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  • 1. Berglund, M.
    et al.
    Rymell, L.
    Peuker, M.
    Wilhein, T.
    Hertz, Hans M.
    KTH, Superseded Departments, Physics.
    Compact water-window transmission X-ray microscopy2000In: Journal of Microscopy, ISSN 0022-2720, E-ISSN 1365-2818, Vol. 197, p. 268-273Article in journal (Refereed)
    Abstract [en]

    We demonstrate sub-100 nm resolution water-window soft X-ray full-field transmission microscopy with a compact system. The microscope operates at lambda = 3.37 nm and is based on a 100 Hz table-top regenerative debris-free droplet-target laser-plasma X-ray source in combination with normal-incidence multilayer condenser optics for sample illumination. High-spatial-resolution imaging is performed with a 7.3% efficiency nickel zone plate and a 1024 x 1024 pixel CCD detector. Images of dry test samples are recorded with exposure times of a few minutes and show features smaller than 60 nm.

  • 2.
    Carlsson, Kjell
    et al.
    KTH, Superseded Departments, Physics.
    Liljeborg, A.
    Andersson, R. M.
    Brismar, Hjalmar
    Confocal pH imaging of microscopic specimens using fluorescence lifetimes and phase fluorometry: influence of parameter choice on system performance2000In: Journal of Microscopy, ISSN 0022-2720, E-ISSN 1365-2818, Vol. 199, p. 106-114Article in journal (Refereed)
    Abstract [en]

    We investigate the performance of confocal pH imaging when using phase fluorometry and fluorophores with pH-dependent lifetimes. In these experiments, the specimen is illuminated by a laser beam, whose intensity is sinusoidally modulated. The lifetime-dependent phase shift in the fluorescent signal is detected by a lock-in amplifier, and converted into a pH value through a calibration procedure. A theoretical investigation is made of how the different system parameters will influence the results concerning sensitivity and noise. Experiments carried out with the fluorophore SNAFL-2 support these theoretical predictions. It is found that, under realistic experimental conditions, we can expect a pH change of 0.1 units to be easily detected in an 8-bit digital image. However, the pixel-to-pixel root mean square noise is often of the order of one pH unit. This comparatively high level of noise has its origin in photon quantum noise. pH measurements on living cells show a systematic deviation from expected values. This discrepancy appears to be the result of fluorophore interaction with various cell constituents, and is the subject of further investigation.

  • 3.
    Das, Prabir Kumar
    et al.
    KTH, School of Architecture and the Built Environment (ABE), Transport Science, Highway and Railway Engineering.
    Kringos, Niki
    KTH, School of Architecture and the Built Environment (ABE), Transport Science, Highway and Railway Engineering.
    Birgisson, Björn
    KTH, School of Architecture and the Built Environment (ABE), Transport Science, Highway and Railway Engineering.
    Microscale investigation of thin film surface ageing of bitumen2014In: Journal of Microscopy, ISSN 0022-2720, E-ISSN 1365-2818, Vol. 254, no 2, p. 95-107Article in journal (Refereed)
    Abstract [en]

    This paper investigates the mechanism of bitumen surface ageing, which was validated utilizing the atomic force microscopy and the differential scanning calorimetry. To validate the surface ageing, three different types of bitumen with different natural wax content were conditioned in four different modes: both ultraviolet and air, only ultraviolet, only air and without any exposure, for 15 and 30 days. From the atomic force microscopy investigation after 15 and 30 days of conditioning period, it was found that regardless the bitumen type, the percentage of microstructure on the surface reduced with the degree of exposure and time. Comparing all the four different exposures, it was observed that ultraviolet radiation caused more surface ageing than the oxidation. It was also found that the combined effect was not simply a summation or multiplication of the individual effects. The differential scanning calorimetry investigation showed that the amount of crystalline fractions in bitumen remain constant even after the systematic conditioning. Interestingly, during the cooling cycle, crystallization of wax molecules started earlier for the exposed specimens than the without exposed one. The analysis of the obtained results indicated that the ageing created a thin film upon the exposed surface, which acts as a barrier and creates difficulty for the wax induced microstructures to float up at the surface. From the differential scanning calorimetry analysis, it can be concluded that the ageing product induced impurities in the bitumen matrix, which acts as a promoter in the crystallization process.

