Electron Spectroscopic Imaging Research Articles

Immunocytochemical approach for surface layer proteins of freeze-substituted Tannerella forsythensis by energy-filtering transmission electron microscopy

Tannerella forsythensis (Bacteroides forsythus), an anaerobic gram-negative potential periodontal pathogens in the progression of periodontitis. IT forsythensis has unique bacterial protein profiles containing major proteins with apparent molecular weight of more than 200-kDa shown by sodium dodecylsulfate-polyacrylamide gel electrophoresis. It is also known to have a typical surface layer (S-layer) consisting of regularly arrayed subunits outside the outer membrane revealed by electron microscopy. On the other hand, electron microscopy showed that the best preservation of structure was obtained when cells were postfixed with OsO4, but this resulted in very low levels of gold particles labeling. Therefore, cells were applied to pieces of filter paper and freeze-substituted by plung-freezing in Liquid propane, substituted in methanol containing 0.5% uranyl acetate, and infiltrated with LR-White resin. We also examined the relation between high molecular weight proteins and S-layer in energy-filtering transmission electron microscopy (EF-TEM) to visualize 3,3'-diaminobenzidene, tetrahydrochloride (DAB) reaction. The three-window method in electron spectroscopic images (ESI) of nitrogen (N) element, reflecting the presence of DAB moieties by the DAB reaction solution, horseradish peroxidase (HRP)-conjugated secondary antibodies instead of immunogold particles obtained by the EF-TEM. The mapping patterns of net N element were restricted to the outermost cell surface.

Ion etching methods for depth profiling of complex three-dimensional samples in combination with scanning Auger electron microscopy

Ion etching of surfaces combined with detection of secondary events (particles or radiation emitted) are used for depth profiling of samples with interesting features at-, near-, or somewhat below the surface. These methods are destructive and relatively slow, and compete with non-destructive methods like Rutherford backscattering spectroscopy, energy-dispersive X-ray spectroscopy in the scanning electron microscope or angle-resolved photoemission spectroscopy, which are non-destructive and relatively faster methods. In this work we have concentrated on the use of noble gas ion sputtering with low-energy beams in combination with electron excited Auger electron spectroscopy and imaging for analysis of nanostructured and microstructured samples. No attempt will be made here to justify this method over the other methods, as their relative merits depend on the nature of the sample and the problem at hand. We have thus chosen to study samples and problems for which this technique would be obvious to use. This work is also aimed at providing practical standards and guidelines (“metrology”) for the use of the technique in the context of industrial nanotechnology. The use of Auger electron spectroscopy instead of photoemission spectroscopy is preferred for laterally non-uniform samples due to the presently better resolution capabilities of electron beams and narrower information depths of typical Auger electron transitions. The use of Auger electrons for concentration sampling, and low-energy beams of noble gas ions for sputtering, reduces the adverse influence of atomic mixing. In this report two systems are intensively studied with sputter profiling in combination with Auger electron spectroscopy and scanning electron imaging: a hard disk and a surface of a stainless-steel sample.

Analytical TEM study of annealed nanocrystalline cobalt–phosphorous electrodeposits

Investigation of thermal stability of two nanocrystalline Co-P alloys shows that P atoms segregate to the grain boundaries upon annealing until precipitation of Co(2)P and CoP precipitates takes place. The P-rich precipitates formed have been investigated by analytical transmission electron microscopy to obtain statistical results of precipitate size, volume fraction and spatial distribution. Electron spectroscopic imaging maps show that the P-rich precipitates are 33 +/- 9 nm in Co-1.1at.%P and 33 +/- 12 nm in Co-3.2at.%P. The main differences between the alloys are the precipitate size distribution (Co-3.2at.%P having broader distribution) and precipitate volume number density (Co-3.2at.%P has 1.8 times more precipitates than Co-1.1at.%P). The volume fraction of precipitates is 3.0% in Co-1.1at.%P and 4.4% in Co-1.1at.%P. Most of the precipitates are of nearly spherical or slightly elongated shape, and only a few have a platelet-like shape as expected from previous tomographic atom probe measurements. Due to the truncation and projection effects, the composition of the precipitates could not be determined.

