Low Voltage Field Emission Scanning Electron Microscopy Research Articles

High-Resolution Field Emission Scanning Electron Microscopy (FESEM) Imaging of Cellulose Microfibril Organization in Plant Primary Cell Walls

We have used field emission scanning electron microscopy (FESEM) to study the high-resolution organization of cellulose microfibrils in onion epidermal cell walls. We frequently found that conventional "rule of thumb" conditions for imaging of biological samples did not yield high-resolution images of cellulose organization and often resulted in artifacts or distortions of cell wall structure. Here we detail our method of one-step fixation and dehydration with 100% ethanol, followed by critical point drying, ultrathin iridium (Ir) sputter coating (3 s), and FESEM imaging at a moderate accelerating voltage (10 kV) with an In-lens detector. We compare results obtained with our improved protocol with images obtained with samples processed by conventional aldehyde fixation, graded dehydration, sputter coating with Au, Au/Pd, or carbon, and low-voltage FESEM imaging. The results demonstrated that our protocol is simpler, causes little artifact, and is more suitable for high-resolution imaging of cell wall cellulose microfibrils whereas such imaging is very challenging by conventional methods.

Materials characterization using high-resolution scanning-electron microscopy and x-ray microanalysis

Low-voltage field-emission scanning-electron microscopy offers the possibility to characterize a wide range of materials. Electron optics in an electron beam column have improved in recent decades and now probe diameters of 1–10 nm can be obtained, allowing a wide range of applications to be explored. This article discusses the applications of low-voltage microscopy, including the characterization of nanoparticles, super-lattice structures, and carbon nanotubes.

Preparation and antibacterial test of chitosan/PAA/PEGDA bi-layer composite membranes

Chitosan/poly(acrylic acid)/poly(ethylene glycol) diacrylate (PEGDA) composite membranes with a bi-layer configuration were prepared and their potential application as an antibacterial material was examined. A two-step process was adopted. A dope consisting of PEGDA, acrylic acid (AA) and a photoinitiator was cast and then UV-cured on a glass substrate to form a mechanically strong, dense membrane. Subsequently, the membrane was coated with a layer of solution composed of chitosan, AA and water. As the majority of AA diffused downwards into the supporting layer underneath, chitosan coagulated with residual AA to form a nano-layer on the top surface by means of UV irradiation. Low-voltage field-emission scanning electron microscopy was used to observe the membrane morphology and to measure the thickness of the top layer. Contact angle measurements indicated a top layer mixed with chitosan and poly(acrylic acid), which was confirmed by chemical composition analysis of X-ray photon spectroscopy. The antibacterial activities of the formed membranes were tested both with respect to a Gram-negative (Escherichia coli) and a Gram-positive (Staphylococcus aureus) bacteria.

Structural evidence for actin-like filaments in Toxoplasma gondii using high-resolution low-voltage field emission scanning electron microscopy.

The protozoan parasite Toxoplasma gondii is representative of a large group of parasites within the phylum Apicomplexa, which share a highly unusual motility system that is crucial for locomotion and active host cell invasion. Despite the importance of motility in the pathology of these unicellular organisms, the motor mechanisms for locomotion remain uncertain, largely because only limited data exist about composition and organization of the cytoskeleton. By using cytoskeleton stabilizing protocols on membrane-extracted parasites and novel imaging with high-resolution low-voltage field emission scanning electron microscopy (LVFESEM), we were able to visualize for the first time a network of actin-sized filaments just below the cell membrane. A complex cytoskeletal network remained after removing the actin-sized fibers with cytochalasin D, revealing longitudinally arranged, subpellicular microtubules and intermediate-sized fibers of 10 nm, which, in stereo images, are seen both above and below the microtubules. These approaches open new possibilities to characterize more fully the largely unexplored and unconventional cytoskeletal motility complex in apicomplexan parasites.

Microstructure of Monoplacophora (Mollusca) shell examined by low-voltage field emission scanning electron and atomic force microscopy.

