Since its initial use to fix fungal hyphae (1), plunge freezing followed by freeze substitution has become the “gold standard” for TEM studies of fungal hyphae and spores. In this presentation we discuss results we have obtained using plunge freezing and freeze substitution to fix various types of spores produced by plant pathogenic fungi. Examples include basidiospores and aeciospores of rust fungi, teliospores of rust and smut fungi and conidia of a variety of ascomycetes and deuteromycetes.

Generally speaking, plunge freezing followed by freeze substitution usually yields best results when spore diameter does not exceed 10-15 μm. However, in a number of cases we (2) have obtained good to excellent results with much larger spores. A key to good freezing appears to be direct exposure of spores to liquid propane during plunging. We have had best results by placing spores on small pieces of dialysis membrane and then plunging these membranes. In most cases the spores remain attached to membranes throughout the freezing, substitution, infiltration and embedment procedures.

Determining the length of the various stages of the nuclear cycle and correlating nuclear cycle events with particular aspects of cell morphology generally require the use of autoradiography. The most useful label is tritiated thymidine, because it is incorporated only into DNA. Unfortunately fungi, unlike most other organisms, do not take incorporate thymidine from medium. In the past, microspectrophotometry has been used to study fungal nuclei, but this technique measures nuclei one at a time. Recent advances in image capture and analysis software have made it possible to use the technique of photomicrodensitometry to quantify DNA in nuclei stained with fluorochromes that bind quantitatively to DNA. We are using this method to study nuclear cycle events in cells of the pathogenic fungus Ustilago maydis.

The basidiomycete fungus Ustilago maydis causes corn smut disease. This fungus is dimorphic—in addition to growing by filaments in corn plants, it can exist saprobically in a yeast-like budding stage.

Entomosporium mespili has emerged as a significant pathogen of red tip (Photinia × fraseri), a popular and widely grown ornamental in the southeastern United States. This fungal pathogen produces its distinctive multi-celled, insect-like asexual spores or conidia (Fig. 1) in structures known as acervuli (Fig. 2) that rupture the surfaces of infected leaves. This study examines the fine structure of these conidia using a combination of scanning and transmission electron microscopy. In the case of transmission electron microscopy, conidia were prepared for study using either plunge freezing or high pressure freezing followed by freeze substitution.

Each mature conidium of E. mespili consists of four to six cells (Fig. 1). These include one apical cell and one basal cell and two to four small lateral cells attached to the basal cell. The apical and lateral cells each possess a long, slender appendage. Excluding these appendages, the length of a mature conidium is usually 20-24μm while the diameters of the apical and basal cells are 8-11μm and 6-9μm respectively.

Cryofixation is becoming more widely used to study host-pathogen relationships in fungal diseases of plants. This presentation describes results we have obtained using high pressure freezing and freeze substitution to study powdery mildew disease of poinsettia ﹛Euphorbia pulcherrima) caused by Oidium sp.

Approximately 0.5 mm leaf disks bearing sporulating colonies of Oidium sp. were excised and placed in a 15% dextran solution contained in brass planchets. Samples were frozen using a Balzer's HPM 010 High Pressure Freezing Machine and substituted according to the procedures of Hoch.6 Thin sections of embedded leaves were cut using a diamond knife, collected on gold slot grids, and placed on formvar-coated racks. Sections were poststained with uranyl acetate and lead citrate and examined using a Zeiss EM 902A transmission electron microscope.

Outstanding preservation of haustoria, the specialized nutrient-absorbing structures produced in host epidermal cells by Oidium, was obtained. Both young, unlobed (Fig. 1) as well as mature, highly lobed (Fig. 2) haustoria were observed.

There are number of dwarf mutants in maize such as d8, dl and nal. The nana plant 1 (nal/nal) is characterized as short erect dwarf; the phenotype does not respond to treatment of giberellins. The recessive nal allele is located on the long arm of chromosome 3 (3L-113). In this study, we report the abnormal development of the stem vasculature and the lack of clearly defined nodes.

Maize (Zea mays L., var. Ohio 43 and nal mutant) plants were grown in 1999 summer nursery at the fieldstation of University of Western Ontario, London Ontario, Canada. Plants at approximately 2 months old were fixed in 1:3 acetic acid/ EtOH. Multiple changes of fixative were made for a period of a month to remove as much pigmentation as possible. The specimen was then washed thoroughly, stained in diluted Schiffs reagent (10%), washed in 0.1M metabisulphate solution (with 1 ml of 1 N HC1 per liter), dehydrated in EtOH and cleared in methyl salicylate.

