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  • 1. Berglund, S.
    et al.
    Magalhaes, I.
    Gaballa, A.
    Vanherberghen, Bruno
    KTH, School of Engineering Sciences (SCI), Applied Physics.
    Uhlin, Michael
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cellular Biophysics. Karolinska Institutet, Sweden.
    Advances in umbilical cord blood cell therapy: the present and the future2017In: Expert Opinion on Biological Therapy, ISSN 1471-2598, E-ISSN 1744-7682, Vol. 17, no 6, p. 691-699Article, review/survey (Refereed)
    Abstract [en]

    Introduction: Umbilical cord blood (UCB), previously seen as medical waste, is increasingly recognized as a valuable source of cells for therapeutic use. The best-known application is in hematopoietic stem cell transplantation (HSCT), where UCB has become an increasingly important graft source in the 28 years since the first umbilical cord blood transplantation (UCBT) was performed. Recently, UCB has been increasingly investigated as a putative source for adoptive cell therapy. Areas covered: This review covers the advances in umbilical cord blood transplantation (UCBT) to overcome the limitation regarding cellular dose, immunological naivety and additional cell doses such as DLI. It also provides an overview regarding the progress in adoptive cellular therapy using UCB. Expert opinion: UCB has been established as an important source of stem cells for HSCT. Successful strategies to overcome the limitations of UCBT, such as the limited cell numbers and naivety of the cells, are being developed, including novel methods to perform in vitro expansion of progenitor cells, and to improve their homing to the bone marrow. Promising early clinical trials of adoptive therapies with UCB cells, including non-immunological cells, are currently performed for viral infections, malignant diseases and in regenerative medicine.

  • 2.
    Blom, Hans
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cellular Biophysics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Widengren, Jerker
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Stimulated Emission Depletion Microscopy2017In: Chemical Reviews, ISSN 0009-2665, E-ISSN 1520-6890, Vol. 117, no 11, p. 7377-7427Article, review/survey (Refereed)
    Abstract [en]

    Despite its short history, diffraction-unlimited fluorescence microscopy techniques have already made a substantial imprint in the biological sciences. In this review, we describe how stimulated emission depletion (STED) imaging originally evolved, how it compares to other optical super-resolution imaging techniques, and what advantages it provides compared to previous golden-standards for biological microscopy, such as diffraction-limited optical microscopy and electron microscopy. We outline the prerequisites for successful STED imaging experiments, emphasizing the equally critical roles of instrumentation, sample preparation, and photophysics, and describe major evolving strategies for how to push the borders of STED imaging even further in life science. Finally, we provide examples of how STED nanoscopy can be applied, within three different fields with particular potential for STED imaging experiments: neuroscience, plasma membrane biophysics, and subcellular clinical diagnostics. In these areas, and in many more, STED imaging can be expected to play an increasingly important role in the future.

  • 3.
    Fogelqvist, Emelie
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics.
    Kördel, Mikael
    KTH, School of Engineering Sciences (SCI), Applied Physics, Biomedical and X-ray Physics.
    Carannante, Valentina
    Önfelt, Björn
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cellular Biophysics.
    Hertz, Hans
    KTH, School of Engineering Sciences (SCI), Applied Physics, Biomedical and X-ray Physics. Karolinska Institutet, Sweden.
    Laboratory cryo x-ray microscopy for 3D cell imaging2017In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 7, article id 13433Article in journal (Refereed)
    Abstract [en]

    Water-window x-ray microscopy allows two-and three-dimensional (2D and 3D) imaging of intact unstained cells in their cryofixed near-native state with unique contrast and high resolution. Present operational biological water-window microscopes are based at synchrotron facilities, which limits their accessibility and integration with complementary methods. Laboratory-source microscopes have had difficulty addressing relevant biological tasks with proper resolution and contrast due to long exposure times and limited up-time. Here we report on laboratory cryo x-ray microscopy with the exposure time, contrast, and reliability to allow for routine high-spatial resolution 3D imaging of intact cells and cell-cell interactions. Stabilization of the laser-plasma source combined with new optics and sample preparation provide high-resolution cell imaging, both in 2D with ten-second exposures and in 3D with twenty-minute tomography. Examples include monitoring of the distribution of carbon-dense vesicles in starving HEK293T cells and imaging the interaction between natural killer cells and target cells.

  • 4.
    Fontana, Jacopo
    et al.
    Karolinska Institutet.
    Khodus, Georgiy
    Uppsala universitet.
    Unnersjö-Jess, David
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cellular Biophysics.
    Blom, Hans
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cellular Biophysics.
    Brismar, Hjalmar
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cellular Biophysics.
    Aperia, Anita
    Karolinska Institutet.
    Temporal calcium activity in metanephric mesenchyme cells regulates kidney branching morphogenesisManuscript (preprint) (Other academic)
    Abstract [en]

    The role of calcium signaling for development of early vertebrates is well documented, but little is known about its role in mammalian embryogenesis. We have used explanted embryonic rat kidneys to study the role of calcium for branching morphogenesis, a process that depends on reciprocal interaction between mesenchymal and epithelial ureteric bud cells. We recorded a spontaneous calcium activity characterized by stochastic and irregular calcium spikes, in the mesenchymal cells. This activity is due to calcium release from the endoplasmic reticulum (ER). Depletion of ER calcium stores results in down-regulation of the calcium activity, retardation of branching morphogenesis and formation of primitive nephrons, but has no effect on cell proliferation. We propose that the excretion of morphogenic factors that mediate the interaction between 26 the mesenchymal and epithelial cells, which initiate branching morphogenesis, is calcium dependent. In support of this we demonstrate expression of the calcium dependent excretory protein synaptotagmin1.