Size effects in van der Waals clusters studied by spin and angle-resolved electron spectroscopy and multi-coincidence ion imaging

We have studied the valence and inner-shell photoionization of free rare-gas clusters by means of angle and spin resolved photoelectron spectroscopy and momentum resolving electron-multi-ion coincidence spectroscopy. The electron measurements probe the evolution of the photoelectron angular distribution and spin polarization parameters as a function of photon energy and cluster size, and reveal a strong cluster size dependence of the photoelectron angular distributions in certain photon energy regions. In contrast, the spin polarization parameter of the cluster photoelectrons is found to be very close to the atomic value for all covered photon energies and cluster sizes. The ion imaging measurements, which probe the fragmentation dynamics of multiply charged van der Waals clusters, also exhibit a pronounced cluster size dependence.

Multiple Correlative Immunolabeling for Light and Electron Microscopy Using Fluorophores and Colloidal Metal Particles

Multiple correlative immunolabeling permits colocalization of molecular species for sequential observation of the same sample in light microscopy (LM) and electron microscopy (EM). This technique allows rapid evaluation of labeling via LM, prior to subsequent time-consuming preparation and observation with transmission electric microscopy (TEM). The procedure also yields two different complementary data sets. In LM, different fluorophores are distinguished by their respective excitation and emission wavelengths. In EM, colloidal metal nanoparticles of different elemental composition can be differentiated and mapped by energy-filtering transmission electron microscopy with electron spectroscopic imaging. For the highest level of spatial resolution in TEM, colloidal metal particles were conjugated directly to primary antibodies. For LM, fluorophores were conjugated to secondary antibodies, which did not affect the spatial resolution attainable by fluorescence microscopy but placed the fluorophore at a sufficient distance from the metal particle to limit quenching of the fluorescence signal. It also effectively kept the fluorophore at a sufficient distance from the colloidal metal particles, which resulted in limiting quenching of the fluorescent signal. Two well-defined model systems consisting of myosin and alpha-actinin bands of skeletal muscle tissue and also actin and alpha-actinin of human platelets in ultrathin Epon sections were labeled using both fluorophores (Cy2 and Cy3) as markers for LM and equally sized colloidal gold (cAu) and colloidal palladium (cPd) particles as reporters for TEM. Each sample was labeled by a mixture of conjugates or labels and observed by LM, then further processed for TEM.

Sub-cellular localization of manganese in the basal ganglia of normal and manganese-treated rats: An electron spectroscopy imaging and electron energy-loss spectroscopy study

We have studied at the ultrastructural level the presence of manganese (Mn) in rat basal ganglia, which are target regions of the brain for Mn toxicity. The rats underwent a moderate level of Mn exposure induced per os for 13 weeks. Mn was detected by means of electron spectroscopy imaging (ESI) and electron energy-loss spectroscopy (EELS) analyses on perfusion fixed samples embedded in resin. While no significant contamination by exogenous Mn occurred during the processing procedures, less than 50% of endogenous Mn was lost during fixation and dehydration of the brain samples. The residual Mn ions in the samples appeared as discrete particles, localized in selected sub-cellular organelles in a cell, suggesting that no significant translocation had occurred in the surrounding area. In control rats, the Mn sub-cellular localization and relative content were the same in neurons and astrocytes of rat striatum and globus pallidus: the Mn level was highest in the heterochromatin and in the nucleolus, intermediate in the cytoplasm, and lowest in the mitochondria ( p < 0.001). After chronic Mn treatment , while no ultrastructural damage was detected in the neurons and glial cells, the largest rate of Mn increase was noted in the mitochondria of astrocytes (+700%), an intermediate rate in the mitochondria of neurons (+200%), and the lowest rate in the nuclei (+100%) of neurons and astrocytes; the Mn level in the cytoplasm appeared unchanged. EELS analysis detected the specific spectra of Mn L 2,3 (peak at Δ E = 665 eV) in such organelles, confirming the findings of ESI. Although a consistent loss of Mn occurred during the processing of tissue samples, ESI and EELS can be useful methods for localization of endogenous Mn in embedded tissues. The high rate of Mn sequestration in the mitochondria of astrocytes in vivo may partly explain the outstanding capacity of astrocytes to accumulate Mn, and their early dysfunction in Mn neurotoxicity. The high level of Mn in the heterochromatin and nucleoli of neurons and astrocytes in basal conditions and its further increase after Mn overload should provide insight into new avenues of investigating the role of Mn in the normal brain and a baseline for future Mn toxicity studies.

