Journal of Microscopy

Photoconversion of DAPI following UV or violet excitation can cause DAPI to fluoresce with blue or cyan excitation.

Publication Date: 2012 Jan 31 PMID: 22288651
Authors: Piterburg, M. - Panet, H. - Weiss, A.
Journal: J Microsc

4'-6-Diamidino-2-phenylindole is a fluorescent dye commonly used to visualize deoxyribonucleic acid or cell nuclei in fixed cell preparations, and is often used together with fluorescein or green fluorescent protein, which can be excited without exciting 4'-6-Diamidino-2-phenylindole. It is assumed that when using typical fluorescein or green fluorescent protein filter cubes, 4'-6-Diamidino-2-phenylindole will not be observed. In this paper, we show that following observation of 4'-6-Diamidino-2-phenylindole using UV or violet excitation, it may become sensitive to the blue/cyan excitation used in fluorescein/green fluorescent protein filter cubes. This has serious implications for the use of 4'-6-Diamidino-2-phenylindole together with widely used green fluorophores in double labelling experiments.

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Novel combination of orientation measurements and transmission microscopy for experimental determination of grain boundary miller indices in silicon and other semiconductors.

Publication Date: 2012 Jan 23 PMID: 22268685
Authors: Funke, C. - Behm, T. - Helbig, R. - Schmid, E. - Wurzner, S.
Journal: J Microsc

The determination of grain boundary planes in multicrystalline material has only been restricted to transmission electron microscope investigations (Jang et al., 1992; Elgat et al., 1985) or to metallograpical investigations of the grain boundary (Randle et al., 1993). The first method is expensive, and both are complex and time consuming in grain boundary preparation. This paper proposes the determination of grain boundary planes in semiconductor wafer by a combined application of Electron Back Scatter Diffraction and Infrared Transmission Microscopy. In particular, the new method is demonstrated with directional solidificated multicrystalline silicon.

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Cryo-SEM specimen preparation under controlled temperature and concentration conditions.

Publication Date: 2012 Jan 23 PMID: 22268668
Authors: Issman, L. - Talmon, Y.
Journal: J Microsc

Cryogenic temperature scanning electron microscopy (cryo-SEM) is an excellent technique for imaging liquid and semi-liquid materials of high vapour pressure, which are highly viscous or contain large (>0.5 mum) aggregates, in which nanometric details are to be studied. However, so far there have been no adequate tools for controlled cryo-specimen preparation. The specimen preparation stage is critical, because most of those samples are very sensitive to concentration and temperature changes, leading to nanostructural artefacts in the specimens. We designed and built a system for easy and reliable cryo-SEM specimen preparation under controlled conditions of fixed temperature and humidity. We describe this new methodology, and demonstrate its applicability, by showing imaging data of three liquid material systems. We have studied carbon nanotubes (CNTs) dispersions in superacid. We also characterized a number of systems made of water/isooctane/nonionic and cationic surfactant that showed different microemulsion phases as function of the system composition and temperature. In all of the examples given, we demonstrate artefact- and contamination-free specimens, which have preserved their native nanostructure. Our new system paves the way for a new methodology for the newly emerging field of cryo-SEM.

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Automatic tracking of individual migrating cells using low-magnification dark-field microscopy.

Publication Date: 2012 Jan 19 PMID: 22260196
Authors: Nenasheva, T. A. - Carter, T. - Mashanov, G. I.
Journal: J Microsc

Many fundamental biological processes, such as the search for food, immunological responses and wound healing, depend on cell migration. Video microscopy allows the magnitude and direction of cell migration to be documented. Here, we present a simple and inexpensive method for simultaneous tracking of hundreds of migrating cells over periods of several days. Low-magnification dark-field microscopy was used to visualize individual cells whereas time-lapse video images were acquired by computer for future analysis. We employed an automated tracking algorithm to identify individual cells on each video image allowing migration paths to be tracked using a nearest neighbour algorithm. To test the method, we followed the time-course of migration of 3T3 fibroblasts, endothelial cells and individual amoeba in the absence of any chemical stimulus gradient. All cell types showed a 'random walk' behaviour in which mean squared displacement in position increased linearly with time. We defined a 'migration coefficient' (D(mig) ), analogous to a diffusion coefficient, which gave an estimate of cell migration rate. D(mig) depended on cell type and temperature. When amoebas were made to undergo chemotaxis, the cells no longer followed a random walk but instead moved at a near constant velocity (V(av) ) towards the chemotactic stimulus.

