The nematode C. elegans is a simple model for genetic studies of cell and tissue development. There is a need to improve the preservation of embryonic and early larval stages, during which nematode tissues elaborate and separate. However, these stages are particularly resistant to fixation and embedment due an impenetrable eggshell and larval cuticle. Their small size at these ages precludes mechanical cutting, which has been used successfully for immersion fixation of older stages. Here we compare the quality of preservation under three rather different regimes: using laserholes to permeabilize the eggshell during the primary fixation step, using microwave energy to enhance the first fixation step, or using fast freezing and freeze substitution to circumvent the standard immersion procedure. Vancoppenolle et al (2000) have recently demonstrated very good results through enzymatic weakening of the eggshell prior to immersion fixation. Their data are comparable to what we achieve by either the laserhole or microwave methods.

Co-localization of proteins and nucleic acid sequences by in situ hybridization and immunohistochemistry is frequently difficult as the procedure necessary to detect the target structure of one technique may negatively affect the target of the other. Structural impairments may also limit the application of the two techniques. to overcome these problems we developed a method to perform in situ hybridization and immunohistochemistry on Chromosome spreads, intact cells and semi-thin sections of methyl methacrylate-embedded tissue. Microwavestimulated antigen retrieval, signal amplification by catalyzed reporter deposition, and fluorescent dyes were used for all techniques, yielding high sensitivity and excellent morphological preservation compared to conventional processing techniques. Co-localization of in situ hybridization and immunohistochemistry probes with high morphological resolution was achieved using computerized image reconstruction. The latter may allow for the co-localization of multiple antigens and a specific DNA sequence at the same tissue level.

In efforts to better understand the cell biology of fungi and their pathogenic interactions with plants, we have pursued the use of cryo-preparative techniques for specimen preservation and immunocytochemistry at the light and electron microscope level. The chemically diverse composition of plant and fungal walls has proven very challenging for immunofluorescence using confocal microscopy. Typically, to allow probe access in these organisms, immunolabeling has required sectioning or enzymatic digestion of cell walls. The ideal method would preserve the spatial fidelity and antigenicity of the target molecule as well as maintain the integrity of the surrounding tissue. Our early work using methacrylate de-embedment, as described for sectioned plant material, was very effective for freeze-substituted fungi. Further improvements, using a combination of slow freezing and freeze-substitution followed by methacrylate de-embedment, permitted whole mount fluorescence antibody labeling of a variety of phylogenetically diverse fungi without enzymatic digestion or sectioning (Fig.l).

There are many strategies for documenting the distribution of gene products or other molecules within cells or tissues. Depending on the required information, techniques can range from cell fractionation to methods that employ electron microscopy: some preclude examination of living cells, and thus can not provide dynamic temporal information. Time sequence data are paramount when gene expression is transient, as is often the case during fungal-pathogen plant-host interactions. Fluorescent dyes and protein tags (e.g., green fluorescent protein, GFP) allow monitoring of specific molecules, organelles or cells over time, for simultaneous recording of both spatial and temporal information. in addition, fluorescent markers are amenable to powerful 3-D analysis using confocal or multi-photon imaging methods. We have achieved good expression of fluorescent proteins in the fungal blast pathogen Magnaporthe grisea, and have generated a functional fusion protein between GFP and P-tubulin (Figs. 1,2).

Resolving the true fine structure and behavior of soft bodied organisms such as arthropod larvae, acari, nematodes, annelids and pupae by conventional scanning electron microscopy has been difficult. Classical preparation techniques, involving immersion fixation, dehydration and critical point drying, cause significant deleterious changes in specimen structure due to effects of surface tension, osmotic stress and mechanical damage. The intact exoskeleton, cuticle or other limiting membranes of these organisms, creates a formidable obstacle to the penetration of fixatives and solvents, which are only effective after a long period of time (hours). During this time, behavioral postures and much of the structural integrity are lost and most organisms detach from their hosts.

In previous studies, our laboratory utilized methods in low temperature freeze stabilization, to preserve and observe the undisturbed fine structure of biological samples. Plunge freezing, although more rapid, resulted in many parasitic organisms dislodging from hosts and being lost.

