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  • 1. Bagawath-Singh, Sunitha
    et al.
    Staaf, Elina
    Stoppelenburg, Arie Jan
    Spielmann, Thiemo
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Kambayashi, Taku
    Widengren, Jerker
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Johansson, Sofia
    Cytokines Induce Faster Membrane Diffusion of MHC Class I and the Ly49A Receptor in a Subpopulation of Natural Killer Cells2016In: Frontiers in Immunology, ISSN 1664-3224, E-ISSN 1664-3224, Vol. 7Article in journal (Refereed)
    Abstract [en]

    Cytokines have the potential to drastically augment immune cell activity. Apart from altering the expression of a multitude of proteins, cytokines also affect immune cell dynamics. However, how cytokines affect the molecular dynamics within the cell membrane of immune cells has not been addressed previously. Molecular movement is a vital component of all biological processes, and the rate of motion is, thus, an inherent determining factor for the pace of such processes. Natural killer (NK) cells are cytotoxic lymphocytes, which belong to the innate immune system. By fluorescence correlation spectroscopy, we investigated the influence of cytokine stimulation on the membrane density and molecular dynamics of the inhibitory receptor Ly49A and its ligand, the major histocompatibility complex class I allele H-2D(d), in freshly isolated murine NK cells. H-2D(d) was densely expressed and diffused slowly in resting NK cells. Ly49A was expressed at a lower density and diffused faster. The diffusion rate in resting cells was not altered by disrupting the actin cytoskeleton. A short-term stimulation with interleukin-2 or interferon- alpha + beta did not change the surface density of moving H-2D(d) or Ly49A, despite a slight upregulation at the cellular level of H-2D(d) by interferon-alpha + beta, and of Ly49A by IL-2. However, the molecular diffusion rates of both H-2D(d) and Ly49A increased significantly. A multivariate analysis revealed that the increased diffusion was especially marked in a subpopulation of NK cells, where the diffusion rate was increased around fourfold compared to resting NK cells. After IL-2 stimulation, this subpopulation of NK cells also displayed lower density of Ly49A and higher brightness per entity, indicating that Ly49A may homo-cluster to a larger extent in these cells. A faster diffusion of inhibitory receptors could enable a faster accumulation of these molecules at the immune synapse with a target cell, eventually leading to a more efficient NK cell response. It has previously been assumed that cytokines regulate immune cells primarily via alterations of protein expression levels or posttranslational modifications. These findings suggest that cytokines may also modulate immune cell efficiency by increasing the molecular dynamics early on in the response.

  • 2.
    Bergstrand, Jan
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Quantum and Biophotonics.
    Liu, Qingyun
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Theoretical Chemistry and Biology.
    Huang, Bingru
    Würth, Christian
    Resch-Genger, Ute
    Zhan, Qiuqiang
    Widengren, Jerker
    KTH, School of Engineering Sciences (SCI), Applied Physics, Quantum and Biophotonics.
    Ågren, Hans
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Theoretical Chemistry and Biology.
    Liu, Haichun
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Theoretical Chemistry and Biology.
    On the decay time of upconversion luminescence2019In: Nanoscale, ISSN 2040-3364, E-ISSN 2040-3372, Vol. 11, no 11, p. 4959-4969Article in journal (Refereed)
    Abstract [en]

    In this study, we systematically investigate the decay characteristics of upconversion luminescence (UCL) under anti-Stokes excitation through numerical simulations based on rate-equation models. We find that a UCL decay profile generally involves contributions from the sensitizer's excited-state lifetime, energy transfer and cross-relaxation processes. It should thus be regarded as the overall temporal response of the whole upconversion system to the excitation function rather than the intrinsic lifetime of the luminescence emitting state. Only under certain conditions, such as when the effective lifetime of the sensitizer's excited state is significantly shorter than that of the UCL emitting state and of the absence of cross-relaxation processes involving the emitting energy level, the UCL decay time approaches the intrinsic lifetime of the emitting state. Subsequently, Stokes excitation is generally preferred in order to accurately quantify the intrinsic lifetime of the emitting state. However, possible cross-relaxation between doped ions at high doping levels can complicate the decay characteristics of the luminescence and even make the Stokes-excitation approach fail. A strong cross-relaxation process can also account for the power dependence of the decay characteristics of UCL.