  • 4.
    Johansson, Göran A.
    et al.
    KTH, Superseded Departments, Physics.
    Khanna, S M
    Nair, A
    Mannstrom, P
    Denbeaux, G
    Ulfendahl, M
    Exploring the use of soft X-ray microscopy for imaging subcellular structures of the inner ear2004In: Journal of Microscopy, ISSN 0022-2720, E-ISSN 1365-2818, Vol. 215, p. 203-212Article in journal (Refereed)
    Abstract [en]

    The soft X-ray microscope at the Lawrence Berkeley National Laboratory was developed for visualization of biological tissue. Soft X-ray microscopy provides high-resolution visualization of hydrated, non-embedded and non-sectioned cells and is thus potentially an alternative to transmission electron microscopy. Here we show for the first time soft X-ray micrographs of structures isolated from the guinea-pig inner ear. Sensory outer hair cells and supporting pillar cells are readily visualized. In the hair cells, individual stereocilia can easily be identified within the apical hair bundle. The underlying cuticular plate is, however, too densely composed or too thick to be clearly visualized, and thus appears very dark. The cytoplasmic structures protruding from the cuticular plates as well as the fibrillar material surrounding and projecting from the cell nuclei can be seen. In the pillar cells the images reveal individual microtubule bundles. Soft X-ray images of the acellular tectorial membrane and thin two-layered Reissner's membrane display a level of resolution comparable to low-power electron microscopy.

  • 5.
    Koeck, P. J. B.
    KTH, School of Technology and Health (STH), Basic Science and Biomedicine, Structural Biotechnology.
    Improved Zernike-type phase contrast for transmission electron microscopy2015In: Journal of Microscopy, ISSN 0022-2720, E-ISSN 1365-2818, Vol. 259, no 1, p. 74-78Article in journal (Refereed)
    Abstract [en]

    Zernike phase contrast has been recognized as a means of recording high-resolution images with high contrast using a transmission electron microscope. This imaging mode can be used to image typical phase objects such as unstained biological molecules or cryosections of biological tissue. According to the original proposal discussed in Danev and Nagayama (2001) and references therein, the Zernike phase plate applies a phase shift of /2 to all scattered electron beams outside a given scattering angle and an image is recorded at Gaussian focus or slight underfocus (below Scherzer defocus). Alternatively, a phase shift of -/2 is applied to the central beam using the Boersch phase plate. The resulting image will have an almost perfect contrast transfer function (close to 1) from a given lowest spatial frequency up to a maximum resolution determined by the wave length, the amount of defocus and the spherical aberration of the microscope. In this paper, I present theory and simulations showing that this maximum spatial frequency can be increased considerably without loss of contrast by using a Zernike or Boersch phase plate that leads to a phase shift between scattered and unscattered electrons of only /4, and recording images at Scherzer defocus. The maximum resolution can be improved even more by imaging at extended Scherzer defocus, though at the cost of contrast loss at lower spatial frequencies. Lay description Zernike phase contrast has been recognized as a means of recording high-resolution images with high contrast using a transmission electron microscope. This imaging mode can be used to image specimens such as unstained biological molecules or sections of biological tissue. According to the original proposal, the Zernike phase plate applies a phase shift of /2 to all scattered electron beams outside a given scattering angle and an image is recorded at or close to focus. The resulting image will be an almost perfect representation of the specimen up to a maximum resolution determined by the energy of the electrons and certain optical parameters of the microscope. In this paper, I present theory and simulations showing that this maximum resolution can be increased considerably without loss of contrast by using a Zernike phase plate that leads to a phase shift between scattered and unscattered electrons of only /4, and recording images somewhat out of focus.

