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  • 1.
    Bertilson, Michael
    et al.
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik.
    von Hofsten, Olov
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik.
    Vogt, Ulrich
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Biomedicinsk fysik och röntgenfysik.
    Holmberg, Anders
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Biomedicinsk fysik och röntgenfysik.
    Christakou, Athanasia E.
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Biomedicinsk fysik och röntgenfysik.
    Hertz, Hans M.
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Biomedicinsk fysik och röntgenfysik.
    Laboratory soft-x-ray microscope for cryotomography of biological specimens2011Inngår i: Optics Letters, ISSN 0146-9592, E-ISSN 1539-4794, Vol. 36, nr 14, s. 2728-2730Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Soft-x-ray cryotomography allows quantitative and high-resolution three-dimensional imaging of intact unstained cells. To date, the method relies on synchrotron-radiation sources, which limits accessibility for researchers. Here we present a laboratory water-window microscope for cryotomography. It is based on a lambda = 2.48nm liquid-jet laser-plasma source, a normal-incidence multilayer condenser, a 30nm zone-plate objective, and a cryotilt sample holder. We demonstrate high-resolution imaging, as well as quantitative tomographic imaging, of frozen intact cells. The reconstructed tomogram of the intracellular local absorption coefficient shows details down to similar to 100nm.

  • 2.
    Bertilson, Michael
    et al.
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Biomedicinsk fysik och röntgenfysik.
    von Hofsten, Olov
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Biomedicinsk fysik och röntgenfysik.
    Vogt, Ulrich
    Holmberg, Anders
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Biomedicinsk fysik och röntgenfysik.
    Christakou, Athanasia
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Biomedicinsk fysik och röntgenfysik.
    Jerlström-Hultqvist, J.
    Svärd, S.
    Hertz, Hans M.
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Biomedicinsk fysik och röntgenfysik.
    Laboratory Soft X-Ray Cryo TomographyManuskript (preprint) (Annet vitenskapelig)
    Abstract [en]

    X-rays allow quantitative high-spatial-resolution three-dimensional (3D) imaging of intact unstained cells. Such 3D imaging is provided by soft x-ray lens-based methods (water-window cryo tomography) and hard x-ray lens-less methods (coherent diffraction imaging) are emerging. However, both methods rely on high-brightness synchrotron-radiation sources, which limit the accessibility of a wider scientific community. Here we show 3D water-window cryo tomography with a laboratory-source-based microscope arrangement. The system relies on a λ=2.48-nm liquid-jet laser-plasma source, normal- incidence multilayer condenser optics, 30-nm zone-plate optics, and a cryo sample chamber. We demonstrate imaging of intact unstained yeast, protozoan parasites and mammalian cells. 3D images show noise-limited features close to ~100 nm and intra-cellular structure is classified based on the local absorption coefficient. A comprehensive theoretical model of the tomographic imaging system allows optimization of system parameters and a quantitative estimate of the 3D imaging accuracy. The model includes issues such as non-geometric projections of the thick samples and stray light, and is applicable to laboratory as well as synchrotron-based x-ray microscopes. The model shows that laboratory x-ray cryo tomography will allow quantitative 3D imaging with ~30-nm (half-period) resolution over a full 5 µm object.

     

  • 3.
    Christakou, Athanasia
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Biomedicinsk fysik och röntgenfysik.
    Ultrasound-assisted Interactions of Natural Killer Cells with Cancer Cells and Solid Tumors2014Doktoravhandling, med artikler (Annet vitenskapelig)
    Abstract [en]

    In this Thesis, we have developed a microtechnology-based method for culturing and visualizing high numbers of individual cells and cell-cell interactions over extended periods of time. The foundation of the device is a silicon-glass multiwell microplate (also referred as microchip) directly compatible with fluorescence microscopy. The initial microchip design involved thousands of square wells of sizes up to 80 µm, for screening large numbers of cell-cell interactions at the single cell level. Biocompatibility and confinement tests proved the feasibility of the idea, and further investigation showed the conservation of immune cellular processes within the wells. Although the system is very reliable for screening, limitations related to synchronization of the interaction events, and the inability to maintain conjugations for long time periods, led to the development of a novel ultrasonic manipulation multiwell microdevice.

    The main components of the ultrasonic device is a 100-well silicon-glass microchip and an ultrasonic transducer. The transducer is used for ultrasonic actuation on the chip with a frequency causing half-wave resonances in each of the wells (2.0-2.5 MHz for wells with sizes 300-350 µm). Therefore, cells in suspension are directed by acoustic radiation forces towards a pressure node formed in the center of each well. This method allows simultaneous aggregation of cells in all wells and sustains cells confined within a small area for long time periods (even up to several days).