Polystyrene−Silica Colloidal Nanocomposite Particles Prepared by Alcoholic Dispersion Polymerization

Micrometer-sized silica-stabilized polystyrene latex particles and submicrometer-sized polystyrene−silica nanocomposite particles have been prepared by dispersion polymerization of styrene in alcoholic media in the presence of a commercial 13 or 22 nm alcoholic silica sol as the sole stabilizing agent. Micrometer-sized near-monodisperse silica-stabilized polystyrene latexes are obtained when the polymerization is initiated with a nonionic AIBN initiator. These particles are stabilized by silica particles that are present on the latex surface at submonolayer concentration. The total silica content is no greater than 1.1 wt %, which corresponds to a silica sol incorporation efficiency of less than 1.3%. Reduction of the initial silica sol concentration led to a systematic increase in the mean latex diameter. In contrast, submicrometer-sized polystyrene-silica nanocomposite particles are obtained when the polymerization is initiated with a cationic azo initiator. The silica contents of these nanocomposite particles are significantly higher, ranging up to 29 wt %. Zeta potential measurements, XPS, and electron spectroscopy imaging by transmission electron microscopy (ESI/TEM) studies reveal a well-defined core−shell morphology for these particles, whereby the core is polystyrene and the shell comprises the silica sol. After calcination, these nanocomposite particles can form hollow silica capsules. Variation of the initial silica sol and initiator concentration has relatively little effect on the final particle size and silica content of these polystyrene−silica nanocomposite particles, but indicates silica sol incorporation efficiencies up to 72%.

Correlation of superconducting properties and microstructure of MgB 2 wires and tapes

Within the “Hipermag” project, MgB 2 wires and tapes were prepared by the powder in tube method using different precursor powders and processing technologies. Either pre-reacted MgB 2 (ex-situ), mixture of MgH 2 + 2B (in-situ) or mechanically alloyed (MA) MgB 2 is used. In this work superconducting properties of a long length 14-filament tape with standard ex-situ powder, MA tapes, and hydrostatically extruded wires with in-situ MgB 2 powder with SiC nanoinclusions, are correlated with their microstructure investigated using advanced electron microscopy techniques. Apart from the conventional diffraction imaging, EDX elemental maps in SEM and TEM and electron spectroscopic imaging (ESI) in TEM have been used to study the MgB 2 phase formation and granularity. The critical current densities of the wires and tapes were measured at 4.2 and 20 K for different magnetic fields. Wires and tapes prepared by different technologies show large differences by orders of magnitude in their critical current densities. MgB 2 colony formation is found as a universal phenomenon with several grains forming a dense colony. Such colonies are embedded in a matrix of reduced density introducing granularity in MgB 2 wires and tapes. Wires and tapes that do not show colony formation contain a large fraction of B-rich phases and their critical current density is limited by the MgB 2 phase formation.