  • 6.
    Koeck, P. J. B.
    et al.
    KTH, School of Technology and Health (STH), Basic Science and Biomedicine, Structural Biotechnology.
    Karshikoff, A.
    Limitations of the linear and the projection approximations in three-dimensional transmission electron microscopy of fully hydrated proteins2015In: Journal of Microscopy, ISSN 0022-2720, E-ISSN 1365-2818, Vol. 259, no 3, p. 197-209Article in journal (Refereed)
    Abstract [en]

    We establish expressions for the linear and quadratic terms in the series expansion of the phase and the phase and amplitude object description of imaging thin specimens by transmission electron microscopy. Based on these expressions we simulate the corresponding contributions to images of unstained protein complexes of varying thickness and arrive at an estimate for how much each term contributes to the contrast of the image. From this we can estimate a maximum specimen thickness for which the weak phase and the weak amplitude and phase object approximation (and therefore linear imaging) is still reasonably accurate. When discussing thick specimens it is also necessary to consider limitations due to describing the image as a filtered projection of the specimen, since the different layers of the specimen are not imaged with the same defocus value. We therefore compared simulations based on the projection approximation with the more accurate multislice model of image formation. However, we find that the errors due to nonlinear image contributions are greater than those due to the defocus gradient for the defocus values chosen for the simulations. Finally, we study how the discussed nonlinear image contributions and the defocus gradient affect the quality of three-dimensional reconstructions. We find that three-dimensional reconstructions reach high resolution when at the same time exhibiting localized systematic structural errors. Non-Technical Abstract Cryo transmission electron microscopy and three-dimensional reconstruction can be used to determine a three-dimensional model of a protein molecule. In the mathematical methods used for three-dimensional reconstruction assumptions are made about a linear relationship between the images recorded in the electron microscope and the objects being imaged. In this paper we investigate with computer simulations at what specimen thickness these assumptions start breaking down and what sort of errors can be expected in the three-dimensional reconstructions when the assumptions are not valid anymore.

  • 7. Norlén, L.
    et al.
    Öktem, Ozan
    KTH, School of Engineering Sciences (SCI), Centres, Center for Industrial and Applied Mathematics, CIAM. KTH, School of Engineering Sciences (SCI), Mathematics (Dept.).
    Skoglund, U.
    Molecular cryo-electron tomography of vitreous tissue sections: current challenges2009In: Journal of Microscopy, ISSN 0022-2720, E-ISSN 1365-2818, Vol. 235, no 3, p. 293-307Article in journal (Refereed)
    Abstract [en]

    Electron tomography of vitreous tissue sections (tissue TOVIS) allows the study of the three-dimensional structure of molecular complexes in a near-native cellular context. Its usage is, however, limited by an unfortunate combination of noisy and incomplete data, by a technically demanding sample preparation procedure, and by a disposition for specimen degradation during data collection. Here we outline some major challenges as experienced from the application of TOVIS to human skin. We further consider a number of practical measures as well as theoretical approaches for its future development.

  • 8. Poulikakos, L
    et al.
    Partl, Manfred
    Empa, Swiss Federal Laboratories for Materials Testing and Research, Laboratory for Road Engineering/Sealing Components, Dübendorf, Switzerland.
    Investigation of Porous Asphalt Microstructure using Optical and Electron Microscopy2010In: Journal of Microscopy, ISSN 0022-2720, E-ISSN 1365-2818, Vol. 240, no 2, p. 145-154Article in journal (Refereed)
    Abstract [en]

    P>Direct observations of porous asphalt concrete samples in their natural state using optical and electron microscopy techniques led to useful information regarding the microstructure of two mixes and indicated a relationship between microstructure and in situ performance. This paper presents evidence that suboptimal microstructure can lead to premature failure thus making a first step in defining well or suboptimal performing pavements with a bottom-up approach (microstructure). Laboratory and field compaction produce different samples in terms of the microstructure. Laboratory compaction using the gyratory method has produced more microcracks in mineral aggregates after the binder had cooled. Well-performing mixes used polymer-modified binders, had a more homogeneous void structure with fewer elongated voids and better interlocking of the aggregates. Furthermore, well-performing mixes showed better distribution of the mastic and better coverage of the aggregates with bitumen. Low vacuum scanning electron microscopy showed that styrene butadiene styrene polymer modification in binder exists in the form of discontinuous globules and not continuous networks. A reduction in the polymer phase was observed as a result of aging and in-service use.