Cadmium and zinc are transition elements belonging to group II B in periodical table and they share similar chemical properties. However, their morphological and physiological effects on plants are quite difference. Cadmium not only consequences changes of plant structure at the light and electron microscopic levels, but also results in the induction of phytochelatin synthesis. In the present studies, The distribution of cadmium in different parts of plants was measured and its effects on plant structure were also examined.

Three to six-week-old Zinnia elegans were grown Hoagland's solution containing different concentrations of cadmium from one to two weeks. Atomic absorption spectroscopic studies showed that the distribution of cadmium was mainly in root. The accumulation of cadmium in stems was higher than that of in leaves. In leaves, the ability of cadmium accumulation in young leaves was much higher than that of old leaves.

The embryo sac (ES) of Plumbago zeylanica, unlike most other flowering plants, lacks synergidsthe cell type that usually receives the pollen tube and the male gametes. Normally, synergids store copious amounts of calcium, estimated to exceed 15% by weight; this is believed to attract pollen tubes, which penetrate the ES, and may trigger the release of the sperm cells within one of the two synergids. If high concentrations of calcium are truly required for fertilization, the ES of Plumbago should also contain significant quantities. Synergids in normal flowering plants are preprogrammed for cell death, receiving the contents of the pollen tube directly in their cells, whereas synergid-lacking angiosperms apparently do not have such an apoptotic cell in the ES. Potassium pyroantimonate labeling was used to localize principally loosely-bound calcium, because it is a relatively mobile form of the Ca2+ ion that is available for redistribution during fertilization events.

The appressoria formation of Colletotrichum graminicola was monitored by scanning electron microscopy (SEM) coupled with an X Ray microanalysis system (EDS - Oxford Instrument LINK ISIS). Recently formed appressoria, an infection structure of plant pathogenic fungi, firmly glues itself to an artificial surface (polystyrene) as a consequence of the production of an adhesive material (AM). The nature of this material was already demonstrated to be mainly constituted of a glycoprotein (Sugui et al, PMPP, 1998). The objective of this work was to verify the involvement of divalent ions, specially calcium, in the process as whole.

The AM was isolated and purified from conidia that germinated on polystyrene Petri dishes. The primary AM was dialyzed against three liters of distilled water before being lyophilized. Subsequently, the material was placed on top of a carbon tape and observed by scanning electron microscopy under high vacuum (Fig. 1 and 2A). On the other hand, the same material was submitted to X Ray microanalysis without coating.

Mature sperm cells of Ceratopteris richardii are spiraled over three revolutions and contain a locomotory apparatus with approximately 70 flagella attached at the cell anterior (Fig. 1). Abundant organelles are dispersed along the inner surface of an elongated, coiled nucleus (Figs. 1, 3, 8). The cytoskeleton comprises a network of microfilaments that encases the nucleus (Fig. 6) and two distinct microtubule arrays: a microtubular ribbon (spline) and flagella with anchoring basal bodies (Fig. 3). This study uses light, fluorescence, transmission electron (TEM), and scanning electron microscopy (SEM) to characterize cell organization in a motility impaired sperm mutant line of Ceratopteris. Because motility mutations likely involve the cytoskeleton, emphasis was placed on observing microtubule and actin arrays in the mutant compared to wild-type sperm cells. Protocols follow Hoffman and Vaughn for TEM and Schmitt and Renzaglia for SEM. Microfilament arrays in relation to the nucleus were examined by rhodamine-phalloidin and DAPI fluorescence.

The accommodation of strain in metal films can lead to complex phenomena, both structurally and chemically. It can generically lead to the formation of dislocations. These dislocations play an important role in defining the evolution and response of the film to further growth and external forces. The structures of the dislocations are directly analogous to bulk dislocations, but their properties are very different due to the presence of the surface. In addition, the strain can greatly affect the chemical properties of the film, leading to alternative strain relief mechanisms involving vacancy formation resulting from reaction with gases such as sulfur and oxygen. Using STM and LEEM, we have investigated these phenomena in the prototype systems of Cu and Ag on Ru(0001).