  • 3.
    Bergstrand, Jan
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Rönnlund, Daniel
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Widengren, Jerker
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Wennmalm, Stefan
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Scanning inverse fluorescence correlation spectroscopy2014In: Optics Express, ISSN 1094-4087, E-ISSN 1094-4087, Vol. 22, no 11, p. 13073-13090Article in journal (Refereed)
    Abstract [en]

    Scanning Inverse Fluorescence Correlation Spectroscopy (siFCS) is introduced to determine the absolute size of nanodomains on surfaces. We describe here equations for obtaining the domain size from cross-and auto-correlation functions, measurement simulations which enabled testing of these equations, and measurements on model surfaces mimicking membranes containing nanodomains. Using a confocal microscope of 270 nm resolution the size of 250 nm domains were estimated by siFCS to 257 +/- 12 nm diameter, and 40 nm domains were estimated to 65 +/- 26 nm diameter. Applications of siFCS for sizing of nanodomains and protein clusters in cell membranes are discussed.

  • 4.
    Bergstrand, Jan
    et al.
    Royal Inst Technol KTH, Dept Appl Phys, Albanova Univ Ctr, Expt Biomol Phys, SE-10691 Stockholm, Sweden.
    Xu, Lei
    Royal Inst Technol KTH, Dept Appl Phys, Albanova Univ Ctr, Expt Biomol Phys, SE-10691 Stockholm, Sweden.
    Miao, Xinyan
    Royal Inst Technol KTH, Dept Appl Phys, Albanova Univ Ctr, Expt Biomol Phys, SE-10691 Stockholm, Sweden.
    Li, Nailin
    Karolinska Inst, Dept Med Solna, Karolinska Univ Hosp Solna, Clin Pharmacol, L7 03, SE-17176 Stockholm, Sweden.
    Oktem, Ozan
    Royal Inst Technol KTH, Dept Math, Lindstedsvagen 25, SE-10044 Stockholm, Sweden.
    Franzen, Bo
    Karolinska Inst, Dept Oncol Pathol, Karolinska Univ Hosp, K7,Z1 00, S-17176 Stockholm, Sweden.
    Auer, Gert
    Karolinska Inst, Dept Oncol Pathol, Karolinska Univ Hosp, K7,Z1 00, S-17176 Stockholm, Sweden.
    Lomnytska, Marta
    Karolinska Inst, Dept Oncol Pathol, Karolinska Univ Hosp, K7,Z1 00, S-17176 Stockholm, Sweden;Acad Univ Hosp, Dept Obstet & Gynaecol, SE-75185 Uppsala, Sweden.
    Widengren, Jerker
    Royal Inst Technol KTH, Dept Appl Phys, Albanova Univ Ctr, Expt Biomol Phys, SE-10691 Stockholm, Sweden.
    Super-resolution microscopy can identify specific protein distribution patterns in platelets incubated with cancer cells2019In: Nanoscale, ISSN 2040-3364, E-ISSN 2040-3372, Vol. 11, no 20, p. 10023-10033Article in journal (Refereed)
    Abstract [en]

    Protein contents in platelets are frequently changed upon tumor development and metastasis. However, how cancer cells can influence protein-selective redistribution and release within platelets, thereby promoting tumor development, remains largely elusive. With fluorescence-based super-resolution stimulated emission depletion (STED) imaging we reveal how specific proteins, implicated in tumor progression and metastasis, re-distribute within platelets, when subject to soluble activators (thrombin, adenosine diphosphate and thromboxane A2), and when incubated with cancer (MCF-7, MDA-MB-231, EFO21) or non-cancer cells (184A1, MCF10A). Upon cancer cell incubation, the cell-adhesion protein P-selectin was found to re-distribute into circular nano-structures, consistent with accumulation into the membrane of protein-storing alpha-granules within the platelets. These changes were to a significantly lesser extent, if at all, found in platelets incubated with normal cells, or in platelets subject to soluble platelet activators. From these patterns, we developed a classification procedure, whereby platelets exposed to cancer cells, to non-cancer cells, soluble activators, as well as non-activated platelets all could be identified in an automatic, objective manner. We demonstrate that STED imaging, in contrast to electron and confocal microscopy, has the necessary spatial resolution and labelling efficiency to identify protein distribution patterns in platelets and can resolve how they specifically change upon different activations. Combined with image analyses of specific protein distribution patterns within the platelets, STED imaging can thus have a role in future platelet-based cancer diagnostics and therapeutic monitoring. The presented approach can also bring further clarity into fundamental mechanisms for cancer cell-platelet interactions, and into non-contact cell-to-cell interactions in general.