  • 5.
    Fontana, Jacopo Maria
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cellular Biophysics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Yin, Huijuan
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cellular Biophysics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Chen, Yun
    Florez, Ricardo
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cellular Biophysics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Brismar, Hjalmar
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cellular Biophysics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Fu, Ying
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cellular Biophysics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Transport and release of colloidal 3-mercaptopropionic acid-coated CdSe-CdS/ZnS core-multishell quantum dots in human umbilical vein endothelial cells2017In: International Journal of Nanomedicine, ISSN 1176-9114, E-ISSN 1178-2013, Vol. 12, p. 8615-8629Article in journal (Refereed)
    Abstract [en]

    Colloidal semiconductor quantum dots (QDs) have been extensively researched and developed for biomedical applications, including drug delivery and biosensing assays. Hence, it is pivotal to understand their behavior in terms of intracellular transport and toxicological effects. In this study, we focused on 3-mercaptopropionic acid-coated CdSe-CdS/ZnS core-multishell quantum dots (3MPA-QDs) converted from the as-grown octadecylamine-coated quantum dots (ODA-QDs) and their direct and dynamic interactions with human umbilical vein endothelial cells (HUVECs). Live cell imaging using confocal fluorescence microscopy showed that 3MPAQDs first attached to and subsequently aggregated on HUVEC plasma membrane similar to 25 min after QD deposition. The aggregated QDs started being internalized at similar to 2 h and reached their highest internalization degree at similar to 24 h. They were released from HUVECs after similar to 48 h. During the 48 h period, the HUVECs responded normally to external stimulations, grew, proliferated and wound healed without any perceptible apoptosis. Furthermore, 1) 3MPA-QDs were internalized in newly formed LysoTracker-stained early endosomes; 2) adenosine 5'-triphosphateinduced [Ca2+](i) modulation caused a transient decrease in the fluorescence of 3MPA-QDs that were attached to the plasma membrane but a transient increase in the internalized 3MPA-QDs; and 3) fluorescence signal modulations of co-stained LysoTracker and QDs induced by the lysosomotropic agent Gly-Phe-beta-naphthylamide were spatially co-localized and temporally synchronized. Our findings suggest that 3MPA-QDs converted from ODA-QDs are a potential nontoxic fluorescent probe for future use in clinical applications. Moreover, the photophysical strategy and techniques reported in this work are easily applicable to study of direct interactions between other nanoparticles and live cells; contributing to awareness and implementation of the safe applications of nanoparticles.

  • 6. Gan, Zhixing
    et al.
    Xu, Hao
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cellular Biophysics. KTH, Centres, Science for Life Laboratory, SciLifeLab. Ningbo University, China.
    Photoluminescence of Diphenylalanine Peptide Nano/Microstructures: From Mechanisms to Applications2017In: Macromolecular rapid communications, ISSN 1022-1336, E-ISSN 1521-3927, Vol. 38, no 22, article id 1700370Article, review/survey (Refereed)
    Abstract [en]

    Diphenylalanine (Phe-Phe, FF) molecules, which can self-assemble into highly ordered nano/microstructures, have increasingly aroused intense interests due to their special optical properties. In this review, recent advances in photoluminescence (PL) of supramolecular architectures of FF-based peptide and the underlying mechanisms are highlighted. Mainly deep ultraviolet emission at around 285 nm and/or blue emission at approximate to 450 nm are observed in various FF peptide structures and its derivatives, which are primarily interpreted by quantum confinement effects, shallow radiative traps, and electron delocalization via hydrogen bonds in beta-sheet structures. Furthermore, current applications of such fluorescent peptide nano/microstructures are also reviewed here, e.g., probing the number of water molecules confined in FF, temperature sensing, and visualization of deep ultraviolet beam. Yet, the PL mechanism is still under fierce debate and the application based on fluorescence is constantly under exploration. Thus, this review is endeavored to boost future explorations on the PL of the bioinspired FF peptide nano/microstructures.

  • 7. Kaucka, Marketa
    et al.
    Zikmund, Tomas
    Tesarova, Marketa
    Gyllborg, Daniel
    Hellander, Andreas
    Jaros, Josef
    Kaiser, Jozef
    Petersen, Julian
    Szarowska, Bara
    Newton, Phillip T.
    Dyachuk, Vyacheslav
    li, Lei
    Qian, Hong
    Johansson, Anne-Sofie
    Mishina, Yuji
    Currie, Joshua D.
    Tanaka, Elly M.
    Erickson, Alek
    Dudley, Andrew
    Brismar, Hjalmar
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cellular Biophysics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Southam, Paul
    Coen, Enrico
    Chen, Min
    Weinstein, Lee S.
    Hampl, Ales
    Arenas, Ernest
    Chagin, Andrei S.
    Fried, Kaj
    Adameyko, Igor
    Oriented clonal cell dynamics enables accurate growth and shaping of vertebrate cartilage2017In: eLIFE, E-ISSN 2050-084X, Vol. 6, article id e25902Article in journal (Refereed)
    Abstract [en]

    Cartilaginous structures are at the core of embryo growth and shaping before the bone forms. Here we report a novel principle of vertebrate cartilage growth that is based on introducing transversally-oriented clones into pre-existing cartilage. This mechanism of growth uncouples the lateral expansion of curved cartilaginous sheets from the control of cartilage thickness, a process which might be the evolutionary mechanism underlying adaptations of facial shape. In rod-shaped cartilage structures (Meckel, ribs and skeletal elements in developing limbs), the transverse integration of clonal columns determines the well-defined diameter and resulting rod-like morphology. We were able to alter cartilage shape by experimentally manipulating clonal geometries. Using in silico modeling, we discovered that anisotropic proliferation might explain cartilage bending and groove formation at the macro-scale.