Exocytotic Process as a Novel Model for Mineralization by Osteoblasts In Vitro and In Vivo Determined by Electron Microscopic Analysis

The process of biomineralization has been examined during osteoblastic differentiation of bone marrow stroma cells (BMSCs) from embryonic chick in culture and in periosteum itself by a number of different techniques including transmission and scanning electron microscopy. In cell culture of BMSCs at days 20-25, crystals were accumulated extracellularly in the collagen matrix, resulting in large plate-like crystallites and noncollagen associated on the culture disk surface. In contrast, up to days 10-18, mainly intracellular mineralization was visible by numerous needle-like crystal structures in the cell cytoplasm and in vacuoles. After 20-30 days, the crystal content of these vacuoles is released, most probably by membrane fusion to the outside of the cells. Energy-dispersive X-ray analysis (EDX), electron spectroscopic imaging, and electron energy loss spectroscopy demonstrated that Ca, O, and P are located in the intra- and extracellular needle-like crystals. From EDX spectra a Ca/P ratio of 1.3 was estimated for the intracellular structures and a Ca/P ratio of 1.5, for the extracellular material (for comparison, the Ca/P ratio in tibiae is 1.6). X-ray diffraction and quantitative infrared spectral analyses also demonstrated an increase of crystalline bone apatite along the mineralization process. In addition to the finding in vitro, the presence of intracellular needle-like crystals in vacuoles could be demonstrated in vivo in osteoblastic cells of the periosteum in tibia of day 11. The results are in favor of a novel model for mineralization by osteoblasts, in which amorphous Ca/P material is directly secreted via an exocytotic process from vacuoles of the osteoblast, deposited extracellularly, propagated into the collagen fibril matrix, and matured to hydroxyapatite.

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BioTechniquesVol. 42, No. 3 WebWatchOpen AccessWebWatchKevin Ahern†Kevin Ahern††- Kevin Ahern - Please send web site recommendations to ahernk@orst.eduSearch for more papers by this authorPublished Online:16 May 2018https://doi.org/10.2144/000112426AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinkedInRedditEmail Book o’ WormsThe free-living nematode Caenorhabditis elegans has been the subject of intense scrutiny that is inversely proportional to its small cell numbers (about 1000). A model organism, the worms have not only inspired developmental biologists for over 30 years, they've also provided fodder for an informative collection of web sites, most of which contain the word “worm.” The latest of these, Wormbook, borrows from the open source movement, providing in chapter form peer-reviewed articles based on the biology of the organism. You can learn about genetics and genomics in 13 easy lessons or discover the roots of neurobiology and behavior at their simplest levels. If you're new to the discipline, a scan through the extensive methods section may be of most benefit. The entire “can of worms” comprising the site is available too in the form of a (gulp) 387 megabyte compressed file.@ www.wormbook.orgScoping It OutThe visible spectrum paints on our retina all of the colors of the rainbow, but it is virtually useless for imaging the nanoscopic world of the cells comprising it. Fortunately, the mighty electron overcomes the limitations of mere photons, permitting researchers to visualize otherwise unseen nanoscopic objects. Electron microscopy, which celebrated its 75th anniversary in 2006, has evolved over the years into a surprisingly diverse set of technologies, each optimized to depict or analyze nanoscale objects. To study structure, for example, one can employ high-resolution transmission techniques, scanning transmission, or electron diffraction. Adaptations to permit analysis of composition (energy-dispersive X-ray spectroscopy or electron loss spectroscopy), morphology (scanning electron microscopy or SEM), and elemental mapping [electron spectroscopic imaging and scanning transmission electron microscopy (STEM), or SEM combined with X-ray spectroscopy] have moved from being techno-curiosities to important research tools, as visitors will discover at Frank Krumeich's informative site.@ www.microscopy.ethz.chCount Down UnderCovering 70% of the earth's surface and providing more than 90% of its biosphere, the oceans teem with the most significant uncharacterized biological domains remaining on earth. Though our knowledge of these ecological treasures is still surprisingly limited, there is hope. The Census of Marine Life (COML) project is a decade-long international effort to understand more of the past, present, and future of this enormous ecosystem. Now in its seventh year, the census is active in identifying threatened species and in helping authorities to plan the management of these resources.@ www.coml.org/coml.htmSalad DazeThe radish—a garden staple, a part of any good salad, an important food crop, a major agricultural pest on six continents, and an invasive species in California, is about to gain another description—sequenced genome. Yes, the lowly radish will soon join the ranks of the “big guys.” The honor is appropriate, as noted at Jeff Conner's informative page, hosted at Michigan State University. The radish is a distant relative of Arabidopsis, and its sequence will likely yield evolutionary insights between the species. In addition, the plant is important for studies of transgene escape and has provided valuable perspectives to the process of natural selection through male and female fitness. A view of the blueprint of the organism's genome is likely to prove additional understanding of these processes at the molecular level.@ radish.plantbiology.msu.edu/index.php/Main_PageKinase KollectionThe nucleoside monophosphate kinases are essential for the production of nucleoside diphosphates, which, in turn, are precursors of the triphosphate building blocks of RNA and DNA. The bacterial enzymes catalyzing these reactions, which have an impressive amount of sequence and structural similarity, can be divided into five families—one each for the adenylate, cytidylate, guanylate, thymidylate, and uridylate nucleotides. 3-D views of these catalysts and their multiply aligned amino sequences are available for your perusal at this well-designed site.@ www.ces.clemson.edu/compbio/databases/kinasesFiguresReferencesRelatedDetails Vol. 42, No. 3 Follow us on social media for the latest updates Metrics History Published online 16 May 2018 Published in print March 2007 Information© 2007 Author(s)PDF download