  • 5.
    Bergstrand, Jan
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Quantum and Biophotonics.
    Xu, Lei
    KTH, School of Engineering Sciences (SCI), Applied Physics, Quantum and Biophotonics. Royal Inst Technol KTH, Dept Appl Phys, Albanova Univ Ctr, Expt Biomol Phys, SE-10691 Stockholm, Sweden..
    Miao, Xinyan
    KTH, School of Engineering Sciences (SCI), Applied Physics, Quantum and Biophotonics. Royal Inst Technol KTH, Dept Appl Phys, Albanova Univ Ctr, Expt Biomol Phys, SE-10691 Stockholm, Sweden..
    Li, Nailin
    Karolinska Inst, Dept Med Solna, Karolinska Univ Hosp Solna, Clin Pharmacol, L7 03, SE-17176 Stockholm, Sweden..
    Öktem, Ozan
    KTH, School of Engineering Sciences (SCI), Mathematics (Dept.), Mathematics (Div.).
    Franzen, Bo
    Karolinska Inst, Dept Oncol Pathol, Karolinska Univ Hosp, K7,Z1 00, S-17176 Stockholm, Sweden..
    Auer, Gert
    Karolinska Inst, Dept Oncol Pathol, Karolinska Univ Hosp, K7,Z1 00, S-17176 Stockholm, Sweden..
    Lomnytska, Marta
    Karolinska Inst, Dept Oncol Pathol, Karolinska Univ Hosp, K7,Z1 00, S-17176 Stockholm, Sweden.;Acad Univ Hosp, Dept Obstet & Gynaecol, SE-75185 Uppsala, Sweden.;Uppsala Univ, Inst Women & Child Hlth, SE-75185 Uppsala, Sweden..
    Widengren, Jerker
    KTH, School of Engineering Sciences (SCI), Applied Physics, Quantum and Biophotonics.
    Super-resolution microscopy can identify specific protein distribution patterns in platelets incubated with cancer cells2019In: Nanoscale, ISSN 2040-3364, E-ISSN 2040-3372, Vol. 11, no 20, p. 10023-10033Article in journal (Refereed)
    Abstract [en]

    Protein contents in platelets are frequently changed upon tumor development and metastasis. However, how cancer cells can influence protein-selective redistribution and release within platelets, thereby promoting tumor development, remains largely elusive. With fluorescence-based super-resolution stimulated emission depletion (STED) imaging we reveal how specific proteins, implicated in tumor progression and metastasis, re-distribute within platelets, when subject to soluble activators (thrombin, adenosine diphosphate and thromboxane A2), and when incubated with cancer (MCF-7, MDA-MB-231, EFO21) or non-cancer cells (184A1, MCF10A). Upon cancer cell incubation, the cell-adhesion protein P-selectin was found to re-distribute into circular nano-structures, consistent with accumulation into the membrane of protein-storing alpha-granules within the platelets. These changes were to a significantly lesser extent, if at all, found in platelets incubated with normal cells, or in platelets subject to soluble platelet activators. From these patterns, we developed a classification procedure, whereby platelets exposed to cancer cells, to non-cancer cells, soluble activators, as well as non-activated platelets all could be identified in an automatic, objective manner. We demonstrate that STED imaging, in contrast to electron and confocal microscopy, has the necessary spatial resolution and labelling efficiency to identify protein distribution patterns in platelets and can resolve how they specifically change upon different activations. Combined with image analyses of specific protein distribution patterns within the platelets, STED imaging can thus have a role in future platelet-based cancer diagnostics and therapeutic monitoring. The presented approach can also bring further clarity into fundamental mechanisms for cancer cell-platelet interactions, and into non-contact cell-to-cell interactions in general.