  • 8.
    Krishnan, Kalaiselvan
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics. KTH.
    Fritz, Nicolas
    Karolinska Institutet.
    Huličiak, Miroslav
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cellular Biophysics.
    Brismar, Hjalmar
    KTH, School of Engineering Sciences (SCI), Applied Physics.
    Study of energy consumption by Na+/K+ ATPase using PercevalHRManuscript (preprint) (Other academic)
    Abstract [en]

    Adenosine triphosphate (ATP) is an important macro molecule that is the prime supplier of energy to run cell metabolism and to regulate several physiological processes in a cell. Despite its central role, the number of non-invasive methods to study ATP on a single cell level in real time are limited. ATPases use energy derived from ATP hydrolysis to maintain cell membrane potential by regulating ion gradients across the plasma membrane. It is generally believed that the Na+/K+-ATPase (NKA) which belongs to the P-type ATPase superfamily represent the main energy consumer among the ATPases. In this study, we set out to quantify ATP consumption by NKA on a single cell level in human embryonic kidney cells (HEK293a) using PercevalHR, a genetically encoded fluorescent biosensor that reports changes in the ATP:ADP during live cell imaging. We demonstrate that ATP hydrolysis by NKA is faster at physiological temperatures (35 -37°C) compared to room temperature. K+ free KREBS pre-treatment increased the ATP consumption by NKA. The inhibition of NKA and SERCA reduced the overall ATP hydrolysis in HEK293a cells demonstrating that these ATPases consume a substantial amount of all ATP produced in the cell. We found that the inhibition of mitochondrial respiration reduced basal ATP:ADP in rat primary proximal tubule cells (PTC) but not in HEK293a cells. In contrast, an inhibition of glycolysis gave a more rapid reduction in basal ATP:ADP in HEK293a compared to PTC, illustrating the Warburg effect in cell lines, where the metabolism has been adapted to a high glucose and low oxygen environment. We demonstrate the use of PercevalHR as a robust tool for studies of dynamic energy consumption by NKA in both cell lines and primary cells. 

  • 9. Kvedaraite, Egle
    et al.
    Jess, David Unnersjö
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cellular Biophysics.
    Gavhed, Desiree
    Idestrom, Maja
    Svensson, Mattias
    Lourda, Magda
    Henter, Jan-Inge
    When Langerhans Met Crohn: A Clinical Case Report and a First Three-Dimentional Reconstruction of Human Gut2017In: Pediatric Blood & Cancer, ISSN 1545-5009, E-ISSN 1545-5017, Vol. 64, p. S20-S20Article in journal (Refereed)
  • 10. Nilsson, J.
    et al.
    Granrot, I.
    Mattsson, J.
    Omazic, B.
    Uhlin, Michael
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cellular Biophysics.
    Thunberg, Sarah
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cellular Biophysics.
    Functionality testing of stem cell grafts to predict infectious complications after allogeneic hematopoietic stem cell transplantation2017In: Vox Sanguinis, ISSN 0042-9007, E-ISSN 1423-0410, Vol. 112, no 5, p. 459-468Article in journal (Refereed)
    Abstract [en]

    Background and Objectives Allogeneic hematopoietic stem cell transplantation (HSCT) is a routine clinical procedure performed to treat patients with haematological malignancies, primary immune deficiencies or metabolic disorders. Infections during lymphopenia after allogeneic HSCT are associated with high mortality and morbidity. Typical infectious agents are Epstein-Barr virus, cytomegalovirus, herpes simplex virus, varicella-zoster virus and fungi. The study aim was to evaluate whether measurement of the responses of antigen-specific T-cells, recognizing infectious pathogens would correlate to protective functions in the stem cell recipient post-transplant. Materials and MethodsTwenty-one grafts were analysed by flow cytometry and cells were stimulated in vitro with relevant infectious antigens, followed by evaluation of T-cell proliferation and cytokine production. Results were compared to the recipients' clinical records 1-year post-transplantation. ResultsWe show that an extensive repertoire of transferred antigen-specific T-cells from allogeneic donor grafts against infectious agents, involved in post-transplant infections, are linked to an absence of infectious complications for the recipient up-to 1-year post-transplant. The protective effect was associated with antigen-specific T-cell proliferation and IL-1 secretion. Conclusion Our results suggest that assaying T-cell function before HSCT could determine individual risks for infectious complications and thus aid in clinical decision-making regarding prophylactic and pre-emptive anti-infective therapy.

  • 11.
    Olofsson, K.
    et al.
    KTH.
    Carannante, V.
    Frisk, T.
    KTH.
    Kushiro, K.
    Takai, M.
    Önfelt, B.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cellular Biophysics.
    Wiklund, Martin
    KTH, School of Engineering Sciences (SCI), Applied Physics, Biomedical and X-ray Physics.
    Unanchored micro-tumors in an ultrasonic actuated multi-well microplate with protein repellent coating2016In: 20th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2016, Chemical and Biological Microsystems Society , 2016, p. 409-410Conference paper (Refereed)
    Abstract [en]

    In this paper we demonstrate an improved tissue engineering method producing 100 three-dimensional (3D) HepG2 cell structures in parallel based on a combination of ultrasonic actuation and polymer coating in a multi-well microplate. By the use of a polymer coating in the plates, the method creates non-adherent tumor models of controlled size and shape which introduces both a more flexible 3D culture system as well as improved quality of the 3D tumor relative to previous studies [1].