The shell of Micropilina arntzi (Mollusca: Monoplacophora), a primitive molluscan class, was examined by using field emission scanning electron microscopy (FESEM) at low voltage and atomic force microscopy (AFM). The use of these two techniques allowed the observation of fine details of Micropilina arntzi shell and contributed to bring new features concerning the study of molluscan shell microtexture. Imaging with low-voltage FESEM provided well-defined edge contours of shell structures, while analyzing the sample with AFM gave information about the step height of stacked internal structures as well as the dimension of the particles present in their surface at a nanometric level. The shell microstructure of Monoplacophora species presents different patterns and may be a taxonomic implication in the systematic studies of the group.

Unconventional specimen preparation techniques using high resolution low voltage field emission scanning electron microscopy to study cell motility, host cell invasion, and internal cell structures in Toxoplasma gondii.

Apicomplexan parasites employ complex and unconventional mechanisms for cell locomotion, host cell invasion, and cell division that are only poorly understood. While immunofluorescence and conventional transmission electron microscopy have been used to answer questions about the localization of some cytoskeletal proteins and cell organelles, many questions remain unanswered, partly because new methods are needed to study the complex interactions of cytoskeletal proteins and organelles that play a role in cell locomotion, host cell invasion, and cell division. The choice of fixation and preparation methods has proven critical for the analysis of cytoskeletal proteins because of the rapid turnover of actin filaments and the dense spatial organization of the cytoskeleton and its association with the complex membrane system. Here we introduce new methods to study structural aspects of cytoskeletal motility, host cell invasion, and cell division of Toxoplasma gondii, a most suitable laboratory model that is representative of apicomplexan parasites. The novel approach in our experiments is the use of high resolution low voltage field emission scanning electron microscopy (LVFESEM) combined with two new specimen preparation techniques. The first method uses LVFESEM after membrane extraction and stabilization of the cytoskeleton. This method allows viewing of actin filaments which had not been possible with any other method available so far. The second approach of imaging the parasite's ultrastructure and interactions with host cells uses semithick sections (200 nm) that are resin de-embedded (Ris and Malecki, 1993) and imaged with LVFESEM. This method allows analysis of structural detail in the parasite before and after host cell invasion and interactions with the membrane of the parasitophorous vacuole as well as parasite cell division.

Ultrastructure of Bone Mineralization and Osseointegration with Hydroxyapatite-Coated Bone Implants in Vivo

The need to optimize adhesion between the femoral stem and surrounding tissue has led to the development of plasma-sprayed HA (PSHA) coatings as attachment facilitators for hip prostheses. The mechanisms underlying this facilitation are incompletely understood, and the long-term efficacy of HA coatings in maintaining bonding to bone remains largely unexplored. in this study, two in vivo models—canine and human—were employed for study by scanning electron microscopy (SEM) and transmission electron microscopy (TEM) to elucidate the sequence of early bone mineralization and the longer term fate of HA coatings. High-resolution SEM and TEM of whole bone and anorganic bone were employed to establish the structure of mature bone for comparison.Whole and Anorganic Bone.Anorganic bone derived from bovine trabecular bone was examined in low-voltage field-emission SEM(LV-FESEM) and high-resolution TEM to establish the morphology of the mineral component of bone [1,2]. Trabeculae were seen to comprise oriented fiber bundles (Fig. 1), each fiber comprising an array of oriented apatite platelets (Fig. 2) arranged like a sheared stack of dominoes with approximately the 64-nm stagger of collagen hole zones.