    The biological target of investigation in this Thesis is the natural killer (NK) cells and their functional properties. NK cells belong to the lymphatic group and they are important factors for host defense and immune regulation. They are characterized by the ability to interact with virus infected cells and cancer cells upon contact, and under suitable conditions they can induce target cell death. We have utilized the ultrasonic microdevice to induce NK-target cell interactions at the single cell level. Our results confirm a heterogeneity within IL-2 activated NK cell populations, with some cells being inactive, while others are capable to kill quickly and in a consecutive manner.

    Furthermore, we have integrated the ultrasonic microdevice in a temperature regulation system that allows to actuate with high-voltage ultrasound, but still sustain the cell physiological temperature. Using this system we have been able to induce formation of up to 100 solid tumors (HepG2 cells) in parallel without using surface modification or hydrogels. Finally, we used the tumors as targets for investigating NK cells ability to infiltrate and kill solid tumors. 

    To summarize, a method is presented for investigating individual NK cell behavior against target cells and solid tumors. Although we have utilized our technique to investigate NK cells, there is no limitation of the target of investigation. In the future, the device could be used for any type of cells where interactions at the single cell level can reveal critical information, but also to form solid tumors of primary cancer cells for toxicology studies.

  • 4.
    Christakou, Athanasia E.
    et al.
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik.
    Ohlin, Mathias
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik.
    Kadri, N.
    Frisk, Thomas
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik.
    Önfelt, Björn
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik. Karolinska Institute, Sweden.
    Wiklund, Martin
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik.
    Characterization of natural killer cells' cytotoxic heterogeneity using an array of sono-cages2012Inngår i: Proceedings of the 16th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2012, Chemical and Biological Microsystems Society , 2012, s. 1555-1557Konferansepaper (Fagfellevurdert)
    Abstract [en]

    Using a multi-well device as an array of sono-cages for single cell analysis, we quantify for the first time the heterogeneity of natural killer (NK) cells' cytotoxic response against cancer cells. We report a fraction of inactive NK cells within the tested population (36%), as well as the existence of few 'serial killers' that eliminate up to six targets during 4 hours. We also characterize the multi-well acoustic device in terms of trapping efficiency at different actuation voltages, using adherent and non-adherent cell lines. We show that the acoustic forces applied on the cells can be compared to forces of biological processes (i.e. cell adherence).

  • 5.
    Christakou, Athanasia. E.
    et al.
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik.
    Ohlin, Mathias
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik.
    Khorshidi, Mohammad Ali
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik.
    Frisk, Thomas
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik.
    Vanherberghen, Bruno
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik.
    Önfelt, Björn
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik.
    Wiklund, Martin
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik.
    Aggregation and long-term positioning of cells by ultrasound in a multi-well microchip for high-resolution imaging of the natural killer cell immune synapse2011Inngår i: 15th International Conference on Miniaturized Systems for Chemistry and Life Sciences 2011, MicroTAS 2011, 2011, s. 329-331Konferansepaper (Fagfellevurdert)
    Abstract [en]

    In this study we investigate the ability of Natural Killer (NK) cells to form ultrasound-mediated intercellular contacts with target cells in a multi-well microdevice by high-resolution confocal-microscopy imaging of inhibitory immune synapses. Furthermore, we compare the NK-Target cell cluster migration with and without ultrasound actuation. Experiments indicate that clusters of cells are positioned and maintained centered in the wells for 17 hours when they are exposed continuously to ultrasound. Our system can be used for both screening high numbers of events in low resolution and also for high resolution imaging of long term cell-cell interactions.

  • 6.
    Christakou, Athanasia E.
    et al.
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Biomedicinsk fysik och röntgenfysik.
    Ohlin, Mathias
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Biomedicinsk fysik och röntgenfysik.
    Vanherberghen, Bruno
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik.
    Khorshidi, Mohammad Ali
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik.
    Kadri, Nadir
    Frisk, Thomas
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Cellens fysik.
    Wiklund, Martin
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Biomedicinsk fysik och röntgenfysik.
    Önfelt, Björn
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Cellens fysik. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Live cell imaging in a micro-array of acoustic traps facilitates quantification of natural killer cell heterogeneity2013Inngår i: Integrative Biology, ISSN 1757-9694, E-ISSN 1757-9708, Vol. 5, nr 4, s. 712-719Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Natural killer (NK) cells kill virus-infected or cancer cells through the release of cytotoxic granules into a tight intercellular contact. NK cell populations comprise individual cells with varying sensitivity to distinct input signals, leading to disparate responses. To resolve this NK cell heterogeneity, we have designed a novel assay based on ultrasound-assisted cell-cell aggregation in a multiwell chip allowing high-resolution time-lapse imaging of one hundred NK-target cell interactions in parallel. Studying human NK cells' ability to kill MHC class I deficient tumor cells, we show that approximately two thirds of the NK cells display cytotoxicity, with some NK cells being particularly active, killing up to six target cells during the assay. We also report that simultaneous interaction with several susceptible target cells increases the cytotoxic responsiveness of NK cells, which could be coupled to a previously unknown regulatory mechanism with implications for NK-mediated tumor elimination.