  • 12.
    Olofsson, Per Erik
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cellular Biophysics.
    Microscopy-based single-cell in vitro assays for NK cell function in 2-D and 3-D2017Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Natural killer (NK) cells are effector cells of the innate immune system that are responsible for mediating cellular cytotoxicity against virally infected or neoplastically transformed cells. NK cell subsets are defined by their expression of certain cell-surface markers, and are usually related to activation and developmental status.

    However, how distinct NK cell phenotypes correlate with behavior in NK-target interactions is less widely characterized. There is therefore a need to study NK cell behavior down at the single-cell level. One aim of this thesis is to approach methods that quantitatively describe these single-cell-level behavioral differences of NK cells.

    Additionally, the ability of NK cells to migrate through the extracellular matrix (ECM) microenvironment is crucial for NK cell trafficking and immune surveillance. Traditional imaging studies of NK cell migration and cytotoxicity do not properly reproduce the structural and mechanical cues that shape the migratory response of NK cells in vivo.

    Therefore, it is desirable to implement 3-D in vitro migration and killing assays that better mimic in vivo conditions. Another aim of this thesis is to develop a microwell-based assay for 3-D time-lapse imaging of NK cell migration and cytotoxicity.

    Using a newly developed single-cell imaging and screening assay, we trap small populations of NK and target cells inside microwells, where they are imaged over extended periods of time. We have performed experiments on resting, IL-2-activated, educated, and non-educated NK cells and quantified their migration behavior and cytotoxicity. One major discovery was that a small population of NK cells mediate a majority of the cytotoxicity directed against target cells. A particularly cytotoxic group of cells, termed serial killers, displayed faster and more effective cytotoxicity. Serial killers were more prevalent in IL-2-activated and educated NK cells, but were also present in a small fraction of resting and non-educated NK cells. IL-2-activated and educated NK cells displayed more dynamic migration behavior than resting and non-educated NK cells. Additionally, IL-2-activated and educated NK cells spent more time in NK–target cell conjugates and post-conjugation attachment than resting and non-educated NK cells.

    To more closely approximate in vivo conditions, we have combined our microwell assay with an interstitial ECM-like matrix. The microwells allow for long-term imaging of NK–target cell interactions within a confined 3-D volume. NK cells were tracked and interactions with target cells were scored for duration and outcome. The developed microwell-based assay is suitable for 3-D time-lapse imaging of NK cell migration and cytotoxicity. As it allows for experiments with human cells, it could be used as a complement to in vivo imaging.

    We have quantified NK cell behavioral heterogeneity and developed tools that can be used to further study and elucidate differences in the behavior of single immune cells. These tools advance current methods for single-cell analysis, which will likely play an even more important role in the study of immune responses in the future.

  • 13.
    Olofsson, Per
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cellular Biophysics.
    Magnusson, Klas E. G.
    KTH, School of Electrical Engineering (EES), Signal Processing.
    Frisk, Thomas
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cellular Biophysics.
    Jaldén, Joakim
    KTH, School of Electrical Engineering (EES), Signal Processing.
    Önfelt, Björn
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cellular Biophysics.
    A collagen-based microwell migration assay to study NK—target cell interactionsManuscript (preprint) (Other academic)
    Abstract [en]

    Natural killer (NK) cell cytotoxicity is highly dependent on the ability of NK cells to migrate through the extracellular matrix (ECM) microenvironment. Traditional imaging studies of NK cell migration and cytotoxicity have utilized 2-D surfaces, which do not properly reproduce the structural and mechanical cues that shape the migratory response of NK cells in vivo. In addition, current in vivo imaging does not allow for the accurate long-term single-cell imaging required to dissect the functional heterogeneity of NK cell populations, and importantly, it does not allow studies of human cells. Therefore, it is desirable to implement in vitro migration and killing assays that better mimic in vivo conditions.

    We have combined a microwell assay that allows long-term imaging and tracking of small, well-defined populations of NK cells with an interstitial ECM-like matrix to more closely approximate in vivo conditions. The microwells, which are loaded with a gel mixture containing NK and target cells, allows for long-term imaging of NK–target cell interactions within a confined 3-D volume. The microwells were optically sectioned by confocal fluorescence microscopy once every 2 min for 12 h. NK cells were tracked by the Baxter Algorithms to assess motility parameters and interactions with target cells were manually scored for duration and outcome.

    We found marked differences in motility between individual cells with a significant fraction of the cells moving slowly and being confined to a small area within the matrix, while other cells moved more freely, probably reflecting local variations in the matrix structure and inherent difference in motility between individual cells. A majority of NK cells also exhibited transient variation in their mobility alternating between periods of migration arrest and random movement. NK cells that alternated between different modes of migration switched on average once every 3 h.

    NK cells made fewer and shorter contacts with target cells than in comparable 2-D assays. The difference was particularly pronounced for the process of post-conjugation attachment when NK and target cells separate. The timing of this process is likely influenced by a biomechanical component only present in 3-D environments where the cells are offered multiple anchor points with the matrix that can be used to generate the forces needed to pull apart.

