Journal of Microscopy

Low temperature and anhydrous electron microscopy techniques to observe the infection process of the bacterial pathogen Xanthomonas fragariae on strawberry leaves.

Publication Date: 2010 Sep 1 PMID: 20701664
Authors: Allan-Wojtas, P. - Hildebrand, P. D. - Braun, P. G. - Smith-King, H. L. - Carbyn, S. - Renderos, W. E.
Journal: J Microsc

Preserving the structural arrangement of the components of a bacterial infection process within a plant for microscopy study is a technical challenge because of the different requirements of each component for optimal preservation and visualization. We used low temperature scanning electron microscopy (cryo-SEM), anhydrous fixation at ambient temperature and freeze-substitution for transmission electron microscopy to examine fractured and sectioned strawberry leaves infected with Xanthomonas fragariae. Cryo-SEM images of fractured samples showed the bacterial colonization of mesophyll air spaces in the leaf, limited by the vascular bundles and the orientation and packing of bacteria in extracellular polysaccharide. Transmission electron microscopy of samples fixed using osmium tetroxide dissolved in FC-72 solvent at ambient temperature showed that the entire plant/bacteria/extracellular polysaccharide system was preserved in situ, and showed plasmolysis of mesophyll cells and disruption of organelles. In freeze-substitution samples, osmium tetroxide in FC-72 solvent gave superior preservation of the extracellular polysaccharide as compared to a conventional cocktail. In addition, strands believed to be xanthan were preferentially contrasted to show their density and orientation around the bacterial cells. We conclude that anhydrous fixation using osmium tetroxide in FC-72 at ambient temperature gave the best preservation of the entire system, and freeze-substitution using this same fixative enhanced the visualization of strands in the biofilm.

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Electron back-scattering diffraction (EBSD) measurements of antigorite lattice-preferred orientations (LPO).

Publication Date: 2010 Sep 1 PMID: 20701663
Authors: van de Moortele, B. - Bezacier, L. - Trullenque, G. - Reynard, B.
Journal: J Microsc

Lattice preferred orientations of serpentines induce a strong anisotropy of various properties in serpentine bearing-rocks. Lattice preferred orientations had so far been obtained only by X-ray diffraction techniques. We have applied electron back-scattering diffraction to the measurement of the lattice preferred orientations of antigorite in a naturally deformed high-pressure serpentinite. This technique is very sensitive to sample preparation that can lead to surface amorphization in the case of serpentine. A polishing procedure is described that avoids amorphization and allows accurate electron back-scattering diffraction measurements with optimized experimental conditions in a variable pressure scanning electron microscope. Results indicate that deformation leads to lattice preferred orientations characterized by extremely strong c-axis clustering perpendicular to the foliation, as expected for a layered silicate. In the foliation plane, a significant clustering of the a-axis is observed and tentatively attributed to intracrystalline deformation mechanisms. These data suggest that antigorite deforms mostly by gliding along the basal plane of the layered phyllosilicate structure, but that gliding may occur along directions favouring a-axis alignment. Electron back-scattering diffraction appears to be a reliable method for determining phyllosilicate lattice preferred orientations in deformed rocks, with potential applications for determining anisotropy of properties like seismic velocities or thermal and electrical conductivities.

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Domain structures of ultrafine grained ferromagnets achieved by severe plastic deformation or melt quenching.

Publication Date: 2010 Sep 1 PMID: 20701662
Authors: Korznikova, G. F.
Journal: J Microsc

Lorentz electron microscopy was used to analyse the domain structure in some ferromagnets having an ultrafine grained structure produced by high pressure torsion (pure Ni, Fe, Co, Mn(55.5)Al(45.5) C(0.5)) and by melt quenching (Fe(83)Nd(13)B(4)). For low anisotropy ferromagnets (Ni, Fe, Co), it has been established that the domain structure does not depend strongly on grain size. In high anisotropy ferromagnets alloy (Mn(55.5)Al(45.5) C(0.5) and Fe(83)Nd(13)B(4)), grain refinement causes changes in the domain structure configuration and affects the magnetization mechanism.

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Scintillation SE detector for variable pressure scanning electron microscopes.