Shadowing is widely used for biological macromolecules and their complexes structure determination . It has been shown that tungsten shadowing produces fine granularity and good structural detail resolution. We have developed a working prototype of an electron beam evaporator (“electron gun“) based on the principle described by V. Vasiliev. The electron gun has characteristics that allow us to deposit high quality films of carbon or even pure tungsten. in addition we have constructed a prototype sample handling apparatus for freeze drying and shadowing. The prototype allows tilting of +/- 90° in 2° increments, rotation (if desired) at 6 rpm, controlled heating at a rate of 1-2° per min, and cooling using a liquid nitrogen trap attached to the device. The system is vacuumed by 480 1/sec maglev turbomolecular pump with scroll pump at the first stage. It produces oil-free vacuum in 2-5*10-7 torr range, which we believe, is sufficient for good quality shadowing.

Classic studies of gill vasculature indicate the gross anatomy of elasmobranch gills is similar in most species, and in general, resembles those of teleosts. However, studies of fine gill structure examining arterio-arterial pathways in several species of batoid elasmobranchs (Order: Rajiiformes, Suborder: Myliobatoidei) have reported some structural differences. These differences include the presence, in urolophids (Urolophus jamaicensis, U. mucosus, U. paucimaculatus), of 1) a tip channel, possibly an extension of the afferent filament artery that runs along the top of the filament corpus cavernosum, and 2) a vascular arcade, a vessel which connects the afferent filament arteries of a hemibranch near the tip of each filament (FIG. I). Until recently these structures were believed to be unique to the urolophids as they were not previously found in either rajids (Raja erinacea, R. clavata) or the shark Scyliorhinus canicula. Animals of mixed species and sexes were collected, perfused, and acrylic casts made of the circulatory system.

Dynamin is a large GTPase essential for various intracellular processes such as synaptic vesicle recycling, caveolae internalization and trafficking into and out of the Golgi. It is also involved in receptor-mediated endocytosis, and is believed to assemble at the necks of clathrin-coated pits and assist in pinching vesicles from the plasma membrane upon GTP binding and hydrolysis.

Purified recombinant dynamin self-assembles into rings and spirals in low salt conditions [1]. A dynamin mutant lacking the c-terminal proline rich domain (APRD) also assembles into rings and spirals, however unlike wild type dynamin APRD constricts in the presence of GTP analogous such as GMP-PCP [2] or GTPγS. to explore differences in the behavior of the wild type and mutant dynamin we dialyzed them into different salt solutions containing various types of nucleotides and studied their assembly over time using negative staining and cryo-TEM.

In the early 1960s, concerns about artifacts in preparing biological material for electron microscopy led to a new technique whereby samples are rapidly frozen, fractured under high vacuum, the fractured surfaces shadowed and replicated with a thin metal-carbon coat, and the cleaned replica examined in a transmission electron microscope. Pioneered by Moor and Muhlethaler subcellular structures are revealed with extraordinary three-dimensional clarity at near-molecular resolution. Furthermore, it was observed and proven at model systems as well as biological membranes that lipid bilayers split along their hydrophobic interior during freeze-fracture procedure. Therefore, freeze-fracture electron microscopy (FFEM) has the unique advantage of accessing the hydrophobic interior of biological (FIG.l, 5 and 6) as well as artificial bilayers (FIG. 2, 4, 5, and 6). Here it permits study of pattern generated by intrinsic proteins as well as lipids (FIG. 1 and 2).

Transmission electron microscopy (TEM) has traditionally served as an important tool for understanding the physical properties of many materials. Recent improvements in “ex-situ” techniques have further strengthened the role of TEM as a unique characterization method.

The use of ExxonMobilfs dedicated reactor system for studying metal oxidation at moderate temperatures has been reported. However, high temperature oxidation studies have been limited by the use of holey carbon support films, which oxidize and disintegrate during these experiments. Consequently, a new method was developed to replace the holey carbon support film with a holey metal oxide support film that both maintains its structural integrity during high temperature oxidation and remains electron transparent.

The new support film was prepared using a commercially available, holey carbon coated TEM grid as a template (Figure 1). An Al metal film (∼10nm thick) was deposited onto the holey carbon coated side of the TEM grid using a magnetron sputtering system.