    The developed microwell-based assay is suitable for 3-D time-lapse imaging of NK cells migration and cytotoxicity. As it allows for experiments with human cells, it could be used as a complement to in vivo imaging to study the influence of e.g. education and cytokine activation on NK cell heterogeneity in migration and cytotoxicity.

  • 14.
    Reuss, Matthias
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Förds, F.
    Blom, Hans
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cellular Biophysics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Öktem, Ozan
    KTH, School of Engineering Sciences (SCI), Mathematics (Dept.), Mathematics (Div.).
    Högberg, B.
    Brismar, Hans
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cellular Biophysics. KTH, Centres, Science for Life Laboratory, SciLifeLab. Karolinska Institutet, Sweden.
    Measuring true localization accuracy in super resolution microscopy with DNA-origami nanostructures2017In: New Journal of Physics, ISSN 1367-2630, E-ISSN 1367-2630, Vol. 19, no 2, article id 025013Article in journal (Refereed)
    Abstract [en]

    A common method to assess the performance of (super resolution) microscopes is to use the localization precision of emitters as an estimate for the achieved resolution. Naturally, this is widely used in super resolution methods based on single molecule stochastic switching. This concept suffers from the fact that it is hard to calibrate measures against a real sample (a phantom), because true absolute positions of emitters are almost always unknown. For this reason, resolution estimates are potentially biased in an image since one is blind to true position accuracy, i.e. deviation in position measurement from true positions. We have solved this issue by imaging nanorods fabricated with DNA-origami. The nanorods used are designed to have emitters attached at each end in a well-defined and highly conserved distance. These structures are widely used to gauge localization precision. Here, we additionally determined the true achievable localization accuracy and compared this figure of merit to localization precision values for two common super resolution microscope methods STED and STORM.

  • 15. Rådestad, E.
    et al.
    Egevad, L.
    Jorns, C.
    Mattsson, J.
    Sundberg, B.
    Nava, S.
    Ericzon, B. -G
    Henningsohn, L.
    Levitsky, V.
    Uhlin, Michael
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cellular Biophysics. Karolinska Institutet, Sweden.
    Characterization of infiltrating lymphocytes in human benign and malignant prostate tissue2017In: OncoTarget, ISSN 1949-2553, E-ISSN 1949-2553, Vol. 8, no 36, p. 60257-60269Article in journal (Refereed)
    Abstract [en]

    Immune checkpoint blockade has shown promising results in numerous cancer types. However, in prostate cancer (PC), absent or limited responses have been reported. To investigate further, we compared the phenotype of infiltrating T-cells isolated from prostate tissue from patients with PC (n = 5), benign prostatic hyperplasia (BPH) (n = 27), BPH with concurrent PC (n = 4) and controls (n = 7). The majority of T-cells were CD8+ and had a CCR7-CD45RO+ effector memory phenotype. However, the yield of T-cells isolated from PC lesions was on average 20-fold higher than that obtained from control prostates. Furthermore, there were differences between the prostate conditions regarding the percentage of T-cells expressing several activation markers and co-inhibitory receptors. In conclusion, many prostateinfiltrating T-cells express co-inhibitory receptors PD-1 and LAG-3, regardless of prostate condition. Despite the observed increase in counts and percentages of PD- 1+ T-cells in PC, the concomitant demonstration of high percentage of PD-1+ T-cells in control prostates suggests that PD-1 may play a role in controlling the homeostasis of the prostate rather than in contributing to PC-associated immune-suppression. Thus, PD-1 may not be a good candidate for checkpoint blockade in PC and these data are relevant for evaluation of clinical trials and in designing future immunotherapeutic approaches of PC.

  • 16. Stikvoort, Arwen
    et al.
    Chen, Yang
    Radestad, Emelie
    Torlen, Johan
    Lakshmikanth, Tadepally
    Bjorklund, Andreas
    Mikes, Jaromir
    Achour, Adnane
    Gertow, Jens
    Sundberg, Berit
    Remberger, Mats
    Sundin, Mikael
    Mattsson, Jonas
    Brodin, Petter
    Uhlin, Michael
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cellular Biophysics. Karolinska Institute, Sweden; Karolinska University Hospital, Sweden.
    Combining Flow and Mass Cytometry in the Search for Biomarkers in Chronic Graft-versus-Host Disease2017In: Frontiers in Immunology, ISSN 1664-3224, E-ISSN 1664-3224, Vol. 8, article id 717Article in journal (Refereed)
    Abstract [en]

    Chronic graft-versus-host disease (cGVHD) is a debilitating complication arising in around half of all patients treated with an allogeneic hematopoietic stem cell transplantation. Even though treatment of severe cGVHD has improved during recent years, it remains one of the main causes of morbidity and mortality in affected patients. Biomarkers in blood that could aid in the diagnosis and classification of cGVHD severity are needed for the development of novel treatment strategies that can alleviate symptoms and reduce the need for painful and sometimes complicated tissue biopsies. Methods that comprehensively profile complex biological systems such as the immune system can reveal unanticipated markers when used with the appropriate methods of data analysis. Here, we used mass cytometry, flow cytometry, enzyme-linked immunosorbent assay, and multiplex assays to systematically profile immune cell populations in 68 patients with varying grades of cGVHD. We identified multiple subpopulations across T, B, and NK-cell lineages that distinguished patients with cGVHD from those without cGVHD and which were associated in varying ways with severity of cGVHD. Specifically, initial flow cytometry demonstrated that patients with more severe cGVHD had lower mucosal-associated T cell frequencies, with a concomitant higher level of CD38 expression on T cells. Mass cytometry could identify unique subpopulations specific for cGVHD severity albeit with some seemingly conflicting results. For instance, patients with severe cGVHD had an increased frequency of activated B cells compared to patients with moderate cGVHD while activated B cells were found at a reduced frequency in patients with mild cGVHD compared to patients without cGVHD. Moreover, results indicate it may be possible to validate mass cytometry results with clinically viable, smaller flow cytometry panels. Finally, no differences in levels of blood soluble markers could be identified, with the exception for the semi-soluble combined marker B-cell activating factor/B cell ratio, which was increased in patients with mild cGVHD compared to patients without cGVHD. These findings suggest that interdependencies between such perturbed subpopulations of cells play a role in cGVHD pathogenesis and can serve as future diagnostic and therapeutic targets.