  • 7.
    Christakou, Athanasia E.
    et al.
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Cellens fysik.
    Ohlin, Mathias
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik.
    Önfelt, Björn
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Cellens fysik.
    Wiklund, Martin
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Biomedicinsk fysik och röntgenfysik.
    Solid tumor spheroid formation by temperature-controlled high voltage ultrasound in a multi-well microdevice2014Inngår i: 18th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2014, Chemical and Biological Microsystems SocietyChemical and Biological Microsystems Society , 2014, s. 573-575Konferansepaper (Fagfellevurdert)
    Abstract [en]

    In the present work we demonstrate effective 3D growth of human hepatocellular carcinoma (HCC) HepG2 cell spheroids in parallel in a multi-well microdevice actuated with high voltage ultrasound in a temperature-controlled system. We compare the spheroid formation during continuous ultrasound exposure for one week where we formed spheroids in 59% of the wells, with the spheroid formation without ultrasound actuation, where we obtained 0% spheroids. Furthermore, we present an application of the tumor spheroids for investigating natural killer (NK) cells behavior against solid tumors.

  • 8.
    Christakou, Athanasia E.
    et al.
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Biomedicinsk fysik och röntgenfysik.
    Ohlin, Mathias
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Biomedicinsk fysik och röntgenfysik.
    Önfelt, Björn
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Cellens fysik.
    Wiklund, Martin
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Biomedicinsk fysik och röntgenfysik.
    Ultrasonic three-dimensional cell culture on chip for dynamic studies of tumor immune surveillance by natural killer cellsManuskript (preprint) (Annet vitenskapelig)
  • 9.
    Christakou, Athanasia E.
    et al.
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Biomedicinsk fysik och röntgenfysik.
    Ohlin, Mathias
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Biomedicinsk fysik och röntgenfysik.
    Önfelt, Björn
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Biomedicinsk fysik och röntgenfysik.
    Wiklund, Martin
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Biomedicinsk fysik och röntgenfysik.
    Ultrasound-assisted three-dimensional tumor formation in a multi well microplate for monitoring natural killer cell functional behaviorManuskript (preprint) (Annet vitenskapelig)
  • 10.
    Christakou, Athanasia
    et al.
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik.
    Ohlin, Mathias
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik.
    Önfelt, Björn
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik.
    Wiklund, Martin
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik.
    Characterization of natural killer cell immune surveillance against solid liver tumors2015Inngår i: MicroTAS 2015 - 19th International Conference on Miniaturized Systems for Chemistry and Life Sciences, Chemical and Biological Microsystems Society , 2015, s. 915-917Konferansepaper (Fagfellevurdert)
    Abstract [en]

    We demonstrate a method for investigating natural killer (NK) cell aggression against ultrasound-assisted human hepatocellular carcinoma (HCC) HepG2 solid tumors in a multi-well microdevice. We quantify the activity of human primary IL-2 activated NK cells against HepG2 tumors for up to five days and we present the correlation between NK cell numbers versus average tumor volume and final tumor outcome (growth or defeat). We suggest future applications on formation of tumors originated from primary tumors cells and other tumor components as well as primary NK originating from the patient for use in personalized immunotherapy.

