Description: Publication date: February 2014 Source: Ultramicroscopy, Volume 137 Author(s): Peter Hawkes Recent books and conference proceedings are examined.

Description: Publication date: March 2014 Source: Ultramicroscopy, Volume 138 Author(s): Robert M. Glaeser , Holger Müller As shown by Matsumoto and Tonomura, the phase shift imposed on an electron beam by an electrostatic phase plate is constant for all (straight) electron trajectories passing through a circular aperture, provided that (1) the electric field goes to zero at distances far above and below the aperture and (2) the value of the phase shift at the boundary (i.e. perimeter of the aperture) is constant [5] . We now point out that the result can be valid for any shape of the hole in the aperture, and, furthermore, it requires only that the electric field is equal and opposite at large distances above and below the aperture, respectively. We also point out that the conditions of validity of the Matsumoto–Tonomura approximation constrain the phase shift across the open aperture to a quadratic algebraic form when the phase shift is not constant around the perimeter. Finally, it follows that the projection approximation for calculating the phase shift must fail for strong phase shifts of higher than quadratic form. These extensions of the original result of Matsumoto and Tonomura give further insight to the analysis of charging phenomena observed with apertures that are designed to produce contrast in in-focus images of weak phase objects.

Description: Publication date: Available online 10 December 2013 Source: Ultramicroscopy Author(s): K.S. Karvinen , S.O.R. Moheimani While conventional techniques in dynamic mode atomic force microscopy typically involve the excitation of the first flexural mode of a microcantilever, situations arise where the excitation of higher modes may result in image artefacts. Strong nonlinear coupling between the cantilever modes in liquid environments may result in image artefacts, limiting the accuracy of the image. Similar observations have been made in high-speed contact mode AFM. To address this issue, we propose the application of the modulated-demodulated control technique to attenuate problematic modes to eliminate the image artefacts. The modulated-demodulated control technique is a high-bandwidth technique, which is well suited to the control of next generation of high-speed cantilevers. In addition to potential improvements in image quality, a high-bandwidth controller may also find application in multifrequency AFM experiments. To demonstrate the high-bandwidth nature of the control technique, we construct an amplitude modulation AFM experiment in air utilizing low amplitude setpoints, which ensures that harmonic generation and nonlinear coupling of the modes result in image artefacts. We then utilize feedback control to highlight the improvement in image quality. Such a control technique appears extremely promising in high-speed atomic force microscopy and is likely to have direct application in AFM in liquids.

Description: Publication date: February 2014 Source: Ultramicroscopy, Volume 137 Author(s): Andrew M. Thron , Peter Greene , Kai Liu , Klaus van Benthem Dewetting of ultra-thin Ni films deposited on SiO 2 layers was observed, in cross-section, by in situ scanning transmission electron microscopy. Holes were observed to nucleate by voids which formed at the Ni/SiO 2 interface rather than at triple junctions at the free surface of the Ni film. Ni islands were observed to retract, in attempt to reach equilibrium on the SiO 2 layer. SiO 2 layers with 120 nm thickness were found to limit in situ heating experiments due to poor thermal conductivity of SiO 2 . The formation of graphite was observed during the agglomeration of ultra-thin Ni films. Graphite was observed to wet both the free surface and the Ni/SiO 2 interface of the Ni islands. Cr forms surface oxide layers on the free surface of the SiO 2 layer and the Ni islands. Cr does not prevent the dewetting of Ni, however it will likely alter the equilibrium shape of the Ni islands.

Description: Publication date: February 2014 Source: Ultramicroscopy, Volume 137 Author(s): Sachin L. Shrestha , Andrew J. Breen , Patrick Trimby , Gwénaëlle Proust , Simon P. Ringer , Julie M. Cairney The identification and quantification of the different ferrite microconstituents in steels has long been a major challenge for metallurgists. Manual point counting from images obtained by optical and scanning electron microscopy (SEM) is commonly used for this purpose. While classification systems exist, the complexity of steel microstructures means that identifying and quantifying these phases is still a great challenge. Moreover, point counting is extremely tedious, time consuming, and subject to operator bias. This paper presents a new automated identification and quantification technique for the characterisation of complex ferrite microstructures by electron backscatter diffraction (EBSD). This technique takes advantage of the fact that different classes of ferrite exhibit preferential grain boundary misorientations, aspect ratios and mean misorientation, all of which can be detected using current EBSD software. These characteristics are set as criteria for identification and linked to grain size to determine the area fractions. The results of this method were evaluated by comparing the new automated technique with point counting results. The technique could easily be applied to a range of other steel microstructures.

