Microwave energy (MWE) as a method of rapid tissue processing is gaining increasing support as an alternative to routine chemical processing in diagnostic laboratory environments. Chemical fixatives and conventional fixation times used for standardized preservation of tissues can result in serious alterations in morphology as a consequence of solubilization and conformational changes of proteins and lipids. These untoward changes typically result in compromised antigenicity of many tissue proteins. With MWE processing, tissue antigens can be distinctly better preserved, antigen retrieval made more replicate and histochemical and immunochemical reactions made ultrafast. The present study had two objectives: to compare MWE processed skin to conventionally processed skin for ultrastructural integrity; to compare MWE expression of antigenicity of selected skin proteins which are usually sensitive to conventional chemical processing. Skin proteins selected were bullous pemphigoid antigen (BPA) and laminin.

The average sperm nucleus is only 8 micrometers long, but contains 1 meter of DNA. We have previously described how the sperm cell is able to package this DNA into the small volume of the sperm head. The only DNA structure that exists in both somatic cells and sperm nuclei is the organization of DNA loop domains that are attached at their bases to the nuclear skeleton, the nuclear matrix. We compared the structure of one gene in sperm nuclei to that of spermatogenic and adult cells.

Individual loop domains of the 5S rRNA gene cluster were visualized by fluorescent in situ hybridization (FISH). Nuclei were isolated from hamster spermatozoa, spermatogonia, pachytene spermatocytes, round spermatids, and adult liver cells. They were then extracted to remove the protamines or histones, fixed, hybridized to a biotinylated 5S rDNA probe, then viewed by epifluorescence microscopy.

Myosin is composed of two high-molecular weight heavy chains and four low-molecular weight hght chains. In both vertebrate and invertebrate skeletal muscle, each myosin heavy chain is associated with two myosin light chains. In skeletal muscle myosins studied by X-ray diffraction, each myosin heavy chain binds one of each of two distinct classes of hght chains. Thus, while isoform distributions may vary within and between fibers, the spatial distribution of each class of light chain should be uniform within the A band and between sarcomeres and fibers. Since no such study exists for crustacean myosin, we investigated the spatial distribution of the hght chains within the superficial flexor muscle (SFM) of the lobster, Homarus americanus, using immunoelectron microscopy. The SFM contains two classes of myosin hght chains, termed “alpha” (Mr = 21,000 to 23,500) and “beta” (Mr = 18,000 to 18,500). Immunocytochemical electron microscopic results suggest that the alpha light chains are not uniformly distributed at the subsarcomere level.

Cisplatin, a commonly used broad spectrum chemotherapeutic drug, has been proven effective in the treatments of bladder, lung, ovarian, head and neck, testicular and breast cancers. One of the major side effects of this drug is its gastrointestinal toxicity, which includes severe nausea, vomiting and in case of rats bloating of the stomach and gastric ulceration. In case of rats, it manifests as bloating of the stomach and gastric ulceration since the rats don't have vomiting reflex. This has been attributed to hypocalcemia induced by cisplatin treatment which results in the suppression of acetylcholine and constitutive nitric oxide release inducing a bloating of the stomach. This has further been shown to induce an increase in the acid content of the stomach. There are no studies available on any changes of gastric protection after cisplatin treatment Gastrin has been shown to have gastroprotective effects against injury by maintaining the gastric mucosal integrity and gastric mucosal blood flow.

Poly-[(trans-1,2-diaminocyclohexane) platinumj-carboxyamylose (“poly-plat“) is a second generation analog of cisplatin which enhances the immune system with greater efficacy in vitro and in vivo “Poly-plat” contains 1/5 the platinum of CDDP and demonstrates less toxicity. In order to understand the mechanism of action of this compound an in vitro and in vivo study was performed. Swiss Webster mice and isolated murine peritoneal macrophages were treated with “poly-plat” (10 mg/kg). The Swiss Webster mice were given bolus injections and sacrificed at 2 and 12 days. Peritoneal macrophages were then isolated and allowed to incubate in culture for 24 h. Peritoneal macrophages were also isolated from normal mice and treated with the drugs for 2 h. After treatments the macrophages were placed in fresh media and allowed to incubate 24 h. Supematants were isolated at various times during culture for immunocytochemical analysis.

Both in vitro and in vivo studies showed enhanced immunostimulation after their respective treatments.