  • 9. Rullgard, H.
    et al.
    Ofverstedt, L. -G
    Masich, S.
    Daneholt, B.
    Öktem, Ozan
    KTH, School of Engineering Sciences (SCI), Mathematics (Dept.). KTH, School of Engineering Sciences (SCI), Centres, Center for Industrial and Applied Mathematics, CIAM.
    Simulation of transmission electron microscope images of biological specimens2011In: Journal of Microscopy, ISSN 0022-2720, E-ISSN 1365-2818, Vol. 243, no 3, p. 234-256Article in journal (Refereed)
    Abstract [en]

    We present a new approach to simulate electron cryo-microscope images of biological specimens. The framework for simulation consists of two parts; the first is a phantom generator that generates a model of a specimen suitable for simulation, the second is a transmission electron microscope simulator. The phantom generator calculates the scattering potential of an atomic structure in aqueous buffer and allows the user to define the distribution of molecules in the simulated image. The simulator includes a well defined electron-specimen interaction model based on the scalar Schrodinger equation, the contrast transfer function for optics, and a noise model that includes shot noise as well as detector noise including detector blurring. To enable optimal performance, the simulation framework also includes a calibration protocol for setting simulation parameters. To test the accuracy of the new framework for simulation, we compare simulated images to experimental images recorded of the Tobacco Mosaic Virus (TMV) in vitreous ice. The simulated and experimental images show good agreement with respect to contrast variations depending on dose and defocus. Furthermore, random fluctuations present in experimental and simulated images exhibit similar statistical properties. The simulator has been designed to provide a platform for development of new instrumentation and image processing procedures in single particle electron microscopy, two-dimensional crystallography and electron tomography with well documented protocols and an open source code into which new improvements and extensions are easily incorporated.

  • 10.
    Stollberg, Heide
    et al.
    KTH, Superseded Departments, Physics.
    Boutet de Monvel, Jacques
    Holmberg, Anders
    KTH, Superseded Departments, Physics.
    Hertz, Hans M.
    KTH, Superseded Departments, Physics.
    Wavelet-based image restoration for compact x-ray microscopy2003In: Journal of Microscopy, ISSN 0022-2720, E-ISSN 1365-2818, Vol. 211, no 2, p. 154-160Article in journal (Refereed)
    Abstract [en]

    Compact water-window X-ray microscopy with short exposure times will always be limited on photons owing to sources of limited power in combination with low-efficency X-ray optics. Thus, it is important to investigate methods for improving the signal-to-noise ratio in the images. We show that a wavelet-based denoising procedure significantly improves the quality and contrast in compact X-ray microscopy images. A non-decimated, discrete wavelet transform (DWT) is applied to original, noisy images. After applying a thresholding procedure to the finest scales of the DWT, by setting to zero all wavelet coefficients of magnitude below a prescribed value, the inverse DWT to the thresholded DWT produces denoised images. It is concluded that the denoising procedure has potential to reduce the exposure time by a factor of 2 without loss of relevant image information.

  • 11.
    Stollberg, Heide
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Biomedical and X-ray Physics.
    Guttmann, Peter
    Institut für Röntgenphysik, Georg-August-Universität Göttingen, Berlin.
    Takman, Per
    KTH, School of Engineering Sciences (SCI), Applied Physics, Biomedical and X-ray Physics.
    Hertz, Hans
    KTH, School of Engineering Sciences (SCI), Applied Physics, Biomedical and X-ray Physics.
    Size-selective colloidal-gold localization in transmission x-ray microscopy2007In: Journal of Microscopy, ISSN 0022-2720, E-ISSN 1365-2818, Vol. 225, no 1, p. 80-87Article in journal (Refereed)
    Abstract [en]

    Colloidal gold is a useful marker for functional-imaging experiments in transmission X-ray microscopy. Due to the low contrast of gold particles with small diameters it is necessary to develop a powerful algorithm to localize the single gold particles. The presented image-analysis algorithm for identifying colloidal gold particles is based on the combination of a threshold with respect to the local absorption and shape discrimination, realized by fitting a Gaussian profile to the identified regions of interest. The shape discrimination provides the possibility of size-selective identification and localization of single colloidal gold particles down to a diameter of 50 nm. The image-analysis algorithm, therefore, has potential for localization studies of several proteins simultaneously and for localization of fiducial markers in X-ray tomography.