  • 11.
    Christakou, Athanasia
    et al.
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Biomedicinsk fysik och röntgenfysik.
    Ohlin, Mathias
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Biomedicinsk fysik och röntgenfysik.
    Önfelt, Björn
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Cellens fysik. Karolinska Inst, Dept Microbiol Tumor & Cell Biol, Stockholm, Sweden.
    Wiklund, Martin
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Biomedicinsk fysik och röntgenfysik.
    Ultrasonic three-dimensional on-chip cell culture for dynamic studies of tumor immune surveillance by natural killer cells2015Inngår i: Lab on a Chip, ISSN 1473-0197, E-ISSN 1473-0189, Vol. 15, nr 15, s. 3222-31Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    We demonstrate a simple method for three-dimensional (3D) cell culture controlled by ultrasonic standing waves in a multi-well microplate. The method gently arranges cells in a suspension into a single aggregate in each well of the microplate and, by this, nucleates 3D tissue-like cell growth for culture times between two and seven days. The microplate device is compatible with both high-resolution optical microscopy and maintenance in a standard cell incubator. The result is a scaffold- and coating-free method for 3D cell culture that can be used for controlling the cellular architecture, as well as the cellular and molecular composition of the microenvironment in and around the formed cell structures. We demonstrate the parallel production of one hundred synthetic 3D solid tumors comprising up to thousands of human hepatocellular carcinoma (HCC) HepG2 cells, we characterize the tumor structure by high-resolution optical microscopy, and we monitor the functional behavior of natural killer (NK) cells migrating, docking and interacting with the tumor model during culture. Our results show that the method can be used for determining the collective ability of a given number of NK cells to defeat a solid tumor having a certain size, shape and composition. The ultrasound-based method itself is generic and can meet any demand from applications where it is advantageous to monitor cell culture from production to analysis of 3D tissue or tumor models using microscopy in one single microplate device.

  • 12.
    Fogelqvist, Emelie
    et al.
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Biomedicinsk fysik och röntgenfysik.
    Selin, Mårten
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Biomedicinsk fysik och röntgenfysik.
    Martz, Dale H.
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Biomedicinsk fysik och röntgenfysik.
    Christakou, Athanasia E.
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Biomedicinsk fysik och röntgenfysik.
    Hertz, Hans M.
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Biomedicinsk fysik och röntgenfysik.
    The Stockholm laboratory cryo x-ray microscope: towards cell-cell interaction studies2013Inngår i: 11th International Conference On X-Ray Microscopy (XRM2012), Institute of Physics (IOP), 2013, s. 012054-Konferansepaper (Fagfellevurdert)
    Abstract [en]

    We describe recent improvements in the Stockholm laboratory x-ray microscope and the first experiments aiming towards studies of cell-cell interaction. The shorter exposure time due to a higher brightness laser-plasma source will become of large importance for tomography while the reproducible cryo preparation of few-cell samples is essential for the interaction studies.

  • 13. Forslund, E.
    et al.
    Guldevall, Karolin
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Cellens fysik.
    Olofsson, Per E.
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Cellens fysik.
    Frisk, Thomas
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Cellens fysik.
    Christakou, Athanasia E.
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Biomedicinsk fysik och röntgenfysik.
    Wiklund, Martin
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Biomedicinsk fysik och röntgenfysik.
    Önfelt, Björn
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Cellens fysik.
    Novel microchip-based tools facilitating live cell imaging and assessment of functional heterogeneity within NK cell populations2012Inngår i: Frontiers in Immunology, ISSN 1664-3224, E-ISSN 1664-3224, Vol. 3, nr OCT, s. 300-Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Each individual has a heterogeneous pool of NK cells consisting of cells that may be specialized towards specific functional responses such as secretion of cytokines or killing of tumor cells. Many conventional methods are not fit to characterize heterogeneous populations as they measure the average response of all cells. Thus, there is a need for experimental platforms that provide single cell resolution. In addition, there are transient and stochastic variations in functional responses at the single cell level, calling for methods that allow studies of many events over extended periods of time. This paper presents a versatile microchip platform enabling long-term microscopic studies of individual NK cells interacting with target cells. Each microchip contains an array of microwells, optimized for medium or high-resolution time-lapse imaging of single or multiple NK and target cells, or for screening of thousands of isolated NK-target cell interactions. Individual NK cells confined with target cells in small microwells is a suitable setup for high-content screening and rapid assessment of heterogeneity within populations, while microwells of larger dimensions are appropriate for studies of NK cell migration and sequential interactions with multiple target cells. By combining the chip technology with ultrasonic manipulation, NK and target cells can be forced to interact and positioned with high spatial accuracy within individual microwells.This setup effectively and synchronously creates NK-target conjugates at hundreds of parallel positions in the microchip. Thus, this facilitates assessment of temporal aspects of NK-target cell interactions, e.g., conjugation, immune synapse formation, and cytotoxic events.The microchip platform presented here can be used to effectively address questions related to fundamental functions of NK cells that can lead to better understanding of how the behavior of individual cells add up to give a functional response at the population level.