Oocyte meiotic maturation occurs without the aid of centrioles in the mouse; instead the division spindles are organized by a number of microtubule organizing centers (MTOC). Immunofluorescence microscopy using anti-γ tubulin shows that multiple MTOCs migrate to surround the central nucleus (germinal vesicle, GV) during early maturation.. However, at the EM level, recognizable MTOCs have never been seen prior to the nucleation of microtubules (MT) which occurs near the time of nuclear envelope breakdown. Recently, I described multivesicular aggregates (MVAs) as the organelles most likely to represent immature MTOCs prior to MT nucleation4. These are first seen at time zero by EM as two large aggregates in the cortex, with sizes averaging 10um. MVA subsequently fragment into smaller units as they migrate centrally.

Materials and Methods: Female ICR mice were injected IP with 100 iu of chorionic gonadotropin (Sigma) to stimulate follicle growth, and ovaries were removed 48 hours later.

Confocal laser scanning microscopy is an excellent method to study nerve cell morphology and the three-dimensional distribution and interrelationship of dentrites and axons in the central nervous system. The cerebellum has been taking as a model of a gray center.

The FM4-64, a member of the family of fluorescent dyes, has been applied to the cerebellar cortex to evaluate its properties as an intracellular stain and intracortical tracer. Slabs of hamster cerebellum,5 mm thick, were incubated in 10,30 and 100 μm solutions of FM4-64 in sodium phosphate buffer and observed in a slow scan confocal laser scanning microscope. Mossy and climbing fibers were traced in the cerebellar white and gray substances .They exhibited high fluorescence signal at the level of myelin sheath.

Mossy fibers were identified in the granular layer by their typical rosette formation and dichotomous bifurcation pattern. Climbing fibers bundles were observed crossing the granular layer and giving collateral branches around Golgi cell bodies.

The average sperm nucleus is only 8 micrometers long, but contains 1 meter of DNA. We have previously described how the sperm cell is able to package this DNA into the small volume of the sperm head. The only DNA structure that exists in both somatic cells and sperm nuclei is the organization of DNA loop domains that are attached at their bases to the nuclear skeleton, the nuclear matrix. We compared the structure of one gene in sperm nuclei to that of spermatogenic and adult cells.

Individual loop domains of the 5S rRNA gene cluster were visualized by fluorescent in situ hybridization (FISH). Nuclei were isolated from hamster spermatozoa, spermatogonia, pachytene spermatocytes, round spermatids, and adult liver cells. They were then extracted to remove the protamines or histones, fixed, hybridized to a biotinylated 5S rDNA probe, then viewed by epifluorescence microscopy. The 5S rDNA in liver nuclei was organized into a single large loop domain (Fig. 1),

Environmental Scanning Electron Microscopy has opened up the potential to study biological events in their native occurence. While still in the early stages of exploration, the ability to view samples in their native state with the resolution provided by scanning electron microscopy is highly desirable and will provide insights in dynamic processes which previously had only been possible by interpretation with indirect methods or by static images obtained with transmission electron microscopy.

In preliminary experiments to explore the potential of studying native events with the ESEM we have investigated sea urchin embryos after deciliation with chloral hydrate. The Philips XL-30 ESEM instrument was used to obtain images of lightly fixed (0.1% glutaraldehyde in sea water), lightly coated embryos that had been treated as follows. Chloral hydrate was added at a concentration of 0.1% to blastulae of Lytechinus pictus at 24 hours after fertilization. Observation with light microscopy indicates that cilia become gradually shed into the sea water within 10 to 20 hours after chloral hydrate application.

Sea urchins have been used for over a century as a remarkable animal model system in which to study cell, molecular, and developmental biology. The studies presented here have used sea urchin eggs and embryos for pioneering experiments to explore the effects of microgravity on the cytoskeleton during a space flight on the space shuttle Endeavor. The culture conditions followed those described previously utilizing the Aquatic Research Facility (ARF) to fertilize and culture eggs and embryos up to the pluteus stage under controlled temperature (12°C) and fixation conditions. To achieve a final fixation with 0.5% glutaraldehyde and 4μM taxol, concentrated fixation fluid was injected at preselected time points to preserve microtubules, centrioles, centrosomes, microfilaments, mitochondria, and cell membranes.

The analysis of the results revealed that the centriole-centrosome complex during cell division and cilia formation showed alterations in samples that had been exposed to microgravity while control cells cultured in a centrifuge at lg in space and those cultured on ground appeared normal.