  • 6.
    Bergstrand, Jan
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Quantum and Biophotonics.
    Xu, Lei
    KTH, School of Engineering Sciences (SCI), Applied Physics, Quantum and Biophotonics.
    Miao, Xinyan
    KTH, School of Engineering Sciences (SCI), Applied Physics, Quantum and Biophotonics.
    Li, Nailin
    Karolinska Institutet, Department of Medicine, Karolinska University Hospital, L7:03, SE-171 76 Stockholm, Sweden.
    Öktem, Ozan
    KTH, School of Engineering Sciences (SCI), Mathematics (Dept.), Mathematics (Div.).
    Franzén, Bo
    Karolinska Institutet, Department of Medicine, Karolinska University Hospital, L7:03, SE-171 76 Stockholm, Sweden.
    Auer, Gert
    Karolinska Institutet, Department of Medicine, Karolinska University Hospital, L7:03, SE-171 76 Stockholm, Sweden.
    Lomnytska, Marta
    Karolinska Institutet, Department of Medicine, Karolinska University Hospital, L7:03, SE-171 76 Stockholm, Sweden.
    Widengren, Jerker
    KTH, School of Engineering Sciences (SCI), Applied Physics, Quantum and Biophotonics.
    Super-resolution microscopy can identify specific protein distribution patterns in platelets incubated with cancer cellsManuscript (preprint) (Other academic)
    Abstract [en]

    Protein contents in platelets are frequently changed upon tumor development and metastasis. However, how cancer cells can influence protein-selective redistribution and release within platelets, thereby promoting tumor development, remains largely elusive. With fluorescence-based super-resolution stimulated emission depletion (STED) imaging we reveal how specific proteins, implicated in tumor progression and metastasis, re-distribute within platelets, when subject to soluble activators (thrombin, adenosine-diphosphate and thromboxaneA2), and when incubated with cancer (MCF-7, MDA-MB-231, EFO21) or non-cancer cells (184A1, MCF10A). Upon cancer cell incubation, the cell-adhesion protein P-selectin was found to re-distribute into circular nano-structures, consistent with accumulation into the membrane of protein-storing alpha-granules within the platelets. These changes were to a significantly lesser extent, if at all, found in platelets incubated with normal cells, or in platelets subject to soluble platelet activators. From these patterns, we developed a classification procedure, whereby platelets exposed to cancer cells, to non-cancer cells, soluble activators as well as non-activated platelets all could be identified in an automatic, objective manner. We demonstrate that STED imaging, in contrast to electron and confocal microscopy, has the necessary spatial resolution and labelling efficiency to identify protein distribution patterns in platelets and can resolve how they specifically change upon different activations. Combined with image analyses of specific protein distribution patterns within the platelets, STED imaging can thus have a role in future platelet-based cancer diagnostics and therapeutic monitoring. The presented approach can also bring further clarity into fundamental mechanisms for cancer cell-platelet interactions, and into non-contact cell-to-cell interactions in general. 

  • 7.
    Blom, Hans
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Chmyrov, Andriy
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Hassler, Kai
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Davis, L.M.
    Widengren, Jerker
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Triplet-State Investigations of Fluorescent Dyes at Dielectric Interfaces Using Total Internal Reflection Fluorescence Correlation Spectroscopy2009In: Journal of Physical Chemistry A, ISSN 1089-5639, E-ISSN 1520-5215, Vol. 113, no 19, p. 5554-5566Article in journal (Refereed)
    Abstract [en]

    The triplet-state kinetics of several fluorescent dyes used in ultrasensitive fluorescence microscopy are investigated using total internal reflection fluorescence correlation spectroscopy (TIR-FCS). A theoretical outline of the correlation analysis and the physical aspects of evanescent excitation and fluorescence emission at dielectric interfaces are given. From this analysis, the rates of intersystem crossing and triplet decay are deduced for fluorescein, ATTO 488, rhodamine 110, rhodamine 123, and rhodamine 6G in aqueous buffer solutions. All investigated dyes show slightly higher triplet rates at the dielectric interface compared to bulk solution measurements. We attribute this enhancement to possible modifications of the dyes’ photophysical properties near a dielectric interface. In the case of rhodamine 6G, the impact of changes in the dye concentration, ionic strength of the solvent, and potassium iodide concentration are also investigated. This leads to a better understanding of the influences of dye−dye, dye−solvent, and dye−surface interactions on the increased triplet intersystem crossing and triplet decay rates. The study shows that analysis of triplet-state kinetics by TIR-FCS not only results in a better understanding of how the photophysical properties of the dyes are affected by the presence of an interface, but also provides a means for probing the microenvironment near dielectric interfaces.