  • 12.
    Stollberg, Heide
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics.
    Pokorny, Milan
    KTH, School of Engineering Sciences (SCI), Applied Physics.
    Hertz, Hans M.
    KTH, School of Engineering Sciences (SCI), Applied Physics.
    A vacuum-compatible wet-specimen chamber for compact x-ray microscopy2007In: Journal of Microscopy, ISSN 0022-2720, E-ISSN 1365-2818, Journal of Microscopy, Vol. 226, no 1, p. 71-73Article in journal (Refereed)
    Abstract [en]

    Soft X-ray microscopy is a powerful tool for investigations of, for example, polymers or soils in their natural liquid environment. This requires a wet-specimen chamber. Compact X-ray microscopy allows the horizontal mounting of such samples, thereby reducing the influence of gravitational forces. We have developed a wet-specimen chamber for such compact X-ray microscope. The chamber is vacuum compatible, which reduces the exposure time. The vacuum sealing is achieved by a combination of mechanical sealing and sealing by bio-compatible glue. With the wet-specimen chamber the specimens can be kept in an aqueous environment in a vacuum of 10(-4) mbar for several hours. Imaging of lipid droplets in water demonstrates the function of the wet-specimen chamber.

  • 13.
    Takman, Per A. C.
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Biomedical and X-ray Physics.
    Stollberg, Heide
    KTH, School of Engineering Sciences (SCI), Applied Physics, Biomedical and X-ray Physics.
    Johansson, Göran A.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Biomedical and X-ray Physics.
    Holmberg, Anders
    KTH, School of Engineering Sciences (SCI), Applied Physics, Biomedical and X-ray Physics.
    Lindblom, Magnus
    KTH, School of Engineering Sciences (SCI), Applied Physics, Biomedical and X-ray Physics.
    Hertz, Hans M.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Biomedical and X-ray Physics.
    Sub 30-nm resolution compact x-ray microscopy2006In: Journal of Microscopy, ISSN 0022-2720, E-ISSN 1365-2818Article in journal (Other academic)
  • 14.
    Takman, Per
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Biomedical and X-ray Physics.
    Stollberg, Heide
    KTH, School of Engineering Sciences (SCI), Applied Physics, Biomedical and X-ray Physics.
    Johansson, Göran A.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Biomedical and X-ray Physics.
    Holmberg, Anders
    KTH, School of Engineering Sciences (SCI), Applied Physics, Biomedical and X-ray Physics.
    Lindblom, Magnus
    KTH, School of Engineering Sciences (SCI), Applied Physics, Biomedical and X-ray Physics.
    Hertz, Hans M.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Biomedical and X-ray Physics.
    High-resolution compact x-ray microscopy2007In: Journal of Microscopy, ISSN 0022-2720, E-ISSN 1365-2818, Vol. 226, no 2, p. 175-181Article in journal (Refereed)
    Abstract [en]

    We demonstrate compact full-field soft X-ray transmission microscopy with sub 60-nm resolution operating at λ= 2.48 nm. The microscope is based on a 100-Hz regenerative liquid-nitrogen-jet laser-plasma source in combination with a condenser zone plate and a micro-zone plate objective for high-resolution imaging onto a 2048 × 2048 pixel CCD detector. The sample holder is mounted in a helium atmosphere and allows imaging of both dry and wet specimens. The microscope design enables fast sample switching and the sample can be pre-aligned using a visible-light microscope. High-quality images can be acquired with exposure times of less than 5 min. We demonstrate the performance of the microscope using both dry and wet samples.

  • 15. Thaler, Marlene
    et al.
    Roy, Soumen
    Qin, Jian
    KTH, School of Information and Communication Technology (ICT), Material Physics, Functional Materials, FNM.
    Bitsche, Mario
    Glueckert, Rudolf
    Fornara, Andrea
    KTH, School of Information and Communication Technology (ICT), Material Physics, Functional Materials, FNM.
    Muhammed, Mamoun
    KTH, School of Information and Communication Technology (ICT), Material Physics, Functional Materials, FNM.
    Salvenmoser, Willi
    Rieger, Gunde
    Visualization and Analysis of Superparamagnetic Ferrogels in the Inner Ear by Light Microscopy and Energy Filtred TEM: a promising approach for advanced local drug deliveryIn: Journal of Microscopy, ISSN 0022-2720, E-ISSN 1365-2818Article in journal (Other academic)
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