Description: Publication date: January 2014 Source: Ultramicroscopy, Volume 136 Author(s): Chris J. Rossouw , Christian Dwyer , Hadas Katz-Boon , Joanne Etheridge Quantitative analysis of lattice resolved images generated by scanning transmission electron microscopy (STEM) requires specification of probe characteristics, such as defocus, aberration and source distribution. In this paper we show that knowledge of such characteristics is unnecessary for quantitative interpretation, if the signal is integrated over a unit cell. Such a condition, whether the result of experimental setup or post-processing of lattice resolved images, reduces the intensity distribution to that of channelling contrast, where the signal for plane wave incidence is averaged over the angular range of the probe, and the result is independent of the probe characteristics. We use a Bloch wave model to show analytically how this applies to all forms of STEM imaging, such as that formed by annular dark field or backscatter detection, as well as characteristic X-ray fluorescence or electron energy loss. As a specific example, we consider how the signal from an annular dark field detector can be used to determine specimen thickness via a transfer curve for the zone axis and scattering geometries employed. This method has advantages over matching lattice images with calculations since these are sensitive to probe coherence and aberration, and saturation of the on-column intensity is approached more rapidly.

Description: Publication date: January 2014 Source: Ultramicroscopy, Volume 136 Author(s): W. Christenson , I. Yermolenko , B. Plochberger , F. Camacho-Alanis , A. Ros , T.P. Ugarova , R. Ros Adsorption of fibrinogen on various surfaces produces a nanoscale multilayer matrix, which strongly reduces the adhesion of platelets and leukocytes with implications for hemostasis and blood compatibility of biomaterials. The nonadhesive properties of fibrinogen matrices are based on their extensibility, ensuing the inability to transduce strong mechanical forces via cellular integrins and resulting in weak intracellular signaling. In addition, reduced cell adhesion may arise from the weaker associations between fibrinogen molecules in the superficial layers of the matrix. Such reduced stability would allow integrins to pull fibrinogen molecules out of the matrix with comparable or smaller forces than required to break integrin–fibrinogen bonds. To examine this possibility, we developed a method based on the combination of total internal reflection fluorescence microscopy, single cell manipulation with an atomic force microscope and microcontact printing to study the transfer of fibrinogen molecules out of a matrix onto cells. We calculated the average fluorescence intensities per pixel for wild-type HEK 293 (HEK WT) and HEK 293 cells expressing leukocyte integrin Mac-1 (HEK Mac-1) before and after contact with multilayered matrices of fluorescently labeled fibrinogen. For contact times of 500 s, HEK Mac-1 cells show a median increase of 57% of the fluorescence intensity compared to 6% for HEK WT cells. The results suggest that the integrin Mac-1-fibrinogen interactions are stronger than the intermolecular fibrinogen interactions in the superficial layer of the matrix. The low mechanical stability of the multilayer fibrinogen surface may contribute to the reduced cell adhesive properties of fibrinogen-coated substrates. We anticipate that the described method can be applied to various cell types to examine their integrin-mediated adhesion to the extracellular matrices with a variable protein composition.