Small particles such as clay minerals, because of its large surface area to volume ratio and their high chemical activity on the particle surface, when added to composites affects (and often enhances) the physical, chemical and mechanical properties of materials immensely. As a result, there is a need to develop a method of characterizing these particles individually. However, physical dispersion of these particles can be very difficult, since they tend to agglomerate to reduce the high surface energy. Disperants of various sorts could be used to disperse/stabilize the system, but they could compromise/damage the integrity of the inherent state of these small particles. This paper presents a comparison study of preparing/dispersing small clay particles for electron microscopy research, and also represents the first report of cryo-microscopy of clay minerals.

Two types of smectites, namely a montmorillonite with cation exchange capacity (CEC= 140) and a saponite (CEC=42) were used for this study.

Interplanetary dust particles (IDPs) collected from the stratosphere are grains of cosmic dust delivered from asteroids and comets. IDPs are composed of complex mixtures of carbon, silicate glass and submicron to nanometer-sized minerals including Fe-Mg silicates, Fe-Ni sulfides, oxides and metal. Bulk SEM and detailed TEM investigations of stratospheric IDPs in the size range of 5 - 15 μm have shown that many IDPs have remarkably high concentrations of carbon - essentially the highest carbon abundances of any known extraterrestrial materials - and often this carbon is intermixed at the nanometer scale with other constituent phases. in order to investigate the properties, occurrence and spatial distribution of carbon in IDPs, we have developed a unique aciddissolution technique using standard microtomed thin-sections to remove the silicate and oxide fractions, thereby leaving a residue primarily of carbon and Fe-Ni sulfides. This technique causes no damage to the delicate microtomed sections or TEM grid and dramatically minimizes the quantity of acids required for etching.

Tin dioxide (SnO2) has been extensively studied and used as gas sensors to detect toxic gases such as CO, NOxand flammable gases like H2.[l] Recently, considerable researches have focused on thin film sensors due to their high performance as well as their integration compatibility with semiconductor technology for making microsensors and sensor arrays. [2] The performance of thin film sensors is remarkably influenced by the way they were fabricated.[3] Among various deposition techniques, pulsed laser deposition (PLD) has shown great prominence in the deposition of a wide variety of oxide thin film materials such as high Tc superconductors, semiconductors and dielectrics. in this work we present our experimental results on tin dioxide films deposited using pulsed laser ablation on sapphire (α -Al2O3) substrates with different surface orientations.

Tin oxide films with a thickness of 100 nm were deposited on the (1012) and (0001) sapphire by pulsed laser ablation of ceramic SnO2 targets.

Besides perfect dislocations, partial dislocations were proposed as effective means for misfit relaxation in the heretostructure system [1]. The microstructure of SrRuO3 films on SrTiO3 and LaA1O3 substrates have been studied. While misfit dislocations could be hardly found at the SrTiO3/SrRuO3 interface [2], high density of defects was observed in the SrRuO3/LaA1O3 interfaces [3]. in this paper, we report the high-resolution electron microscopy study of the SrTiO3/SrRuO3 bilayer films on (100) LaA1O3 substrates. The emphasis is focused on the means of misfit relaxation at the two interfaces.

Figure 1(a) is a low magnification cross-sectional image of a two-layer SrTiO3/SrRuO3 film on LaA1O3 taken along the [110] direction of the substrate. A high density of defects were observed along the SrRuO3/LaA1O3 interface. Figure 1(b) is a quarter of a superposed electron diffraction pattern (EDP) from the SrTiO3/SrRuO3 interface area, while fig. 1(c) is from the SrRuO3/LaA1O3 interface area.

Zinc oxide based coatings are of much interest in science and technology due to their interesting applications, such as in gas sensor devices, transparent electrodes, piezoelectric devices, varistor ,surface acoustic-wave devices ,etc. Thin films of ZnO also have some advantages over indium tin oxide (ITO) and tin oxide, which are usually used as transparent conductors, the former are chemically stable to a hydrogen plasma such as that used in the elaboration of solar cells. Many techniques have been employed to produce zinc oxide based coatings e.g. radio frequency magnetron, spray pyrolysis, sputtering, chemical vapor deposition, sol gel, pulsed laser deposition, etc. Among these techniques, the spray pyrolysis has proved to be a simple, reproducible, and inexpensive method, particularly useful for large area applications.