  • 17. Terriac, Emmanuel
    et al.
    Coceano, Giovanna
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cellular Biophysics.
    Mavajian, Zahra
    KTH, School of Engineering Sciences (SCI), Applied Physics.
    Hageman, Tijmen A. G.
    Christ, Andreas F.
    Testa, Ilaria
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cellular Biophysics.
    Lautenschlaeger, Franziska
    Gad, Annica K. B.
    Vimentin Levels and Serine 71 Phosphorylation in the Control of Cell-Matrix Adhesions, Migration Speed, and Shape of Transformed Human Fibroblasts2017In: CELLS, ISSN 2073-4409, Vol. 6, no 1, article id 2Article in journal (Refereed)
    Abstract [en]

    Metastasizing tumor cells show increased expression of the intermediate filament (IF) protein vimentin, which has been used to diagnose invasive tumors for decades. Recent observations indicate that vimentin is not only a passive marker for carcinoma, but may also induce tumor cell invasion. To clarify how vimentin IFs control cell adhesions and migration, we analyzed the nanoscale (30-50 nm) spatial organization of vimentin IFs and cell-matrix adhesions in metastatic fibroblast cells, using three-color stimulated emission depletion (STED) microscopy. We also studied whether wild-type and phospho-deficient or -mimicking mutants of vimentin changed the size and lifetime of focal adhesions (FAs), cell shape, and cell migration, using live-cell total internal reflection imaging and confocal microscopy. We observed that vimentin exists in fragments of different lengths. Short fragments were mostly the size of a unit-length filament and were mainly localized close to small cell-matrix adhesions. Long vimentin filaments were found in the proximity of large FAs. Vimentin expression in these cells caused a reduction in FAs size and an elongated cell shape, but did not affect FA lifetime, or the speed or directionality of cell migration. Expression of a phospho-mimicking mutant (S71D) of vimentin increased the speed of cell migration. Taken together, our results suggest that in highly migratory, transformed mesenchymal cells, vimentin levels control the cell shape and FA size, but not cell migration, which instead is linked to the phosphorylation status of S71 vimentin. These observations are consistent with the possibility that not only levels, but also the assembly status of vimentin control cell migration.

  • 18.
    Testa, Ilaria
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cellular Biophysics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    RESOLFT Optical Nanoscopy for the Life Sciences2017In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 112, no 3, p. 7A-7AArticle in journal (Other academic)
  • 19.
    Testa, Ilaria
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cellular Biophysics.
    RESOLFT super resolution microscopy in neuronal cells2017In: Optics InfoBase Conference Papers, Optics Info Base, Optical Society of America, 2017, Vol. Part F76Conference paper (Refereed)
    Abstract [en]

    We show that emergent RESOLFT fluorescence nanoscopy enables fast and continuous imaging of living hippocampal cells and tissues with details at the nanoscale by producing raw data images using only ultralow levels of light.

  • 20.
    Unnersjö-Jess, David
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cellular Biophysics.
    High-resolution imaging of kidney tissue samples2017Licentiate thesis, comprehensive summary (Other academic)
    Abstract [en]

    The kidney is one of the most important and complex organs in the human body, filtering hundreds of litres of blood daily. Kidney disease is one of the fastest growing causes of death in the modern world, and this motivates extensive research for better understanding the function of the kidney in health and disease. Some of the most important cellular structures for blood filtration in the kidney are of very small dimensions (on the sub-200 nm scale), and thus electron microscopy has been the only method of choice to visualize these minute structures. In one study, we show for the first time that by combining optical clearing with STED microscopy, protein localizations in the slit diaphragm of the kidney, a structure around 75 nanometers in width, can now be resolved using light microscopy. In a second study, a novel sample preparation method, expansion microscopy, is utilized to physically expand kidney tissue samples. Expansion improves the effective resolution by a factor of 5, making it possible to resolve podocyte foot processes and the slit diaphragm using confocal microscopy. We also show that by combining expansion microscopy and STED microscopy, the effective resolution can be improved further. In a third study, influences on the development of the kidney were studied. There is substantial knowledge regarding what genes (growth factors, receptors etc.) are important for the normal morphogenesis of the kidney. Less is known regarding the physiology behind how paracrine factors are secreted and delivered in the developing kidney. By depleting calcium transients in explanted rat kidneys, we show that calcium is important for the branching morphogenesis of the ureteric tree. Further, the study shows that the calcium-dependent initiator of exocytosis, synaptotagmin, is expressed in the metanephric mesenchyme of the developing kidney, indicating that it could have a role in the secretion of paracrine growth factors, such as GDNF, to drive the branching.