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Silica-rubber microstructure visualised in three dimensions by focused ion beam-scanning electron microscopy.

Publication Date: 2012 Jan 19 PMID: 22260177
Authors: Tunnicliffe, L. B. - Thomas, A. G. - Busfield, J. J.
Journal: J Microsc

A focused ion beam-scanning electron microscope (FIB-SEM) technique for three-dimensional reconstruction and representation of material microstructures was applied to a silica-filled synthetic rubber for the first time. Backscattered electron imaging allowed differentiation between rubber matrix, silica filler and zinc oxide (used as an activator for the sulphur vulcanisation reaction). Subsequent image processing allowed three-dimensional isosurface model generation of the particulate structure within the rubber composite and separation of zinc oxide from the silica filler. The potential for development and application of this technique using finite element analysis modelling is also highlighted.

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Mechanical properties of asphalt binders evaluated by atomic force microscopy.

Publication Date: 2012 Feb PMID: 22251090
Authors: Dourado, E. R. - Simao, R. A. - Leite, L. F.
Journal: J Microsc

Atomic Force Microscopy was employed in order to relate the features observed on the surface of a 50/70 asphalt binder according to its local stiffness and elastic recovery. Indentations were performed in different points of the surface and a significant variation of elasticity was observed between the points on the so-called bee structure and the matrix. Also, indentations varying the maximum force were performed on similar white spots in the bee structure and the recovery was followed up to 1 h after indentation. It was observed that the elastic recovery is very much dependent on the colloidal structure of the bee. The final surface state of the binder, close to the bee for usual bees is not the same as the initial one indicating severe plastic deformation. Also, permanent phase change could be observed for bright spots presented in not well-structured bee arrangements. A surface hardening was observed in the bee region.

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Optimization of EBSD parameters for ultra-fast characterization.

Publication Date: 2012 Feb PMID: 22251089
Authors: Chen, Y. - Hjelen, J. - Gireesh, S. S. - Roven, H. J.
Journal: J Microsc

Ultra-fast pattern acquisition of electron backscatter diffraction and offline indexing could become a dominant technique over online electron backscatter diffraction to investigate the microstructures of a wide range of materials, especially for in situ experiments or very large scans. However, less attention has been paid to optimize the parameters related to ultra-fast electron backscatter diffraction. The present results show that contamination on a clean and unmounted specimen is not a problem even at step sizes as small as 1 nm at a vacuum degree of 6.1 x 10(-5) Pa. There exists an optimum step size at about 50 data acquisition board units. A new and easy method to calculate the effective spatial resolution is proposed. Effective spatial resolution tends to increase slightly as the probe current increases from 10 to 100 nA. The fraction of indexed points decreases slightly as the frame rate increases from 128 patterns per second (pps) to 835 pps by compensating the probe current at the same ratio. The value 96 x 96 is found to be the optimum pattern resolution to obtain optimum speed and image quality. For a fixed position of electron backscatter diffraction detector, the fraction of indexed points as a function of working distance has a maximum value and drops sharply by shortening the working distance and it decreases slowly with increasing the working distance.

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A 2D MEMS mirror with sidewall electrodes applied for confocal MACROscope imaging.