A New High Resolution Field Emission SEM with Variable Pressure Capabilities

To examine non conductive samples in their natural state (i.e. without significant sample preparation) at high resolution in the SEM the technique of low voltage field emission scanning electron microscopy (LVFESEM) is used. Due to the limitation in accelerating voltage (U<1kV) this technique is limited in respect of chemical analysis. Furthermore it is not possible to examine humid and outgassing samples in high vacuum. in recent years the application of variable pressure scanning electron microscopes (VPSEM) became an important technique in materials science as well as in life science. Due to the capability of maintaining a high chamber pressure humid, outgassing and non-conductive samples, can be examined in their natural state without significant sample modification or preparation. Especially compound materials with different electron yields can be imaged without any charging effects (Fig. 2), [2]. This paper describes a high resolution field emission electron microscope, that combines low voltage and variable pressure capabilities.The high pressure capabilities of the instrument are realized by eliminating the high vacuum requirements of SEM in the microscope chamber. This is done by separating the vacuum environment in the chamber from the ultra high vacuum environment in the gun area.

Uncoated Lvsem and Imaging Tof-Sims of Unfixed, Plunge Frozen, Freeze Dried, Fungal and Plant Material

A Cryosorption Freeze Drying (CFD) system was evaluated for its effectiveness in preparing delicate biological materials for both low voltage-field emission scanning electron microscopy (LVFESEM) and imaging liquid metal (Ga) ion beam, static time-of-flight, secondary ion mass spectrometry (TOF-SIMS). The primary goals of these studies were to investigate the retention of both structural and chemical integrity using fresh cryoprepared biological material which had not been exposed to any chemical fixation and which would not be coated by any conductive material in order to obtain information from the native surfaces. Duplicate chemically fixed samples were processed for comparison. LV-FESEM (2-2.5kV) was used to assess the quality of the structural preservation of the freezing and freeze drying (FD) protocols. Imaging static TOF-SIMS was used to investigate the surface chemical compositions of the biological samples.

Effect of Staining on Latex Particle Size Examined by Electron Microscopy

Electron microscopy has been an invaluble tool to the study of polymer morphology, especially latex particles, microphase-separated block copolymers, and polymer blends. However, in order to examine these materials in an electron microscope, the sample often needs to be stained to heighten contrast between locales of different composition in the specimen. Staining usually has the following advantages: heightened contrast; higher polymer glass transition temperature Tg; lowered charging; and reduced radiation damage. However, staining can also introduce artifact. It is reported that the latex particle size is raised and the polybutadiene “sphere” in block copolymers is lowered after staining. This indicates that the staining process may be complex.Recently, low voltage field emission scanning electron microscopy (LVFESEM), especially high-resolution backscattered electron imaging produced with staining, has been used to study latex film formation, latex particle deformation and adhesion on substrates, latex particle deformation, distribution and binding in paper coatings.

Latex Particle Deformation and Adhesion on Substrates Studied by Low Voltage Field Emission SEM

Synthetic polymer latex is widely used in paints, paper coatings, adhesives, and a growing number of other water-borne coatings. All these applications require the latex particles adhere to one another and weld by interdiffusion. The processes of film formation by latex have been studied intensively during the last several decades. The three major stages of the processes are identified: consolidation, particle deformation and coalescence. In contrast, the deformation and adhesion of latex particles on pigment are poorly documented because the needed measurements are difficult to perform at particle level by conventional techniques2. Recently, the ordering and adhesion of styrene-butyl acrylate latex particles on model inorganic surfaces have been studied by atomic force microscopy (AFM). In this work, the deformation and adhesion of latex particles of styrene-butadiene on smooth substrates are examined by low voltage field emission scanning electron microscopy (LVFESEM) with contrast enhancement by staining.