Publication Date: 2010 Sep 1 PMID: 20701661
Authors: Jirak, J. - Nedela, V. - Cernoch, P. - Cudek, P. - Runstuk, J.
Journal: J Microsc

We present results obtained with a new scintillation detector of secondary electrons for the variable pressure scanning electron microscope. A detector design is based on the positioning of a single crystal scintillator within a scintillator chamber separated from the specimen chamber by two apertures. This solution enables us to decrease the pressure to several Pa in the scintillator chamber while the pressure in the specimen chamber reaches values of about 1000 Pa (7.5 Torr). Due to decreased pressure, we can apply a potential of the order of several kV to the scintillator, which is necessary for the detection of secondary electrons. Simultaneously, the two apertures at appropriate potentials of the order of several hundreds of volts create an electrostatic lens that allows electrons to pass from the specimen chamber to the scintillator chamber. Results indicate a promising utilization of this detector for a wide range of specimen observations.

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Limitations of beam damage in electron spectroscopic tomography of embedded cells.

Publication Date: 2010 Sep 1 PMID: 20701660
Authors: Aronova, M. A. - Sousa, A. A. - Zhang, G. - Leapman, R. D.
Journal: J Microsc

Elemental mapping in the energy filtering transmission electron microscope (EFTEM) can be extended into three dimensions (3D) by acquiring a series of two-dimensional (2D) core-edge images from a specimen oriented over a range of tilt angles, and then reconstructing the volume using tomographic methods. EFTEM has been applied to imaging the distribution of biological molecules in 2D, e.g. nucleic acid and protein, in sections of plastic-embedded cells, but no systematic study has been undertaken to assess the extent to which beam damage limits the available information in 3D. To address this question, 2D elemental maps of phosphorus and nitrogen were acquired from unstained sections of plastic-embedded isolated mouse thymocytes. The variation in elemental composition, residual specimen mass and changes in the specimen morphology were measured as a function of electron dose. Whereas 40% of the total specimen mass was lost at doses above 10(6) e(-)/nm(2), no significant loss of phosphorus or nitrogen was observed for doses as high as 10(8) e(-)/nm(2). The oxygen content decreased from 25 + or - 2 to 9 + or - 2 atomic percent at an electron dose of 10(4) e(-)/nm(2), which accounted for a major component of the total mass loss. The specimen thickness decreased by 50% after a dose of 10(8) e(-)/nm(2), and a lateral shrinkage of 9.5 + or - 2.0% occurred from 2 x 10(4) to 10(8) e(-)/nm(2). At doses above 10(7) e(-)/nm(2), damage could be observed in the bright field as well in the core edge images, which is attributed to further loss of oxygen and carbon atoms. Despite these artefacts, electron tomograms obtained from high-pressure frozen and freeze-substituted sections of C. elegans showed that it is feasible to obtain useful 3D phosphorus and nitrogen maps, and thus to reveal quantitative information about the subcellular distributions of nucleic acids and proteins.

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Enhanced angular current intensity from Schottky emitters.

Publication Date: 2010 Sep 1 PMID: 20701659
Authors: Fujita, S. - Wells, T. R. - Ushio, W. - Sato, H. - El-Gomati, M. M.
Journal: J Microsc

Even though the Schottky emitter is a high-brightness source of choice for electron beam systems, its angular current intensity is substantially lower than that of thermionic cathodes, rendering the emitter impractical for applications that require high beam current. In this study, two strategies were attempted to enhance its angular intensity, and their experimental results are reported. The first scheme is to employ a higher extraction field for increasing the brightness. However, the tip shape transformation was found to induce undesirably elevated emission from the facet edges at high fields. The second scheme exploits the fact that the angular intensity is proportional to the square of the electron gun focal length [Fujita, S. & Shimoyama, H. (2005) Theory of cathode trajectory characterization by canonical mapping transformation. J. Electron Microsc. 54, 331-343], which can be increased by scaling-up the emitter tip radius. A high angular current intensity (J(Omega) approximately 1.5 mA sr(-1)) was obtained from a scaled-up emitter. Preliminary performance tests were conducted on an electron probe-forming column by substituting the new emitter for the original tungsten filament gun. The beam current up to a few microamperes was achieved with submicron spatial resolution.

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Photometric calibration for quantitative spectral microscopy under transmitted illumination.