An integrated approach including TEM, SEM and LM was utilized to investigate adhesives produced by the marine fouling diatom Achnanthes longipes. Extracellular adhesive secretion during sequential attachment in living diatom cells was examined using video microscopy. A suite of cryotechniques including high pressure freezing (HPF), freeze-substitution (FS) and high resolution cryo-field-emission SEM (cryo-FESEM) were required for preservation and structural investigation of the hydrophilic polymers synthesized by A. longipes that promote attachment to ship hulls, oil rigs and other submerged structures.

Living cells and associated extracellular biocomposites were rapidly cryo-immoblized and observed using a Hitachi S-900 FESEM equipped with a cryo-stage. This technique allowed resolution of details of polymer organization within the pad, shaft and collar portions of the major adhesive structure termed a stalk (Fig. 1,2). This stalk has been demonstrated to consist mainly of polysaccharide cross-linked by O-linked polypeptides. In addition to permanently adhering the diatom to the substratum, the stalk elevates A. longipes, possibly enhancing the ability to successfully compete with other biofilm dwellers.

Freeze substitution techniques can provide exceptional fixation of many types of small biological materials. Ultrastructural studies on the Foraminiferida (marine rhizopods) traditionally have used standard chemical fixation protocols. The relatively large size (-25 μm - several cm) and the presence of a mineralized shell in most taxa precludes the application of many cryo-techniques. High pressure freezing, however, provides a method for freezing organisms as large as many of the smaller foraminifera often without extensive ice damage. Gamonts (sexual generation) from three representatives of the suborder Astrorhizina ﹛Myxotheca sp., Cribrothalammina alba, and Hyperammina sp.) were selected because they are among the most primitive foraminifera and have only very finely agglutinated shells with non-mineralized, organic cements. Myxotheca sp. and C. alba were originally collected from salt marsh environments on Sapelo Island, Georgia, and Hyperammina sp. was isolated from fine-grained carbonate sediments of the Florida Keys.

Crustacean hemocytes have been characterized on the basis of cell size and nature of cytoplasmic granules. Based on light microscopic morphological analysis and cytochemistry, investigators variously named the hemocyte types (agranular, small-granule, large granule, undifferentiated, hyaline cells, non-explosive, explosive granulocytes, etc.). In his study of the isopod Armadillidium vulgare Faso adopted the terminology of Benjamin and James and referred to the hemocytes as hyaline cells, semi-granulocytes and granulocytes.

In the present investigation we have studied the hemocytes of two isopods, Oniscus asellus and Armadillidium nasatum, using transmission electron microscopy. Hemolymph was collected by penetrating the posterior dorsal exoskeleton of 20 animals of each genus with a microcapillary pipette and drawing 3-5μL per isopod. The samples were processed following a standard technique. Thin sections were collected on 300 mesh copper grids, counterstained with 2% aqueous uranyl acetate and lead citrate, and viewed with a JEOL 1010 electron microscope.

Most plant pathogenic fungi that are obligate parasites produce haustoria which are thought to be involved in nutrient absorption. A haustorium is a specialized hyphal branch that penetrates the host cell wall and invaginates the host cell plasma membrane. The host plasma membrane ensheathing the haustorium is termed the extrahaustorial membrane. This presentation provides examples of different types of haustoria produced by plant pathogenic fungi. Species considered here are 1) Cronartium quercuum f. sp.fusiforme, the cause of fusiform gall rust of pine, 2) Puccinia arachidis, the cause of peanut rust1, 3) Uncinuliella australiana, the cause of powdery mildew of crape myrtle, 4) Exobasidium camelliae, a pathogen of Camellia sasanqua2, and 5) Cercosporidium personatum, the cause of late leaf spot of peanut.

Rust fungi typically require two different host species to complete their life cycles. The dikaryotic phase of the rust life cycle consists of intercellular hyphae that give rise to specialized haustoria known as D-haustoria which are remarkably similar from one species to the next.

The filamentous fungus Aspergillus nidulans partitions its cells by laying down septa at regularly spaced intervals in response to nuclear division. Physiological and genetic studies of the temperature-sensitive sep mutants have been especially useful in dissecting the regulation of septation. Electron microscopic studies of the sep mutants should be equally useful in dissecting the structural intermediates of septation. In preparation for ultrastructural studies of the sep mutants, we have examined septa in wild-type A. nidulans fixed by freeze substitution.

Dialysis membranes were placed on rich medium plates and inoculated with A. nidulans spore suspensions. After 12 hours at 30°C, the dialysis membranes with adhering fungal hyphae were cut into square pieces measuring approximately 5mm on each side. The pieces were plunged into liquid propane and processed according to the procedures of Hoch. Serial sections were cut using a diamond knife and post stained with uranyl acetate and lead citrate.