Publication Date: 2012 Feb PMID: 22092486
Authors: Bai, Y. - Pallapa, M. - Chen, A. - Constantinou, P. - Damaskinos, S. - Wilson, B. C. - Yeow, J. T.
Journal: J Microsc

This paper presents microelectromechanical system micromirrors with sidewall electrodes applied for use as a Confocal MACROscope for biomedical imaging. The MACROscope is a fluorescence and brightfield confocal laser scanning microscope with a very large field of view. In this paper, a microelectromechanical system mirror with sidewall electrodes replaces the galvo-scanner and XYZ-stage to improve the confocal MACROscope design and obtain an image. Two micromirror-based optical configurations are developed and tested to optimize the optical design through scanning angle, field of view and numerical aperture improvement. Meanwhile, the scanning frequency and control waveform of the micromirror are tested. Analysing the scan frequency and waveform becomes a key factor to optimize the micromirror-based confocal MACROscope. When the micromirror is integrated into the MACROscope and works at 40 Hz, the micromirror with open-loop control possesses good repeatability, so that the synchronization among the scanner, XYZ-stage and image acquisition can be realized. A laser scanning microscope system based on the micromirror with 2 mum width torsion bars was built and a 2D image was obtained as well. This work forms the experimental basis for building a practical confocal MACROscope.

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Digital correction of motion artefacts in microscopy image sequences collected from living animals using rigid and nonrigid registration.

Publication Date: 2012 Feb PMID: 22092443
Authors: Lorenz, K. S. - Salama, P. - Dunn, K. W. - Delp, E. J.
Journal: J Microsc

Digital image analysis is a fundamental component of quantitative microscopy. However, intravital microscopy presents many challenges for digital image analysis. In general, microscopy volumes are inherently anisotropic, suffer from decreasing contrast with tissue depth, lack object edge detail and characteristically have low signal levels. Intravital microscopy introduces the additional problem of motion artefacts, resulting from respiratory motion and heartbeat from specimens imaged in vivo. This paper describes an image registration technique for use with sequences of intravital microscopy images collected in time-series or in 3D volumes. Our registration method involves both rigid and nonrigid components. The rigid registration component corrects global image translations, whereas the nonrigid component manipulates a uniform grid of control points defined by B-splines. Each control point is optimized by minimizing a cost function consisting of two parts: a term to define image similarity, and a term to ensure deformation grid smoothness. Experimental results indicate that this approach is promising based on the analysis of several image volumes collected from the kidney, lung and salivary gland of living rodents.

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A pattern recognition method for lattice distortion measurement from HRTEM images.

Publication Date: 2012 Feb PMID: 22044267
Authors: Przybyla, P.
Journal: J Microsc

The idea of the method is to analyse a crystal lattice by creating a grid of quadrilaterals corresponding to repeated cells that are visible in the image. This approach combines image processing elements with a continuum field theory, to create a distortion-independent similarity measure that is used to select the most appropriate among possible lattice configurations. Subsequently, displacement and distortion fields are computed from individual cell positions. The method allows one to obtain these fields even for images where a periodic cell does not necessarily appear as a single dot of intensity in a high-resolution transmission electron microscopy (HRTEM) image, which results in a lower accuracy of commonly used approaches, namely geometric phase and peak finding. The results obtained from this method are verified quantitatively by comparison with known distortion tensor distributions and Burgers vector values on both simulated and real images.

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TEM image analysis and modelling: application to boehmite nanoparticles.

Publication Date: 2012 Feb PMID: 22044223
Authors: Moreaud, M. - Jeulin, D. - Morard, V. - Revel, R.
Journal: J Microsc

Boehmite occurs in the form of nanoparticles. Upon drying, it can form the alumina that is common in catalyst support used in refining and petrochemicals. The topotactic transformation of boehmite alumina led to an interest in the precise shape and size of these nanoparticles which is highly linked to the catalyst activity. Boehmite nanoparticles can be observed by transmission electron microscopy. Although they are highly aggregated, the analysis of transmission electron microscopy images with a specific random model approach, here a dilution model, can give an accurate estimate of their size. To use this approach, electronic noise and diffraction artefacts on the edges of the nanoparticles have to be removed. Covariance measurements on micrographs can be performed. They can be used to fit a model. The fitting uses a novel numerical method to estimate the covariogram of grains. The model can take into account the specific orientations of the nanoparticles. The influence of noise, image filters used to remove noise and diffraction artefacts, as well as all the parameters of the model are all studied in this paper. We propose nanoparticle size estimations procedures based on both single and mixture-of-two particle models.

