides detailed morphological information regarding the human cervical vagus nerve at the level of electrode implant. Results - Eleven pairs of cervical vagus nerves and four pairs of intracranial vagus nerves were analysed by the use of computer software. It was found that the right cervical vagus nerve has an 1.5 times larger effective surface area on average than the left nerve [1,089,492 +/- 98,337 vs 753,915 +/- 102,490 mu m(2), respectively, (P

We have observed that epiphyseal and physeal hypertrophic chondrocytes in BALB/c mice show considerable differences of light microscopic and ultrastructural appearance, even when the cells are at the same stage of differentiation. In addition, cell structure maintenance improved with tissue preparation controlled for osmolarity and for membrane stabilization using 0.5% ruthenium hexammine trichloride (RHT) for both light microscopy (LM) and electron microscopy (EM) or 0.5% lanthanum nitrate for LM. Physeal hypertrophic chondrocytes showed a gradual increase in size closer to the metaphysis and a change in shape as cells elongated along the long axis. The nucleus remained central, with uniformly dispersed chromatin, and the rough endoplasmic reticulum (RER) was randomly dispersed throughout cytoplasm with little to no presence against the cell membrane. Even the lowermost cells showed thin elongated or dilated cisternae of RER and intact cell membranes. Epiphyseal chondrocytes remained circular to oval with no elongation. Nucleus and RER were positioned as a complete transcellular central nucleocytoplasmic column or as an incomplete bud with RER of the column/bud always continuous with RER peripherally against the intact cell membrane. RER was densely packed with parallel cisternae with adjacent cytoplasm empty of organelles but often filled with circular deposits of moderately electron-dense material consistent with fat. Optimal technique for LM involved fixation using glutaraldehyde (GA) 1.3%, paraformaldehyde (PFA) 1% and RHT 0.5% (mOsm 606) embedded in JB-4 plastic and stained with 0.5% toluidine blue. Optimal technique for EM used fixation with GA 1.3%, PFA 1%, RHT 0.5% and cacodylate buffer 0.03 M (mOsm 511) and post-fixation including 1% osmium tetroxide. These observations lead to the possibility that the same basic cell, the hypertrophic chondrocyte, has differing functional mechanisms at different regions of the developing bone.

We have discovered that the immunoreactivity of the fluorophore Alexa Fluor 488 survives glutaraldehyde and osmium tetroxide fixation and epoxy resin embedding and etching. We have developed new localization methods that for the first time take advantage of this property. The antigen is localized in cryosections using suitable primary antibody and an Alexa Fluor 488-conjugated secondary antibody. Cryosection fluorescence can be photographed for later correlation with electron microscopy (EM) findings. The sections are then further fixed with glutaraldehyde and OsO4, if desired and flat-embedded in epoxy resin. Semi-thin sections are etched completely with sodium ethoxide, whereas thin sections are partially etched. Alexa Fluor 488 is then localized with rabbit anti-Alexa Fluor 488 and goat anti-rabbit conjugated to Alexa Fluor 488 [light microscopy (LM)] or to colloidal gold (EM). A second antigen may also be localized using Alexa Fluor 568. When used without postfixation, these methods produce high-resolution semi-thin, or even thin, sections that retain a high level of fluorescence for LM observations. These methods allow highly sensitive immunolocalizations in tissue while preserving cell fine structure through traditional fixation and epoxy embedding. In demonstration of the methods, we describe the localization of the thiazidesensitive sodium/chloride cotransporter and the epithelial sodium channel in rat kidney.

Zebrafish has been used as a powerful model system in biological and biomedical studies studying development and diseases. Comparative, functional, and developmental studies on zebrafish morphology require precise visualization of 3D morphological structures. Few methods that can visualize whole-volume of zebrafish tissues are available because optical bio-imaging methods are limited by pigmentation and hard tissues. To overcome these limitations, the 3D microstructures of a hypercholesterolemic zebrafish model are visualized using synchrotron X-ray micro-computed tomography (SR-μCT). The model spatial resolution ranged from sub- to several microns. The microstructures of various zebrafish organs are observed by combining high-contrast staining (osmium tetroxide and uranyl acetate) and embedding a protocol to enhance the image contrast of soft tissues. Furthermore, blood vessels are identified using a barium sulfate injection technique. The internal organs and cells, such as liver, intestine, oocytes, and adipocytes, of a hypercholesterolemic zebrafish are compared with those of normal organs and cells. The SR-μCT is useful for understanding the pathogenesis of circulatory vascular diseases by detecting the modifications in the 3D morphological structures of the whole body of the zebrafish. This bio-imaging technique can be readily used to study other disease models.