Low-energy-loss EFTEM imaging of thick particles and aggregates

Electron spectroscopy imaging is a powerful tool for the elucidation of colloidal particle morphology and microchemistry, but it normally requires the use of very thin samples, typically less than 50 nm, to avoid the effects of multiple scattering. This work shows that many aspects of the internal morphology of thick particles and aggregates and the chemical component distribution are revealed using low-energy-loss electron imaging in the transmission electron microscope, benefiting from multiple scattering as well as small but significant differences in the low-energy-loss spectra of aggregate constituents. Low-loss images reveal morphological details of thick aggregates made out of colloidal polymers (natural rubber and styrene–acrylic latex) and inorganic particles (silica, montmorillonite, and aluminum phosphate) at a spatial resolution close to that achieved in the bright-field images and much better than in the elemental maps, showing the advantages of the simultaneous use of low-loss images and standard thin-cut elemental maps.

Observation of in vivo DNA in Ice Embedded whole Cyanobacterial Cells by Hilbert Differential Contrast Transmission Electron Microscopy (HDC-TEM)

HDC-TEM has opened a way to visualize the ultrastructure of ice embedded whole cells. The extraordinary advantage of this technique is that it exhibits structures close to the living state while retaining all the in vivo molecular constituents undisturbed. We attempted to identify in vivo DNA by incorporation of BrdU, which conferred electron density to newly synthesized DNA in ice embedded cyanobacterial cells. Localization of Br in the electron dense area in the identical cell was investigated by electron spectroscopic imaging (ESI). Br was also appeared to be associated with polyphosphate bodies, which would indicate a close relationship between newly synthesized DNA and polyphosphate bodies. While ESI indicates the DNA localization, high resolution HDC-TEM reveals the fin efi brous structures in situ .T he combination of ESI with HDC-TEM will be extremely useful to study the in vivo dynamics of DNA synthesis, and its structural and conformational changes close to the living state at high resolution.

Structure and Morphology of Poly(ε-caprolactone)/Chlorinated Polyethylene (PCL/PECl) Blends Investigated by DSC, Simultaneous SAXS/WAXD, and Elemental Mapping by ESI-TEM