  • 14.
    Girnyk, Maksym A.
    et al.
    KTH, Skolan för elektro- och systemteknik (EES), Kommunikationsteori.
    Vehkapera, Mikko
    Rasmussen, Lars Kildehoj
    Christakou, Athanasia
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik.
    Wiklund, Martin
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Biomedicinsk fysik och röntgenfysik.
    Onfelt, Bjorn
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Cellens fysik.
    Orange, Jordan
    Lytic granule convergence is essential for NK cells to promote targeted killing while preventing collateral damage2016Inngår i: Journal of Immunology, ISSN 0022-1767, E-ISSN 1550-6606, Vol. 196Artikkel i tidsskrift (Annet vitenskapelig)
  • 15.
    Guldevall, Karolin
    et al.
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Cellens fysik.
    Frisk, Thomas
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Cellens fysik.
    Vanherberghen, Bruno
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Cellens fysik.
    Khorsidi, Mohammed Ali
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Cellens fysik.
    Manneberg, Otto
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Biomedicinsk fysik och röntgenfysik.
    Christakou, Athanasia
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Cellens fysik.
    Wiklund, Martin
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Biomedicinsk fysik och röntgenfysik.
    Önfelt, Björn
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Cellens fysik.
    Imaging immune surveillance by individual Natural Killer cells isolated in arrays of nanoliter wells2010Konferansepaper (Fagfellevurdert)
  • 16.
    Guldevall, Karolin
    et al.
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Cellens fysik.
    Vanherberghen, Bruno
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Cellens fysik.
    Frisk, Thomas
    Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet.
    Hurtig, Johan
    Department of Chemsitry, University of Washington, Seattle, USA.
    Christakou, Athanasia
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Biomedicinsk fysik och röntgenfysik.
    Manneberg, Otto
    Department of Environmental Health, Harvard School of Public Health, Boston, USA.
    Lindström, Sara
    KTH, Skolan för bioteknologi (BIO), Nanobioteknologi (stängd 20130101).
    Andersson-Svahn, Helene
    KTH, Skolan för bioteknologi (BIO), Nanobioteknologi (stängd 20130101).
    Wiklund, Martin
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Biomedicinsk fysik och röntgenfysik.
    Önfelt, Björn
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Cellens fysik.
    Imaging Immune Surveillance of Individual Natural Killer Cells Confined in Microwell Arrays2010Inngår i: PLOS ONE, ISSN 1932-6203, Vol. 5, nr 11, s. e15453-Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    New markers are constantly emerging that identify smaller and smaller subpopulations of immune cells. However, there is a growing awareness that even within very small populations, there is a marked functional heterogeneity and that measurements at the population level only gives an average estimate of the behaviour of that pool of cells. New techniques to analyze single immune cells over time are needed to overcome this limitation. For that purpose, we have designed and evaluated microwell array systems made from two materials, polydimethylsiloxane (PDMS) and silicon, for high-resolution imaging of individual natural killer (NK) cell responses. Both materials were suitable for short-term studies (<4 hours) but only silicon wells allowed long-term studies (several days). Time-lapse imaging of NK cell cytotoxicity in these microwell arrays revealed that roughly 30% of the target cells died much more rapidly than the rest upon NK cell encounter. This unexpected heterogeneity may reflect either separate mechanisms of killing or different killing efficiency by individual NK cells. Furthermore, we show that high-resolution imaging of inhibitory synapse formation, defined by clustering of MHC class I at the interface between NK and target cells, is possible in these microwells. We conclude that live cell imaging of NK-target cell interactions in multi-well microstructures are possible. The technique enables novel types of assays and allow data collection at a level of resolution not previously obtained. Furthermore, due to the large number of wells that can be simultaneously imaged, new statistical information is obtained that will lead to a better understanding of the function and regulation of the immune system at the single cell level.