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Backscattered electron detection in environmental SEM.

Publication Date: 2012 Feb PMID: 22044175
Authors: Danilatos, G. D.
Journal: J Microsc

An examination of the backscattered electron imaging status in environmental scanning electron microscopy is presented with particular attention to the testing and use of cerium doped yttrium aluminium garnet and yttrium aluminium perovskite scintillation detectors. A comparison is made with plastic scintillating backscattered electron detectors used previously (Nuclear Enterprises type NE102A scintillator). Semi-disk, strip and wedge shapes of these materials have been tested in conjunction with various light-guide geometries. These systems have been combined with two different types of photomultipliers, which also play a critical role in the total detector efficiency. The advantage of increased light output from the monocrystal materials is gained only if matched with suitable light-guides and photomultipliers. The associated problems are discussed and proposals for further work are made for the construction of most efficient backscattered electron detectors in the environmental scanning electron microscope.

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High-resolution cell outline segmentation and tracking from phase-contrast microscopy images.

Publication Date: 2012 Feb PMID: 21999192
Authors: Ambuhl, M. E. - Brepsant, C. - Meister, J. J. - Verkhovsky, A. B. - Sbalzarini, I. F.
Journal: J Microsc

Accurate extraction of cell outlines from microscopy images is essential for analysing the dynamics of migrating cells. Phase-contrast microscopy is one of the most common and convenient imaging modalities for observing cell motility because it does not require exogenous labelling and uses only moderate light levels with generally negligible phototoxicity effects. Automatic extraction and tracking of high-resolution cell outlines from phase-contrast images, however, is difficult due to complex and non-uniform edge intensity. We present a novel image-processing method based on refined level-set segmentation for accurate extraction of cell outlines from high-resolution phase-contrast images. The algorithm is validated on synthetic images of defined noise levels and applied to real image sequences of polarizing and persistently migrating keratocyte cells. We demonstrate that the algorithm is able to reliably reveal fine features in the cell edge dynamics.

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Segmentation of virus particle candidates in transmission electron microscopy images.

Publication Date: 2012 Feb PMID: 21972793
Authors: Kylberg, G. - Uppstrom, M. - Hedlund, K. O. - Borgefors, G. - Sintorn, I. M.
Journal: J Microsc

In this paper, we present an automatic segmentation method that detects virus particles of various shapes in transmission electron microscopy images. The method is based on a statistical analysis of local neighbourhoods of all the pixels in the image followed by an object width discrimination and finally, for elongated objects, a border refinement step. It requires only one input parameter, the approximate width of the virus particles searched for. The proposed method is evaluated on a large number of viruses. It successfully segments viruses regardless of shape, from polyhedral to highly pleomorphic.

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Analytical electron microscopy of black carbon and microaggregated mineral matter in Amazonian dark Earth.

Publication Date: 2012 Feb PMID: 21950566
Authors: Chia, C. H. - Munroe, P. - Joseph, S. D. - Lin, Y. - Lehmann, J. - Muller, D. A. - Xin, H. L. - Neves, E.
Journal: J Microsc

Black carbon (BC) is one of the most stable forms of soil organic matter. Its surface functional groups and structure have been well characterized by a range of analytical methods. However, little is known about the mechanisms of interactions between the BC particles and the surrounding mineral matter. In this paper a range of microscopy techniques, such as transmission electron microscopy and scanning transmission electron microscopy, were used to investigate the possible reactions of BC particles within microaggregates (<2 mm) found in Amazonian dark Earth. Attention is given to the interactions that occur at the interfacial regions between the organic and inorganic phases. Examination of Amazonian dark Earth showed that the carbon-rich phase detected within the BC particles has a significant calcium concentration and a high density of micropores was found at the BC-mineral interface. These observations provide evidence to support suggested mechanisms of interaction between these phases.

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