In this work, the structure and morphology of miscible blends of poly(ε-caprolactone) and chlorinated polyethylene with 48% chlorine weight content (PCL/PECl) were studied by differential scanning calorimetry (DSC), simultaneous small and wide-angle X-ray scattering (SAXS/WAXD), and electron spectroscopy imaging in the transmission electron microscope (ESI-TEM). A unique glass transition temperature was obtained in each blend. In addition to this, the heat capacity and the width of the glass transition did not have a linear behavior with blend compositions. These facts correlate with the presence of microheterogeneities originated from different local compositions and densities of interactions in each blend. A consistent picture of the mode of segregation of PECl in the blend was obtained. For higher concentration of PCL, the volume fraction of lamellar stacks in the samples decreased as a function of the PECl content, indicating preferential interfibrillar localization of the amorphous component. For lower PCL concentration, interespherulitic segregation was the dominant mode. Elemental maps of chlorine confirmed these results and also revealed changes in the concentration of this element depending on its localization in the microstructure of the system. Gradients of chlorine concentration were measured in larger amorphous regions of the 40/60 and 20/80 PCL/PECl blends. Calculations of the one-dimensional correlation function probed the reduction of the lamellar thickness of PCL when the quantity of PECl in the blend was increased. Such a tendency could be rationalized if the reduction of the fold surface free energy was a dominant factor in terms of the reduction of the degree of supercooling in the final crystal thickness.

Morphology and Microchemistry of Colloidal Polymers

AbstractColloidal polymers are highly versatile due to the variety of properties and functions that can be created by changing monomer composition, surfactant, initiator and reaction protocol. Microchemical and morphological observation of the particles and particle aggregates as well as films and monoliths made with them is allowing us to understand the connections between nanosized structural features and macroscopic properties and this is essential for the creation of valuable new polymer materials. Electron spectroscopy imaging (ESI) in the transmission electron microscope (TEM) uses electron energy loss spectroscopy (EELS) to provide a wealth of information on particle constituents and their topological distribution, allowing a number of correlations with the polymer mechanical, thermal, optical and electrical properties. On the other hand, scanning probe microscopy (SPM) techniques allow direct measurement of particle and film properties such as adhesion, stiffness, electrostatic potential as well as rheology information with high spatial resolution, down to 10‐20 nm and under many different experimental conditions. Information from the joint use of these techniques is revealing a wealth of nanostructures within colloidal polymers requiring a revision of many preconceived ideas on these materials.

Synthesis of Biocompatible Poly[2-(methacryloyloxy)ethyl phosphorylcholine]-Coated Magnetite Nanoparticles

A well-defined, double-hydrophilic diblock copolymer comprising poly[2-(methacryloyloxy)ethyl phosphorylcholine]-block-(glycerol monomethacrylate) (PMPC30-PGMA30, where the numbers represent the average degrees of polymerization for each block) was evaluated for the synthesis of colloidally stable ultrafine magnetite sols. Sterically stabilized paramagnetic sols were prepared in aqueous solution by chemical coprecipitation of ferric and ferrous salts in the presence of this block copolymer. The PMPC30-PGMA30-stabilized magnetite sol had a mean transmission electron microscopy (TEM) diameter of 9.4 +/- 1.7 nm and a mean hydrodynamic diameter of 34 nm. This sol exhibited improved colloidal stability with respect to long-term storage and pH variation compared with magnetite sols prepared in the presence of alternative water-soluble homopolymers and diblock copolymers. Fourier transform infrared (FT-IR) spectroscopy, thermogravimetry, electron spectroscopy imaging (ESI), and zeta potential studies indicate that the PMPC30-PGMA30 diblock copolymer was adsorbed onto the surface of the sol via the PGMA30 block, with the PMPC30 chains acting as the stabilizing block. Such sterically stabilized sols are expected to be improved contrast agents for magnetic resonance imaging (MRI) applications.

Processing, crystallization and characterization of polymer derived nano-crystalline Si–B–C–N ceramics

Si–B–C–N ceramics were synthesized from boron modified poly(vinyl)silazanes with the chemical formula (B[C2H4–Si(CH3)NH]3)n. The originally amorphous materials are crystallized at temperatures in the order of 1,800–1,900 °C, which results microstructures with grain sizes significantly below 100 nm. Several parameters of the heat treatment, including temperature, holding time and atmosphere, affect the resulting nanostructures. This and the chemical and phase composition were studied via X-ray diffraction (XRD), transmission electron microscopy (TEM), electron spectroscopic imaging (ESI) and spectrochemical analysis in order to gain an understanding of the mechanisms, which control the crystallization behavior. Ceramic samples were also produced using different particle sizes of the precursor polymer in order to quantify the effect of the varying specific surface on the crystallization behavior.