For example, figure la - d shows a sequence of STM images of a 2 layer Cu film on Ru(0001) during exposure to oxygen. Dislocations present in the film are altered by reaction with oxygen.

Understanding how defects alter physical properties of materials has lead to improvements in materials growth as well as device performance. Transmission electron microscopy (TEM) provides an invaluable tool for structural characterization of defects. Our current knowledge of crystallographic defects, such as dislocations, would not have been possible without TEM. Recently, scanning tunneling microscopy and scanning force microscopy (SFM) have shown the capability of imaging surface defects with atomic or near-atomic resolution in topographic images. What is more important is to gain knowledge on how the presence of a certain type of defects changes the physical properties of materials. For example, how is the carrier lifetime altered near electrically active defects? How does photoresponse vary near grain boundaries? Where are defect levels in the forbidden bandgap? This talk will discuss several examples of how scanning probe microscopies (SPMs) can contribute to this aspect of defect studies in semiconductors.

Possible applications of ferroelectric materials in non-volatile memories, MEMS, microwave ceramics, PTCR devices and sensors draw significant interest to these materials. Operation of most of these devices relies heavily on the surface (FRAM and other thin-film devices) and interface (PTCR, varistors) properties of ferroeiectrics, particularly on the polarization and charge distribution in the surface or interface region. Electrostatic scanning probe techniques such as electrostatic force microscopy (EFM), scanning surface potential microscopy (SSPM) and piezoresponse imaging (PRI) can be successfully employed for the characterization of ferroelectric surfaces on the micron and submicron level. The former technique is based on the detection of the resonant frequency shift of mechanically driven cantilever, which is proportional to gradient of electrostatic force acting on the tip. The latter two techniques are based on the voltage modulation approach, i.e. during imaging the piezoelectric actuator driving the cantilever is disengaged and the AC bias is applied directly to conductive tip.

The stability of polar and neutral MgO surfaces has been addressed by theory and experiment as an ideal example of ionic oxides with rock-salt structure, but with unresolved controversial results. For example, energy-minimization theory predicted that higher energy polar MgO (111) and neutral MgO (110) surfaces could thermally facet into the lowest energy MgO (100) planes. Early low-energy electron diffraction (LEED) experiments reported diffraction spot splitting, interpreted as evidence for thermal faceting to ﹛100﹜ planes. However, a recent study found MgO (111) faceting was caused by chemical etching with nitric or phosphoric acid, and not promoted by annealing. Quantitative atomic force microscopy (AFM) and scanning electron microscopy (SEM) measurement of the facet angles revealed them to be vicinal planes rather than the predicted ﹛100﹜ planes. A recent AFM study reported thermal faceting of MgO (110) into vicinal planes.

Thin films (<100nm) of diblock copolymers are being investigated for various applications including templates for nanopatterning [1], electronic packaging materials, and biomedical applications. In such applications it is essential that stable defect free films be produced repeatedly. Because long and short range forces (like van der Waals) dominate thin polymer films, instabilities are introduced in the films when they are spin-coated onto hydrophobic substrates resulting in dewetting of the film from the substrate. Dewetting is initiated at a nucleation site in a metastable film leading to the formation of a dry patch and proceeds to grow by transport of material away from the nucleation site, forming a lip that surrounds the hole. Highly symmetrical structures form during progression of the dewetting process and completion of the process can be identified when all holes coalesce forming polygons outlined by droplets of the polymer film [2].

Surface plasmons have been used to detect single molecular layers for many years. The most common examples are biosensor systems and the detection of self-assembled monolayers (SAMs). A surface plasmon is a two-dimensional electromagnetic wave that travels on the surface of a noble metal, most commonly gold or silver. This electromagnetic wave is extremely sensitive to changes in the dielectric constant 100 nm above the metal surface. When the dielectric constant above the metal changes due to the addition of a molecular layer, the intensity of the surface plasmon will decrease. In this way monolayers may be indirectly imaged on a surface by mapping the surface plasmon intensity.

Although there are many reasons for imaging ultrathin films or molecular layers over large distances (10-100 μm), there are several difficulties involved. Typically the substrate has a roughness greater than the height of a single monolayer. This can mask the presence of the monolayer on large area scans.