Pompe disease is an autosomal recessive lysosomal storage disorder caused by a deficiency of the lysosomal enzyme acid α-glucosidase, responsible for the degradation of lysosomal glycogen. Absent or low levels of the enzyme leads to lysosomal glycogen accumulation in cardiac and skeletal muscle cells, resulting in progressive muscle weakness and death from cardiac or respiratory failure. Recombinant enzyme replacement and gene therapy are now being investigated as treatment modalities for this disease. A knockout mouse model for Pompe disease, induced by the disruption of exon 6 within the acid α-glucosidase gene, mimics the human disease and has been used to evaluate the efficacy of treatment modalities for clearing glycogen. However, for accurate histopathological assessment of glycogen clearance, maximal preservation of in situ lysosomal glycogen is essential. To improve retention of glycogen in Pompe tissues, several fixation and embedding regimens were evaluated. The best glycogen preservation was obtained when tissues fixed with 3% glutaraldehyde and postfixed with 1% osmium tetroxide were processed into epon-araldite. Preservation was confirmed by staining with the Periodic acid-Schiff's reaction and by electron microscopy. This methodology resulted in high-resolution light microscopy (HRLM) sections suitable for digital quantification of glycogen content in heart and skeletal muscle. Combining this method of tissue fixation with computer-assisted histomorphometry has provided us with what we believe is the most objective and reproducible means of evaluating histological glycogen load in Pompe disease.

Two methods adapted from biological microscopy are described for a new application in imaging the morphology of rubbery latex particles. In the first method, a drop of latex is frozen in liquid nitrogen, sectioned with a diamond knife and vapour-stained with osmium tetroxide, then viewed by transmission electron microscopy. When applied to latexes made by emulsion polymerization of methyl methacrylate in a natural rubber latex seed, inclusions are clearly visible. A chemical fixation method is then described for imaging the morphology of such rubbery latex particles. Glutaraldehyde is added to the latex, followed by osmium tetroxide. The sample is then dehydrated in ethanol, epoxy resin added, and the sample cured, ultramicrotomed, and imaged with transmission electron microscopy. An inclusion morphology is again clearly seen. (C) 2004 Wiley-Liss, Inc.

Techniques for staining (silver, osmium, metal sulfides, ink) and microphotography (epi-illumination) of polished bone surfaces have been developed to visualize the three-dimensional structure of the shafts of mammalian long bones. Bone is a two-compartment system with capillaries and some kinds of connective tissue in one compartment separated from fibers of bone collagen, often forming lamellae, in the other. Laminar bone consists of stacks of lamellae separated by vascular spaces containing capillary network sheets. It is deposited at the periosteal and endosteal surfaces. Osteonic bone, well described in the literature, consists of cylinders of lamellae with central vascular spaces. The primary structure of the shafts of mammalian long bones is laminar and laminae often remain as the main component. Secondary osteons are a replacement within laminae. As laminar bones mature, some of the irregular longitudinal capillary spaces in the network sheets enlarge and become less crooked to form secondary osteons. Parts of the random networks become ordered longitudinal ones, resulting in collapse of those network spaces not converted to osteons. The residual capillaries become bloodless, making the surviving network spaces difficult to resolve. This may account for them being overlooked in descriptions of bone structure. For example, laminar bone occurs with osteonic bone in the human femur, although it is rarely figured. Nearly mature bones switch the kind of primary bone deposited at the peripheral (periosteal) surface from laminar to primary osteonic. J. Morphol. 262:546–565, 2004. © 2004 Wiley-Liss, Inc.