  • 21.
    Unnersjö-Jess, David
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cellular Biophysics.
    Scott, Lena
    Karolinska Institutet.
    Zambrano Sevilla, Sonia
    Karolinska Institutet.
    Patrakka, Jakko
    Karolinska Institutet.
    Blom, Hans
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cellular Biophysics.
    Brismar, Hjalmar
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cellular Biophysics.
    Confocal imaging of slit diaphragm proteins in expanded kidney tissueManuscript (preprint) (Other academic)
    Abstract [en]

    The subtlest element of the kidney, such as the slit diaphragm, has historically only been spatially resolved using electron microscopy due to the nanometer-scale dimensions of these structures. Recently, it was shown that the nanoscale distribution of proteins in the slit diaphragm can be resolved by fluorescence based stimulated emission depletion (STED) microscopy, in combination with optical clearing. Fluorescence microscopy has advantages over electron microscopy in terms of multiplex imaging of different molecular species (i.e. epitopes), and also the amount of volumetric data that can be extracted from a thick sample. However, STED microscopy is still a costly technique commonly not available to all life science researchers. An image technique with which the slit diaphragm proteins in the kidney can be visualized using more standard fluorescence imaging techniques is thus desirable. Recent studies have shown that biological tissue samples can be isotropically expanded while optically cleared, revealing nanoscale localizations of multiple epitopes using confocal microscopy. Here we show that kidney samples can be expanded sufficiently to study the finest elements of the filtration barrier under both healthy and diseased conditions using confocal microscopy. This finding opens up the possibility for any researcher with access to a confocal microscope to study foot process protein distributions on the effective nanometer-scale. We also show that expansion microscopy can be combined with STED microscopy to further increase the effective spatial resolution down to below 20 nm.

  • 22.
    Xie, Sheng
    et al.
    KTH, School of Chemical Science and Engineering (CHE), Chemistry.
    Manuguri, Sesha
    KTH, School of Chemical Science and Engineering (CHE), Chemistry.
    Proietti, Giampiero
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Organic Chemistry.
    Romson, Joakim
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Organic Chemistry.
    Fu, Ying
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cellular Biophysics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Inge, A. K.
    Wu, B.
    Zhang, Yang
    KTH, School of Chemical Science and Engineering (CHE), Chemistry.
    Häll, Daniel
    KTH, School of Chemical Science and Engineering (CHE), Chemistry.
    Ramström, Olof
    KTH, School of Chemical Science and Engineering (CHE), Chemistry.
    Yan, Mingdi
    KTH, School of Chemical Science and Engineering (CHE), Chemistry.
    Design and synthesis of theranostic antibiotic nanodrugs that display enhanced antibacterial activity and luminescence2017In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 114, no 32, p. 8464-8469Article in journal (Refereed)
    Abstract [en]

    We report the modular formulation of ciprofloxacin-based pure theranostic nanodrugs that display enhanced antibacterial activities, as well as aggregation-induced emission (AIE) enhancement that was successfully used to image bacteria. The drug derivatives, consisting of ciprofloxacin, a perfluoroaryl ring, and a phenyl ring linked by an amidine bond, were efficiently synthesized by a straightforward protocol from a perfluoroaryl azide, ciprofloxacin, and an aldehyde in acetone at room temperature. These compounds are propeller-shaped, and upon precipitation into water, readily assembled into stable nanoaggregates that transformed ciprofloxacin derivatives into AIE-active luminogens. The nanoaggregates displayed increased luminescence and were successfully used to image bacteria. In addition, these nanodrugs showed enhanced antibacterial activities, lowering the minimum inhibitory concentration (MIC) by more than one order of magnitude against both sensitive and resistant Escherichia coli. The study represents a strategy in the design and development of pure theranostic nanodrugs for combating drug-resistant bacterial infections.

  • 23.
    Xu, Hao
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cellular Biophysics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Brismar, Hjalmar
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cellular Biophysics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Fu, Ying
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cellular Biophysics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Influence of surface states on blinking characteristics of single colloidal CdSe-CdS/ZnS core-multishell quantum dot2017In: Journal of Colloid and Interface Science, ISSN 0021-9797, E-ISSN 1095-7103, Vol. 505, p. 528-536Article in journal (Refereed)
    Abstract [en]

    We carefully characterized the fluorescence blinking of single colloidal CdSe-CdS/ZnS core-multishell quantum dots (QDs) with different surface modifications, including octadecylamine (ODA) coated QDs dispersed in chloroform, aqueous 3-mercaptopropionic acids (3MPA) coated QDs in HEPES solution treated by Ca2+ ions and ethylene glycol tetraacetic acid (EGTA, Ca2+ chelator), and aqueous 3MPA-QDs treated by glycerol. It was found that the on- and off-state probability density distributions displayed different rules. The off-state probability density distributions of all QDs complied well with the inverse power law, while the on-state probability density distributions bended upwards in log-log scale, and the degree of the upwards-bending correlated strongly with QD surface modification and fluorescence brightness of the single QD. Further autocorrelation analysis revealed that the fluorescence time series of a single QD was more random when the single QD showed a stronger fluorescence. Realistic numerical simulations with input parameters from quantum mechanical calculations showed that the QD exciton was first generated by an excitation photon; It radiatively recombined to give QD's fluorescence response, i.e., the on-state, which displayed the upwards-bended on-state probability density distribution profile; The electron and/or the hole of the photoexcited exciton in the QD core, after tunneling to the QD surface, randomly walked through the two-dimensional network of the QD surface states, resulting in the off-state probability density distribution profile of the inverse power law. Surface modification modified the QD surface-state network, in turn modifying the on/off probability density distribution profiles. Our findings provide us with a novel highway of applying colloidal QDs to study microscopic physical, and chemical, processes in many fields including in vivo and in vitro imaging, sensing and labelling.