The main features of the spray pyrolytic system are fully described elsewhere. We have used a spray system attached onto mobile stage that provides a sweeping movement to the spray nozzle.

Mesoscopic silica is synthesized by using a self-assembling organic species to direct the structure of an inorganic matrix. It has attracted much interest because of its potential for use in a wide variety of catalysis, separations and nanotechnology applications. The commercial success of this material depends on the ability to simultaneously tailor the channel size and organization. A more complete understanding of the mechanism by which the material forms should lead to improved control over the structure. Small angle X-ray diffraction studies have shown that there is an “induction period” which precedes the observation of well-packed channels in films grown at the air-water interface. However, scattering data alone are unable to establish how the development of order might occur. Direct observation of the evolution is necessary to determine the mechanism by which well-packed, nanometer-sized channels form.

Continuous mesoporous silica thin films were grown at the air-water interface.

Thin films of amorphous silicon oxide and silicon nitride are routinely used as gate dielectrics in silicon-based microelectronic devices. It is valuable to be able to image them and measure their thicknesses quickly and accurately. This brief note describes conditions that can be used to obtain accurate and reproducible TEM images of oxide-nitride-oxide (ONO) thin films.

Obtaining adequate contrast differences between oxide and nitride is not trivial because they have the same average atomic number, and both phases are amorphous. As stoichiometric compounds, both SiO2 and Si3N4 would have average atomic numbers equal to 10. For SiO2, (14+2(8))/3=10, and for Si3N4, (3(14)+4(7))/7=10. Thus, the atomic number contrast between these two is weak or nonexistent. Similarly, the amorphous character prevents the use of conventional diffraction contrast techniques.

However, the density of Si3N4 (3.2 g/cm3) is considerably greater than the density of SiO2 (2.6 g/cm3), reflecting the higher average coordination of N compared with O.

Diamond-like carbon (DLC) has been used as thin films to protect the surface of magnetic disks in hard disk drives. Recently, in order to improve the recording density of the hard disks, DLC films need to be thinner than 10 nm in thickness. Therefore, it is necessary to develop the accurate thickness measurement technique for thin DLC films. in this study, to establish the thickness measurement technique for DLC films using electron energy-loss spectroscopy (EELS) and electron holography, we estimate the mean free path for inelastic scattering (ƛp) and the mean inner potential (U0). Through the evaluation of ƛp and U0, we compare the accuracy of EELS and electron holography on thickness measurement of DLC films.

The DLC films in this study are deposited on silicon wafers by chemical vapor deposition (CVD), sputtering and ion beam deposition (IBD) methods.

Microstructurally engineered oxide coatings have good potential as lubricants for aerospace applications over a wide range of environmental conditions1. Using composite microstructures, wear rates and friction coefficients may be reduced compared to monolithic coatings. Here, we report the characterization of ZrO2-Au composite coatings as new candidate lubricants. Au is added to ZrO2 to reduce friction and improve toughness. The composite films were grown on steel and silicon disks at room temperature (RT) by pulsed laser ablation of a ZrO2/5 wt. % Y2O3 target, with simultaneous operation of a magnetron source to add Au. The laser beam was operated at 250mJ, 40 Hz, and the magnetron was operated at 1.5-3.5 W cm-2 in 2.7 Pa argon. Varying the magnetron power produced coatings with different concentrations of Au. The coatings were prepared for transmission electron microscope (TEM) analysis by tripod polishing and ion milling on a cold stage.

Over the years, the study of the oxidation of nanoparticles of iron by transmission electron microscopy (TEM), Mossbauer spectroscopy and X-ray diffraction has established that nanoparticles of iron have a core-shell morphology in which the iron core is enclosed by shell of polycrystalline shell of ultrasmall γ-Fe2O3 and Fe3O4 crystallites. Recently, passivated nanoparticles of iron prepared by gas condensation of plasma evaporated vapor in Tianjin University exhibit remarkable resistance to further oxidation and corrosion in air and water. We have showed by TEM that these nanoparticles of iron are protected by a 4 nm epitaxial shell of γ-Fe2O3. The epitaxial orientation relationship, established by convergent beam electron diffraction from a nanoparticle, is as follows:

The [001] diffraction pattern of the oxide is rotated by 45° about a cubic axis relative to that of iron.