Processes at dislocation sites are frequently predominant mechanisms of surface evaporation or growth and thus determine resulting surface morphologies. Dislocation-induced evaporation patterns are also a convenient object for observation of dynamics of step motion on crystal surfaces, as such patterns have “fixed” origins, being pinned at the point of dislocation emergence. In the present work, observations of evaporation spirals formed on ﹛111﹜ and ﹛001﹜ surfaces of MgAl2O4 spinel are reported.

2x2x1 mm samples of single-crystal MgAl2O4 spinel of ﹛111﹜ and ﹛001﹜ orientation were annealed in a spinel crucible in vacuum (10-4—10-5 Torr) in a Centorr™ furnace at 1800°C for 8 hours. SPM images were collected in air in contact mode on a Nanoscope III (Digital Instruments, Santa Barbara, CA), using Si3N4 cantilevers (Ultralevers, Park Inst., Sunnyvale, CA) with a nominal spring constant of 0.12 N/m. Selected sample surfaces were examined for impurities by energy-dispersive X-ray spectroscopy (EDS) and X-ray photoemission spectroscopy (XPS).

Sintering and many problems in adsorption and catalysis require an understanding of the surface structures of crystals, which have been subjected to various types of heat treatment. For example, heat treatment of m-plane sapphire surfaces can cause a transformation from a flat vicinal surface into those of a faceted hill-and-valley structure. Many technologically interesting materials form with the garnet structure, including YIG, YAG, GGG and many silicates. However, there has been relatively little discussion of the structure and behavior of such surfaces. This paper illustrates the use of visible-light microscopy (VLM), atomic-force microscopy (AFM) and scanning electron microscopy (SEM) for studying the ﹛111﹜ surface of gadolinium gallium garnet (GGG or Gd3Ga5O12).

2x2x1 mm samples of monocrystalline GGG with ﹛111﹜ surface orientation were heat treated in air at 1575°C for 24 hours in order to study the processes a surface undertakes while reaching its equilibrium state.

The local chemistry of many surface reactions is unknown, since no techniques exist for identifying atom types at individual defect sites on extended crystal surfaces. An STM has therefore been constructed which allows atomic clusters of interest to be transferred into a time-of-flight spectrometer for species identification. Atoms are first transferred from the STM sample to the tip, using a small voltage pulse. The sample is then removed, and these atoms ejected into a time-of-flight (TOF) analyzer for mass identification. A 75mm flat Chevron MCP is used as TOF-detector. The output is led to a digital oscilloscope, whose trace is triggered by the tip pulse. Scope, triggering and high voltage are under Labview control, spectra are transferred into the PC and the mass to charge ratio of the ionized species is calculated. The STM has a vertical axis and is based on the Burleigh Inchworm with tube scanner for sample movement.

A combination of electrostatic force microscopy (EFM) and scanning surface potential microscopy (SSPM) is used to quantify nanometer scale field variations for the general case of electronically inhomogeneous surfaces. The specific example illustrated here is the intersection of a Σ5 grain boundary in donor doped SrTiO3 intersecting the (100) surface. ‘The topographic structure is compared to the surface potential, the positive and the negative electrostatic force images in figure 1. The EFM contrast changes sign with the polarity of the applied bias, as expected for an electrostatic attractive/repulsive interaction. There is a significant localization of surface potential at the grain boundary that is manifest as a protrusion in the SPPM image.

To quantify the properties at the surface, rather than above the surface where the measurements are acquired, a relationship connecting the sample-tip interaction is required. It has been shown that models based on simple geometric assumptions do not represent the behavior well.

Reliability of thin polymer films is strongly dependent on interfacial adhesion. This paper concerns the application of indentation combined with atomic force microscopy (AFM) for the evaluation of polymer-non-polymer adhesion. Interfacial toughness calculations have been made using AFM measurement of geometry of delaminations induced by indentation. Fracture surfaces characterized with AFM are also presented here.

The interface chosen for study is that between polystyrene (PS) and a glass substrate. Spin coating has been used to prepare PS films. PS, dissolved in toluene, was spun onto the soda-lime glass and then dried at 60°C for 3h to remove residual solvent. The thickness of the PS film was approximately 660nm. To enhance the driving force for delamination, poly(methyl methacrylate) (PMMA) overlayers were applied on top of the PS films. The PMMA overlayer, of thickness approximately 1.5 μm, was first solvent cast onto a second glass slide, floated off the glass onto deionized water, and then laid on top of a first glass substrate which had been coated with PS.