  • 17.
    Hertz, Hans
    et al.
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Biomedicinsk fysik och röntgenfysik.
    von Hofsten, Olov
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Biomedicinsk fysik och röntgenfysik.
    Bertilson, Mikael
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Biomedicinsk fysik och röntgenfysik.
    Vogt, Ulrich
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Biomedicinsk fysik och röntgenfysik.
    Holmberg, Anders
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Biomedicinsk fysik och röntgenfysik.
    Reinspach, Julia Antonia
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Biomedicinsk fysik och röntgenfysik.
    Martz, Dale
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Biomedicinsk fysik och röntgenfysik.
    Selin, Mårten
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Biomedicinsk fysik och röntgenfysik.
    Christakou, Athanasia
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Biomedicinsk fysik och röntgenfysik.
    Jerlström-Hultqvist, J
    Svärd, S
    Laboratory cryo soft X-ray microscopy2012Inngår i: Journal of Structural Biology, ISSN 1047-8477, E-ISSN 1095-8657, Vol. 177, nr 2, s. 267-272Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Lens-based water-window X-ray microscopy allows two- and three-dimensional (2D and 3D) imaging of intact unstained cells in their near-native state with unprecedented contrast and resolution. Cryofixation is essential to avoid radiation damage to the sample. Present cryo X-ray microscopes rely on synchrotron radiation sources, thereby limiting the accessibility for a wider community of biologists. In the present paper we demonstrate water-window cryo X-ray microscopy with a laboratory-source-based arrangement. The microscope relies on a lambda = 2.48-nm liquid-jet high-brightness laser-plasma source, normal-incidence multilayer condenser optics, 30-nm zone-plate optics, and a cryo sample chamber. We demonstrate 2D imaging of test patterns, and intact unstained yeast, protozoan parasites and mammalian cells. Overview 3D information is obtained by stereo imaging while complete 3D microscopy is provided by full tomographic reconstruction. The laboratory microscope image quality approaches that of the synchrotron microscopes, but with longer exposure times. The experimental image quality is analyzed from a numerical wave-propagation model of the imaging system and a path to reach synchrotron-like exposure times in laboratory microscopy is outlined.

  • 18. Hsu, Hsiang-Ting
    et al.
    Mace, Emily M.
    Carisey, Alexandre F.
    Viswanath, Dixita I.
    Christakou, Athanasia
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Biomedicinsk fysik och röntgenfysik.
    Wiklund, Martin
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Biomedicinsk fysik och röntgenfysik.
    Önfelt, Bjorn
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik.
    Orange, Jordan S.
    NK cells converge lytic granules to promote cytotoxicity and prevent bystander killing2016Inngår i: Journal of Cell Biology, ISSN 0021-9525, E-ISSN 1540-8140, Vol. 215, nr 6, s. 875-889Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Natural killer (NK) cell activation triggers sequential cellular events leading to destruction of diseased cells. We previously identified lytic granule convergence, a dynein-and integrin signal-dependent movement of lysosome-related organelles to the microtubule-organizing center, as an early step in the cell biological process underlying NK cell cytotoxicity. Why lytic granules converge during NK cell cytotoxicity, however, remains unclear. We experimentally controlled the availability of human ligands to regulate NK cell signaling and promote granule convergence with either directed or nondirected degranulation. By the use of acoustic trap microscopy, we generated specific effector-target cell arrangements to define the impact of the two modes of degranulation. NK cells with converged granules had greater targeted and less nonspecific "bystander" killing. Additionally, NK cells in which dynein was inhibited or integrin blocked under physiological conditions demonstrated increased nondirected degranulation and bystander killing. Thus, NK cells converge lytic granules and thereby improve the efficiency of targeted killing and prevent collateral damage to neighboring healthy cells.

  • 19.
    Ohlin, Mathias
    et al.
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik.
    Christakou, Athanasia E.
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik.
    Frisk, Thomas
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik.
    Önfelt, Björn
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik.
    Wiklund, Martin
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik.
    Analysis of trapping and streaming in an ultrasoundactuated multi-well microplate for single-cell studies2012Inngår i: Proceedings of the 16th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2012, Chemical and Biological Microsystems Society , 2012, s. 497-499Konferansepaper (Fagfellevurdert)
    Abstract [en]

    The dynamics of the acoustic streaming and the acoustic positioning performance in an ultrasound-actuated multiwell microplate are investigated by two different ultrasonic frequency actuation schemes: Frequency-modulation and single- frequency actuation. Our results show a significant decrease in size of the field of view when using frequencymodulation compared to single-frequency actuation, which can be used for improving the scanning time for 3D highresolution confocal microscopy by almost one order of magnitude. Furthermore, in the ultrasound-actuated multi-well microplate the high-voltage acoustic streaming show a complex time and temperature dependence and could gain stability by the use of temperature control.

  • 20.
    Ohlin, Mathias
    et al.
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik.
    Christakou, Athanasia E.
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik.
    Frisk, Thomas
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik.
    Önfelt, Björn
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik.
    Wiklund, Martin
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik.
    Controlling acoustic streaming in a multi-well microplate for improving live cell assays2011Inngår i: 15th International Conference on Miniaturized Systems for Chemistry and Life Sciences 2011, MicroTAS 2011, 2011, s. 1612-1614Konferansepaper (Fagfellevurdert)
    Abstract [en]

    Acoustic streaming in a multi-well microplate is investigated using two different ultrasonic actuation frequency-schemes: Single-frequency and frequency-modulation. The streaming is tracked by the use of 1 μm fluorescent polymer beads and micro-particle image velocimetry. The suspension also contained human B cells for studying the acoustic trapping and aggregation performance simultaneously with the acoustic streaming. Our results show a significant difference in the acoustic streaming between the two ultrasonic actuation schemes. A rotational fluid flow speed decreased a factor of 30 when frequency-modulation was applied compared to single-frequency actuation without apparently interfering with the acoustic cell trapping function.