Description: Publication date: Available online 13 November 2013 Source: Ultramicroscopy Author(s): Colin M. Palmer , Jan Löwe The use of slab-like flat specimens for electron cryo-tomography restricts the range of viewing angles that can be used. This leads to the “missing wedge” problem, which causes artefacts and anisotropic resolution in reconstructed tomograms. Cylindrical specimens provide a way to eliminate the problem, since they allow imaging from a full range of viewing angles around the tilt axis. Such specimens have been used before for tomography of radiation-insensitive samples at room temperature, but never for frozen-hydrated specimens. Here, we demonstrate the use of thin-walled carbon tubes as specimen holders, allowing the preparation of cylindrical frozen-hydrated samples of ribosomes, liposomes and whole bacterial cells. Images acquired from these cylinders have equal quality at all viewing angles, and the accessible tilt range is restricted only by the physical limits of the microscope. Tomographic reconstructions of these specimens demonstrate that the effects of the missing wedge are substantially reduced, and could be completely eliminated if a full tilt range was used. The overall quality of these tomograms is still lower than that obtained by existing methods, but improvements are likely in future.

Description: Publication date: January 2014 Source: Ultramicroscopy, Volume 136 Author(s): M. Lentzen In a recent article it is argued that the far-field expansion of electron scattering, a pillar of electron diffraction theory, is wrong (Treacy and Van Dyck, 2012 [1] ). It is further argued that in the first Born approximation of electron scattering the intensity of the electron wave is not conserved to first order in the scattering potential. Thus a “mystery of the missing phase” is investigated, and the supposed flaw in scattering theory is seeked to be resolved by postulating a standing spherical electron wave (Treacy and Van Dyck, 2012 [1] ). In this work we show, however, that these theses are wrong. A review of the essential parts of scattering theory with careful checks of the underlying assumptions and limitations for high-energy electron scattering yields: (1) the traditional form of the far-field expansion, comprising a propagating spherical wave, is correct; (2) there is no room for a missing phase; (3) in the first Born approximation the intensity of the scattered wave is conserved to first order in the scattering potential. The various features of high-energy electron scattering are illustrated by wave-mechanical calculations for an explicit target model, a Gaussian phase object, and for a Si atom, considering the geometric conditions in high-resolution transmission electron microscopy.

Description: Publication date: Available online 12 November 2013 Source: Ultramicroscopy Author(s): N.E. Lonergan , L.D. Britt , C.J. Sullivan Atomic force microscopy (AFM) is a probe-based technique that permits high resolution imaging of live bacterial cells. However, stably immobilizing cells to withstand the probe-based lateral forces remains an obstacle in AFM mediated studies, especially those of live, rod shaped bacteria in nutrient media. Consequently, AFM has been under-utilized in the research of bacterial surface dynamics. The aim of the current study was to immobilize a less adherent Escherichia coli strain in a method that both facilitates AFM imaging in nutrient broth and preserves overall cell viability. Immobilization reagents and buffers were systematically evaluated and the cell membrane integrity was monitored in all sample preparations. As expected, the biocompatible gelatin coated surfaces facilitated stable cell attachment in lower ionic strength buffers, yet poorly immobilized cells in higher ionic strength buffers. In comparison, poly-L-lysine surfaces bound cells in both low and high ionic strength buffers. The benefit of the poly-L-lysine binding capacity was offset by the compromised membrane integrity exhibited by cells on poly-L-lysine surfaces. However, the addition of divalent cations and glucose to the immobilization buffer was found to mitigate this unfavorable effect. Ultimately, immobilization of E. coli cells on poly-L-lysine surfaces in a lower ionic strength buffer supplemented with Mg 2+ and Ca 2+ was determined to provide optimal cell attachment without compromising the overall cell viability. Cells immobilized in this method were stably imaged in media through multiple division cycles. Furthermore, permeability assays indicated that E. coli cells recover from the hypoosmotic stress caused by immobilization in low ionic strength buffers. Taken together, this data suggests that stable immobilization of viable cells on poly-L-lysine surfaces can be accomplished in lower ionic strength buffers that are supplemented with divalent cations to membrane stabilization while minimizing binding interference. The data also indicates that monitoring cell viability as a function of sample preparation is important and should be an integral part of the work flow for determining immobilization parameters. A method for immobilizing a less adherent E. coli mutant for AFM imaging in nutrient broth is presented here in addition to a proposed work flow for developing and optimizing immobilization strategies.