Ivermectin is a drug widely utilized for its anthelminthic activity over a diverse range of animal parasites. It has proved to be particularly useful in the prophylaxis of infection by the heartworm (Dirofilaria immitis) in dogs and cats. Although its application in this respect has been as a filaricide in preventing early growth and maturation of naturally acquired larvae, it is known to have activity against young adults as well. Previous studies have shown that in vitro exposure to ivermectin induces somatic muscle paralysis in the nematode Haemonchus contortus, resulting in pharyngeal dysfunction and disruption of normal ingestion. Experiments were performed to determine the effect of in vivo exposure of adult canine heartworms to this drug.

Adult heartworms were harvested from groups of dogs treated monthly with ivermectin beginning four to five months after inoculation of infective larvae and from untreated control dogs. Live worms from both experimental and control dogs were fixed, embedded, and sectioned for conventional transmission electron microscopy.

Perkinsus marinus is a protistan parasite that infects the Eastern Oyster, Crassostrea virginica, and is one factor responsible for the reduced productivity of oyster fisheries. This parasite (or related species) has a wide distribution and has also been found to infect the oysters in the Tampa Bay region. A significant aspect of this organism has been its enigmatic taxonomic position. Originally classified in two separate fungal genera, it currently is considered an Apicomplexan (Kingdom Protista) based on transmission electron microscopy studies of the zoospore revealing an apical complex. However, rRNA studies have shown that P. marinus may be more closely related to the dinoflagellates. The focus of my study has been on surface features of certain developmental stages that may aid in a definitive taxonomic placement of this organism.

Transmission electron microscopy observations of the trophozoite and hypnospore stages show variation in both the size of the organism and the presence of a cell wall.

Adult Rhabdochona nematodes, commonly parasitizing fish, were present in the digestive tracts of cutthroat trout in Little Cottonwood Creek, Utah. Cutthroat trout, Oncorhyncus clarki, are known to serve as both intermediate and definitive hosts for parasitic nematodes. The larval stage parasitizes almost any tissue of its host, but the adult is always found in the digestive tract. Due to the lack of key morphological features, scanning electron microscopy (SEM) was used to identify specific structures leading to the nematode's taxonomic identification.

Cutthroat trout were obtained using a rod and reel and were dissected the same day. Nematodes were present in all 12 cutthroat trout residing in all parts of the digestive tract. The nematodes, Rhabdochona sp., were prepared for SEM using the following procedures. First, the parasites were fixed in 2% buffered glutaraldehyde, washed in sodium cacodylate buffer, and post fixed in a 1% solution of osmium tetroxide. The samples were then washed in the same buffer system and dehydrated through a graded alcohol series. Critical-point-drying removed the remaining fluids. Finally, the nematodes were placed on specimen stubs, sputter coated with gold, and each specimen examined with a JOEL-840 high resolution scanning electron microscope with micrographs taken at varying magnifications.

Absorption enhancing microtriches of Bothriocephalus sp. were studied in relation to location, size, density, structure, and possible function using SEM.

Carp, C. carpio, were collected by gill nets set in Lake Powell, Utah. Tapeworms were collected from the carp intestine and relaxed in a cold water bath for 12 hours, placed in 1% cacodylate buffered glutaraldehyde, washed in a cacodylate buffer, soaked overnight in 1% OsO4, washed in buffer, dehydrated in an ETOH series, critical point dried, attached to stubs, and sputter coated for four minutes with gold. Bothriocephalus was viewed at varying magnifications using the JEOL 6100 scanning electron microscope. Density counts and measurements were conducted using digitized images and the program Semafore. Images were collected from the scolex and 8 equal body sections. Microtriches were measured from the tip to the base.

The ciliated protozoan Hyalophysa is a symbiont of a variety of crustaceans, including grass shrimp, hermit crabs, and blue crabs. At the molting of the host, the attached protozoans excyst and swim into the exoskeleton to feed by pinocytosis on exuvial fluid. The feeding trophonts expand to many times their original size, transforming into cells that are little more than large food vacuoles with a rim of cytoplasm stretched around the perimeter. After feeding for the only time during their life cycle, they settle on the substrate to encyst and divide into daughter tomites, and in doing so they divide the food vacuole. The tomites then leave the cyst and attach to a crustacean for their next opportunity to feed at molting.

We have followed endocytosis in Hyalophysa using two dyes, nigrosin and congo red, in order to understand the digestive process.