  • 21.
    Ohlin, Mathias
    et al.
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Biomedicinsk fysik och röntgenfysik.
    Christakou, Athanasia E.
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Biomedicinsk fysik och röntgenfysik.
    Frisk, Thomas
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Cellens fysik.
    Önfelt, Björn
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Cellens fysik.
    Wiklund, Martin
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Biomedicinsk fysik och röntgenfysik.
    Influence of acoustic streaming on ultrasonic particle manipulation in a 100-well ring-transducer microplate2013Inngår i: Journal of Micromechanics and Microengineering, ISSN 0960-1317, E-ISSN 1361-6439, Vol. 23, nr 3, s. 035008-Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    We characterize and quantify the performance of ultrasonic particle aggregation and positioning in a 100-well microplate. We analyze the result when operating a planar ultrasonic ring transducer at different single actuation frequencies in the range 2.20-2.40 MHz, and compare with the result obtained from different schemes of frequency-modulated actuation. Compared to our previously used wedge transducer design, the ring transducer has a larger contact area facing the microplate, resulting in lower temperature increase for a given actuation voltage. Furthermore, we analyze the dynamics of acoustic streaming occurring simultaneously with the particle trapping in the wells of the microplate, and we define an adaptive ultrasonic actuation scheme for optimizing both efficiency and robustness of the method. The device is designed as a tool for ultrasound-mediated cell aggregation and positioning. This is a method for high-resolution optical characterization of time-dependent cellular processes at the level of single cells. In this paper, we demonstrate how to operate our device in order to optimize the scanning time of 3D confocal microscopy with the aim to perform high-resolution time-lapse imaging of cells or cell-cell interactions in a highly parallel manner.

  • 22.
    Ohlin, Mathias
    et al.
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Biomedicinsk fysik och röntgenfysik.
    Iranmanesh, Ida Sadat
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Biomedicinsk fysik och röntgenfysik.
    Christakou, Athanasia E.
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Biomedicinsk fysik och röntgenfysik.
    Wiklund, Martin
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Biomedicinsk fysik och röntgenfysik.
    Temperature-controlled MPa-pressure ultrasonic cell manipulation in a microfluidic chip2015Inngår i: Lab on a Chip, ISSN 1473-0197, E-ISSN 1473-0189, Vol. 15, nr 16, s. 3341-3349Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    We study the temperature-independent impact on cell viability of relevant physical parameters during long-term, high-acoustic-pressure ultrasonic exposure in a microfluidic chip designed for ultrasonic-standing-wave trapping and aggregation of cells. We use a light-intensity method and 5 mum polymer beads for accurate acoustic pressure calibration before injecting cells into the device, and we monitor the viability of A549 lung cancer cells trapped during one hour in an ultrasonic standing wave with 1 MPa pressure amplitude. The microfluidic chip is actuated by a novel temperature-controlled ultrasonic transducer capable of keeping the temperature stable around 37 °C with an accuracy better than ±0.2 °C, independently on the ultrasonic power and heat produced by the system, thereby decoupling any temperature effect from other relevant effects on cells caused by the high-pressure acoustic field. We demonstrate that frequency-modulated ultrasonic actuation can produce acoustic pressures of equally high magnitudes as with single-frequency actuation, and we show that A549 lung cancer cells can be exposed to 1 MPa standing-wave acoustic pressure amplitudes for one hour without compromising cell viability. At this pressure level, we also measure the acoustic streaming induced around the trapped cell aggregate, and conclude that cell viability is not affected by streaming velocities of the order of 100 mum s(-1). Our results are important when implementing acoustophoresis methods in various clinical and biomedical applications.