Low-voltage field-emission scanning electron microscopy of non-coated guinea-pig hair cell stereocilia

The stereociliar structures of the guinea-pig cochlear organ of Corti were studied at low-voltage (1–5 kV) with field-emission scanning electron microscope (SEM) using various pre- and post-fixation methods, such as OTOTO (OsO4/thiocarbohydrazide/OsO4/thiocarbohydrazide/OsO4) and TAO (tannic acid/arginine/OsO4), and different dissection procedures of the cochlea. A perfusion and immersion pre-fixation with glutaraldehyde, in combination with removal of the bony wall and stria vascularis from the cochlea, followed by the TAO non-coating treatment, gave the best result at 2 kV acceleration voltage. Due to these new techniques, several interesting delicate structures of the stereocilia, in particular fine surface structures, were detected for the first time using SEM. These findings include the different types of cross-links and tip links, i.e., the fine surface morphology of the stereocilia and their attachments and imprints in the tectorial membrane (TM). One of the most interesting findings in this study is a network of long filamentous structures, which has been identified mainly at the top of the longest stereocilia and the undersurface of the TM and which may represent the glycocalyx. These findings and their possible implications in the process of mechanoelectrical transduction will be discussed.

Structure hierarchy in liquid crystalline polymers

Structure models have been developed for the liquid crystalline polymers (LCPs), showing the existence of fibrillar hierarchies for both the lyotropic aramids and the thermotropic aromatic copolyesters. Hierarchies of structure have also been observed for biological materials. The nature of the smallest nanostructure that aggregates, typically microfibrils, and their interaction, are important in understanding the behavior of the material. This paper discusses the first application of scanning tunneling microscopy (STM) and field emission scanning electron microscopy (FESEM) to image the microfibrils in LCPs, in the 1-10 nm size range, resulting in a new LCP structural model.The structure model proposed earlier, was based on the study of Vectra® thermotropic LCP moldings and extrudates, and Vectran® and Kevlar® fibers. The model resulted from characterization by light microscopy, and transmission and scanning electron microscopy. Recent studies of similar fibers by STM and low voltage FESEM has provided additional insights. Details of single microfibrils and their aggregation into fibrils and macrofibrils was shown.

Effective diameters of protein A-gold and goat anti-rabbit-gold conjugates visualized by field emission scanning electron microscopy.

High-voltage (15-30 kV) field emission scanning electron microscopy (FESEM) was used to evaluate the effects of gold particle size and protein concentration on the formation of protein-gold complexes. Six colloidal gold sols were prepared, ranging in diameter from 7.6 to 39.8 nm. The minimal protecting amounts (m.p.a.) of protein A and goat anti-rabbit antibody (GAR) were experimentally determined. Gold particles were conjugated at the m.p.a., one half the m.p.a., and ten times the m.p.a. for both proteins, and protein-gold complexes prepared for FESEM. The smallest colloidal gold particles required the most protein per milliliter of gold suspension for stabilization. Transmission electron microscopy was found to be the preferred method for accurate sizing of gold particles, whereas FESEM of protein-gold complexes permitted visualization of a protein halo around a spherical gold core. Protein halo width varied significantly with changes in gold particle size. Measurements of protein halos indicated that conjugation with the m.p.a. of protein A resulted in the thickest protein layers for all gold sizes. GAR conjugation with the m.p.a. again produced the thickest protein layers. However, GAR halos were significantly smaller than those obtained with protein A conjugation. The proteins used showed similar adsorption patterns for the larger gold particles. For smaller gold particles, proteins may act differently, and these complexes should be further characterized by low-voltage FESEM.

Low-voltage field-emission scanning electron microscopy applications in nematology

Conventional scanning electron microscopy (CSEM) has long been used to gain structural information on the taxonomy, morphology, host-parasite relationships and predators of plant parasitic nematodes. Although a significant amount of new information has accumulated during the past few years, further gains in structural detail will be hampered because CSEMs have resolutions of 40-70A, 5-20kV accelerating voltages are normally required to excite adequate secondary electrons, and current preparation techniques require specimen coatings of 200-300A.Recently a new SEM, the Hitachi S900, combined a condensor-objective lens system with a field emission electron source. This instrument, known as a field emission (FE) SEM, has a resolution of about 5A or 10 times greater than that of CSEMs, can be used to observe specimens with little or no coating and operates at accelerating voltages as low as 1 or 2 kV while producing electron densities nearly 1000 times brighter than those of CSEMs.