  • 24.
    Yin, Huijuan
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cellular Biophysics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Fontana, Jacopo M.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cellular Biophysics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Solandt, Johan
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cellular Biophysics. KTH, Centres, Science for Life Laboratory, SciLifeLab. AstraZeneca R&D, Sweden.
    Jussi, Johnny Israelsson
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cellular Biophysics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Xu, Hao
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cellular Biophysics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Brismar, Hjalmar
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cellular Biophysics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Fu, Ying
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cellular Biophysics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Quantum dots modulate intracellular Ca2+ level in lung epithelial cells2017In: International Journal of Nanomedicine, ISSN 1176-9114, E-ISSN 1178-2013, Vol. 12, p. 2781-2792Article in journal (Refereed)
    Abstract [en]

    While adverse effects of nanoparticles on lung health have previously been proposed, few studies have addressed the direct effects of nanoparticle exposure on the airway epithelium. In this work, we examine the response of the pulmonary airway to nanoparticles by measuring intracellular Ca2+ concentration ([Ca2+](i)) in the Calu-3 epithelial layer stimulated by 3-mercaptopropionic-acid (3MPA) coated CdSe-CdS/ZnS core-multishell quantum dots (QDs). Simultaneous transient transepithelial electrical resistance (TEER) decrease and global [Ca2+](i) increase in Calu-3 epithelial layer, accompanied by cell displacements, contraction, and expansion, were observed under QD deposition. This suggests that a QD-induced global [Ca2+](i) increase in the Calu-3 epithelial layer caused the transient TEER decrease. The [Ca2+](i) increase was marked and rapid in the apical region, while [Ca2+](i) decreased in the basolateral region of the epithelial layer. TEER transient response and extracellular Ca2+ entry induced by QD deposition were completely inhibited in cells treated with stretched-activated (SA) inhibitor GdCl3 and store-operated calcium entry (SOCE) inhibitor BTP2 and in cells immersed in Ca2+-free medium. The voltage-gated calcium channel (VGCC) inhibitor nifedipine decreased, stabilized, and suppressed the TEER response, but did not affect the [Ca2+](i) increase, due to QD deposition. This demonstrates that the Ca2+ influx activated by QDs' mechanical stretch occurs through activation of both SA and SOCE channels. QD-induced [Ca2+](i) increase occurred in the Calu-3 epithelial layer after culturing for 15 days, while significant TEER drop only occurred after 23 days. This work provides a new perspective from which to study direct interactions between airway epithelium and nanoparticles and may help to reveal the pathologies of pulmonary disease.

  • 25.
    Yin, Huijuan
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cellular Biophysics.
    Shi, Xiafei
    Wang, Hong
    Jin, Wendong
    Li, Yingxin
    Fu, Ying
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cellular Biophysics.
    Photodynamic therapy targeting VCAM-1-expressing human umbilical vein endothelial cells using a PpIX-VCAM-1 binding peptide-quantum dot conjugate2017In: RSC Advances, ISSN 2046-2069, E-ISSN 2046-2069, Vol. 7, no 80, p. 50562-50570Article in journal (Refereed)
    Abstract [en]

    With increasing knowledge of the relevance of vascular cell adhesion molecule 1 (VCAM-1) for tumor growth, metastasis, angiogenesis, and related processes, it has become an attractive anti-tumor strategy to target VCAM-1 expression on the tumor vasculature. We designed a new targeted nanodrug, denoted PVQ, based on a photosensitizer (for the photodynamic effect), VCAM-1 target and quantum dot (QD) carrier, using conjugated water-dispersible colloidal CdSe-CdS/ZnS QDs, protoporphyrin IX (PpIX) photosensitizers, and VCAM-1 binding peptides. Its targeting ability and photodynamic therapy (PDT) efficiency against VCAM-1 expression in human umbilical vein endothelial cells (HUVECs) were then investigated. Conjugates of QD-VCAM-1 binding peptide (VQ), PpIX-VCAM-1 binding peptide (PV), and PVQ prepared using amide coupling were verified by agarose gel electrophoresis, Fourier transform infrared spectroscopy, and fluorescence spectrometry. VCAM-1 expression in HUVECs was induced by TNF-alpha treatment. PVQ conjugates were co-cultured with VCAM-1 expressing (VCAM-1(+)) and non-expressing (VCAM-1(-)) HUVECs, and target imaging, ROS generation, cell death, and apoptosis were analyzed using confocal fluorescence microscopy. VCAM-1 target imaging could not distinguish between VCAM-1(+) and VCAM-1(-) HUVECs after only 6 h of incubation; however it could distinguish between the cells after incubation for 24 h. After incubation for ca. 30 min, PVQ generated a significantly higher yield of ROS (3.6 fold) in VCAM-1(+) HUVECs compared with VCAM-1(-) cells, during 10 min of irradiation at a wavelength of 405 nm, and this was followed by a second rise in ROS at 30 min after irradiation. Moreover, cell destruction was observed clearly in VCAM-1(+) cells treated with PVQ and almost all cells became round after 30 min of irradiation at 405 nm. PVQ-induced PDT effects caused a significant apoptosis (onset and late apoptosis) in VCAM-1(+) HUVECs at 6 h after PDT treatment. In conclusion, PVQ shows a great potential for targeted PDT in cancer therapy.

1 - 25 of 25
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