The Structure of Escherichia coli Signal Recognition Particle Revealed by Scanning Transmission Electron Microscopy

Structural studies on various domains of the ribonucleoprotein signal recognition particle (SRP) have not converged on a single complete structure of bacterial SRP consistent with the biochemistry of the particle. We obtained a three-dimensional structure for Escherichia coli SRP by cryoscanning transmission electron microscopy and mapped the internal RNA by electron spectroscopic imaging. Crystallographic data were fit into the SRP reconstruction, and although the resulting model differed from previous models, they could be rationalized by movement through an interdomain linker of Ffh, the protein component of SRP. Fluorescence resonance energy transfer experiments determined interdomain distances that were consistent with our model of SRP. Docking our model onto the bacterial ribosome suggests a mechanism for signal recognition involving interdomain movement of Ffh into and out of the nascent chain exit site and suggests how SRP could interact and/or compete with the ribosome-bound chaperone, trigger factor, for a nascent chain during translation.

In vivo monitoring of the potassium channel KcsA in Streptomyces lividans hyphae using immuno-electron microscopy and energy-filtering transmission electron microscopy

The previous discovery of the Streptomyces lividans kcsA gene and its overexpression followed by the functional reconstitution of the purified gene product has resulted in new strategies to explore this channel protein in vitro. KcsA has evolved as a general model to investigate the structure/function relationship of ion channel proteins. Using specific antibodies raised against a domain of KcsA lacking membrane-spanning regions, KcsA has now been localized within numerous separated clusters between the outer face of the cytoplasm and the cell envelope in substrate hyphae of the S. lividans wild-type strain but not in a designed chromosomal disruption mutant DeltaK, lacking a functional kcsA gene. Previous findings had revealed that caesium ions led to a block of KcsA channel activity within S. lividans protoplasts fused to giant vesicles. As caesium can be scored by electron energy loss spectroscopy better than potassium, this technique was applied to hyphae that had been briefly exposed to caesium instead of potassium ions. Caesium was found preferentially at the cell envelope. Compared to the DeltaK mutant, the relative level of caesium was approximately 30 % enhanced in the wild-type. This is attributed to the presence of KcsA channels. Additional visualization by electron spectroscopic imaging supported this conclusion. The data presented are believed to represent the first demonstration of in vivo monitoring of KcsA in its original host.

Property Map by Electron Spectroscopy Imaging Series

Extended abstract of a paper presented at Microscopy and Microanalysis 2006 in Chicago, Illinois, USA, July 30 – August 3, 2005

Valence state map of iron oxide thin film obtained from electron spectroscopy imaging series

This paper demonstrates the applicability of electron-spectroscopic imaging (ESI) for valence-state mapping of the iron oxide system. We have previously developed a set of signal-processing methods for an ESI series, to allow mapping of sp 2/sp 3 ratio, dielectric function and energy bandgap. In this study, these methods are applied to generate a valence-state map of an iron oxide thin film (Fe/α-Fe 2O 3). Two problems, data undersampling and a convolution effect associated with extraction of the image-spectrum from the core loss image series, were overcome by using cubic-polynomial interpolation and maximum-entropy deconvolution. As a result, the reconstructed image-spectrum obtained from the ESI series images has a quality as good as that of conventional electron energy-loss spectra. The L 3/ L 2 ratio of the reconstructed ESI spectrum is determined to be 3.30 ± 0.30 and 5.0 ± 0.30 for Fe and α-Fe 2O 3, respectively. Our L 3/ L 2 ratio mapping shows an accurate correspondence across the Cu/Fe/α-Fe 2O 3 region. The effect of delocalization and chromatic aberration on the ESI resolution is discussed and estimated to be about 2 nm for the case of L 3/ L 2 ratio mapping.