  • 23.
    Vanherberghen, Bruno
    et al.
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Cellens fysik.
    Manneberg, Otto
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Biomedicinsk fysik och röntgenfysik.
    Christakou, Athanasia
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Biomedicinsk fysik och röntgenfysik.
    Frisk, Thomas
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Cellens fysik.
    Ohlin, Mathias
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Biomedicinsk fysik och röntgenfysik.
    Hertz, Hans M.
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Biomedicinsk fysik och röntgenfysik.
    Önfelt, Björn
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Cellens fysik.
    Wiklund, Martin
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Biomedicinsk fysik och röntgenfysik.
    Ultrasound-controlled cell aggregation in a multi-well chip2010Inngår i: Lab on a Chip, ISSN 1473-0197, E-ISSN 1473-0189, Vol. 10, nr 20, s. 2727-2732Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    We demonstrate a microplate platform for parallelized manipulation of particles or cells by frequency-modulated ultrasound. The device, consisting of a silicon-glass microchip and a single ultrasonic transducer, enables aggregation, positioning and high-resolution microscopy of cells distributed in an array of 100 microwells centered on the microchip. We characterize the system in terms of temperature control, aggregation and positioning efficiency, and cell viability. We use time-lapse imaging to show that cells continuously exposed to ultrasound are able to divide and remain viable for at least 12 hours inside the device. Thus, the device can be used to induce and maintain aggregation in a parallelized fashion, facilitating long-term microscopy studies of, e.g., cell-cell interactions.

  • 24.
    Vanherberghen, Bruno
    et al.
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Cellens fysik.
    Manneberg, Otto
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Biomedicinsk fysik och röntgenfysik.
    Christakou, Athanasia
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Cellens fysik.
    Frisk, Thomas
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Cellens fysik.
    Önfelt, Björn
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Cellens fysik.
    Wiklund, Martin
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Biomedicinsk fysik och röntgenfysik.
    Highly parallelized cell aggregation by ultrasound for studies of immune cell interaction2009Konferansepaper (Annet vitenskapelig)
  • 25.
    Wiklund, Martin
    et al.
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Biomedicinsk fysik och röntgenfysik.
    Christakou, Athanasia E.
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Biomedicinsk fysik och röntgenfysik.
    Iranmanesh, Ida
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Biomedicinsk fysik och röntgenfysik.
    Ohlin, Mathias
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Biomedicinsk fysik och röntgenfysik.
    Russom, Aman
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Önfelt, Björn
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Cellens fysik.
    On-chip acoustic sample preparation for cell studies and diagnostics2013Inngår i: Proceedings of Meetings on Acoustics: Volume 19, 2013, Acoustical Society of America (ASA), 2013, s. 1-3Konferansepaper (Fagfellevurdert)
    Abstract [en]

    We describe a novel platform for acoustic sample preparation in microchannels and microplates. The utilized method is based on generating a multitude of acoustic resonances at a set of different frequencies in microstructures, in order to accurately control the migration and positioning of particles and cells suspended in fluid channels and chambers. The actuation frequencies range from 30 kHz to 7 MHz, which are applied simultaneously and/or in sweeps. We present two devices: A closed microfluidic chip designed for pre-alignment, size-based separation, isolation, up-concentration and lysis of cells, and an open multi-well microplate designed for parallel aggregation and positioning of cells. Both devices in the platform are compatible with high-resolution live-cell microscopy, which is used for fluorescence-based optical characterization. Two bioapplications are demonstrated for each of the devices: The first device is used for size-selective cell isolation and lysis for DNA-based diagnostics, and the second device is used for quantifying the heterogeneity in cytotoxic response of natural killer cells interacting with cancer cells.

  • 26.
    Wiklund, Martin
    et al.
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Biomedicinsk fysik och röntgenfysik.
    Christakou, Athanasia E.
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Biomedicinsk fysik och röntgenfysik.
    Ohlin, Mathias
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Biomedicinsk fysik och röntgenfysik.
    Iranmanesh, Ida
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Biomedicinsk fysik och röntgenfysik.
    Frisk, Thomas
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Cellens fysik.
    Vanherberghen, Bruno
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik.
    Önfelt, Björn
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Cellens fysik.
    Ultrasound-Induced Cell-Cell Interaction Studies in a Multi-Well Microplate2014Inngår i: Micromachines, ISSN 2072-666X, E-ISSN 2072-666X, Vol. 5, nr 1, s. 27-49Artikkel, forskningsoversikt (Fagfellevurdert)
    Abstract [en]

    This review describes the use of ultrasound for inducing and retaining cell-cell contact in multi-well microplates combined with live-cell fluorescence microscopy. This platform has been used for studying the interaction between natural killer (NK) cells and cancer cells at the level of individual cells. The review includes basic principles of ultrasonic particle manipulation, design criteria when building a multi-well microplate device for this purpose, biocompatibility aspects, and finally, two examples of biological applications: Dynamic imaging of the inhibitory immune synapse, and studies of the heterogeneity in killing dynamics of NK cells interacting with cancer cells.

1 - 26 of 26
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