Publication Date: 2010 Sep 1 PMID: 20701658
Authors: Thigpen, J. - Merchant, F. A. - Shah, S. K.
Journal: J Microsc

Over the past decade, there have been significant developments in the mechanisms for examination of biological and material samples. These developments exploit techniques in light microscopy to elucidate specific parts of cells and tissues, as well as inorganic particles. In recent years, spectral microscopy has become more prevalent for characterization of samples. Simultaneously, sensor technology has progressed as well and modern charge-coupled devices (CCD) cameras are now capable of achieving high spatial resolution and high sensitivity measurements of signals in the optical microscope. One major impediment in obtaining absolute quantitative information of imaged samples is the lack of automated photometric calibration mechanisms for spectral microscopes. In this paper, we present a methodology for achieving photometric calibration of an automated spectral imaging system targeted towards examination of biological samples. By acquiring spatial and spectral data simultaneously, spectral imaging allows one to exploit physical connections between a particle's morphology and its characteristic response to the optical spectrum. In composite biological material, the interpretation of the spectra is a complicated problem. This is because any light source and charge-coupled device camera used for data acquisition does not have a uniform illumination spectra and quantum efficiency, respectively, across the emitted light spectra. To balance the spectral response across individual wavelengths, our method modulates the exposure duration for the charge-coupled device camera during image acquisition. We present an image similarity based method to calibrate the system. Experiments to test the effectiveness of the calibration method under the various image similarity metrics are presented along with results to show the calibrated system's ability to accurately measure spectra based on the measured transmission profiles of optical filters.

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An automated and highly efficient method for counting and measuring fluorescent foci in rod-shaped bacteria.

Publication Date: 2010 Sep 1 PMID: 20701657
Authors: Nielsen, H. J. - Hansen, F. G.
Journal: J Microsc

Direct measurements of cells from photo micrographs are becoming increasingly used when investigating the position and/or distribution of chromosomal loci in bacteria. In general, these measurements have been done manually, and without clear definition of how they are made. Here we present a procedure for standardizing the measurement of cell properties from phase contrast images. Furthermore, we present a program using these standardized methods that can measure the intracellular positions of fluorescent foci in bacterial cells faster and with more precision than manual measurement.

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Microstructural characterization of laser surface melted AISI M2 tool steel.

Publication Date: 2010 Sep 1 PMID: 20701656
Authors: Arias, J. - Cabeza, M. - Castro, G. - Feijoo, I. - Merino, P. - Pena, G.
Journal: J Microsc

We describe the microstructure of Nd:YAG continuous wave laser surface melted high-speed steel, namely AISI M2, treated with different laser scanning speeds and beam diameters on its surface. Microstructural characterization of the remelted surface layer was performed using light optical and scanning electron microscopy and X-ray diffraction. The combination of the three techniques provided new insights into the substantial changes induced by laser surface melting of the steel surface layer. The advantage of the method is that it avoids the difficult and tedious work of preparing samples of this hard material for transmission electron microscopy, which is the technique normally used to study these fine microstructures. A melted zone with a dendritic structure and a partially melted zone with a heterogeneous cellular structure were observed. M(2)C carbides with different morphologies were identified in the resolidified surface layer after laser melting.

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Confined displacement algorithm determines true and random colocalization in fluorescence microscopy.

Publication Date: 2010 Sep 1 PMID: 20701655
Authors: Ramirez, O. - Garcia, A. - Rojas, R. - Couve, A. - Hartel, S.
Journal: J Microsc

The quantification of colocalizing signals in multichannel fluorescence microscopy images depends on the reliable segmentation of corresponding regions of interest, on the selection of appropriate colocalization coefficients, and on a robust statistical criterion to discriminate true from random colocalization. Here, we introduce a confined displacement algorithm based on image correlation spectroscopy in combination with Manders colocalization coefficients M1(ROI) and M2(ROI) to quantify true and random colocalization of a given florescence pattern. We show that existing algorithms based on block scrambling exaggerate the randomization of fluorescent patterns with resulting inappropriately narrow probability density functions and false significance of true colocalization in terms of p values. We further confine our approach to subcellular compartments and show that true and random colocalization can be analysed for model dendrites and for GABA(B) receptor subunits GABA(B)R1/2 in cultured hippocampal neurons. Together, we demonstrate that the confined displacement algorithm detects true colocalization of specific fluorescence patterns down to subcellular levels.

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