  • 8.
    Blom, Hans
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Hassler, Kai
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Chmyrov, Andriy
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Widengren, Jerker
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Electrostatic Interactions of Fluorescent Molecules with Dielectric Interfaces Studied by Total Internal Reflection Fluorescence Correlation Spectroscopy2010In: International Journal of Molecular Sciences, ISSN 1422-0067, E-ISSN 1422-0067, Vol. 11, no 2, p. 368-406Article in journal (Refereed)
    Abstract [en]

    Electrostatic interactions between dielectric surfaces and different fluorophoresused in ultrasensitive fluorescence microscopy are investigated using objective-based TotalInternal Reflection Fluorescence Correlation Spectroscopy (TIR-FCS). The interfacialdynamics of cationic rhodamine 123 and rhodamine 6G, anionic/dianionic fluorescein,zwitterionic rhodamine 110 and neutral ATTO 488 are monitored at various ionic strengthsat physiological pH. As analyzed by means of the amplitude and time-evolution of theautocorrelation function, the fluorescent molecules experience electrostatic attraction orrepulsion at the glass surface depending on their charges. Influences of the electrostaticinteractions are also monitored through the triplet-state population and triplet relaxationtime, including the amount of detected fluorescence or the count-rate-per-moleculeparameter. These TIR-FCS results provide an increased understanding of how fluorophoresare influenced by the microenvironment of a glass surface, and show a promising approachfor characterizing electrostatic interactions at interfaces.

  • 9.
    Blom, Hans
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Ronnlund, Daniel
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Scott, Lena
    Spicarova, Zuzana
    Widengren, Jerker
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Bondar, Alexander
    Aperia, Anita
    Brismar, Hjalmar
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Spatial distribution of Na+-K+-ATPase in dendritic spines dissected by nanoscale superresolution STED microscopy2011In: BMC neuroscience (Online), ISSN 1471-2202, E-ISSN 1471-2202, Vol. 12, p. 16-Article in journal (Refereed)
    Abstract [en]

    Background: The Na+,K+-ATPase plays an important role for ion homeostasis in virtually all mammalian cells, including neurons. Despite this, there is as yet little known about the isoform specific distribution in neurons. Results: With help of superresolving stimulated emission depletion microscopy the spatial distribution of Na+,K+-ATPase in dendritic spines of cultured striatum neurons have been dissected. The found compartmentalized distribution provides a strong evidence for the confinement of neuronal Na+,K+-ATPase (alpha 3 isoform) in the postsynaptic region of the spine. Conclusions: A compartmentalized distribution may have implications for the generation of local sodium gradients within the spine and for the structural and functional interaction between the sodium pump and other synaptic proteins. Superresolution microscopy has thus opened up a new perspective to elucidate the nature of the physiological function, regulation and signaling role of Na+,K+-ATPase from its topological distribution in dendritic spines.

  • 10.
    Blom, Hans
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Rönnlund, Daniel
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Scott, L.
    Westin, L.
    Widengren, Jerker
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Aperia, A.
    Brismar, Hjalmar
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Spatial Distribution of DARPP-32 in Dendritic Spines2013In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 8, no 9, p. e75155-Article in journal (Refereed)
    Abstract [en]

    The phosphoprotein DARPP-32 (dopamine and cyclic adenosine 3́, 5́-monophosphate-regulated phosphoprotein, 32 kDa) is an important component in the molecular regulation of postsynaptic signaling in neostriatum. Despite the importance of this phosphoprotein, there is as yet little known about the nanoscale distribution of DARPP-32. In this study we applied superresolution stimulated emission depletion microscopy (STED) to assess the expression and distribution of DARPP-32 in striatal neurons. Primary culture of striatal neurons were immunofluorescently labeled for DARPP-32 with Alexa-594 and for the dopamine D1 receptor (D1R) with atto-647N. Dual-color STED microscopy revealed discrete localizations of DARPP-32 and D1R in the spine structure, with clustered distributions in both head and neck. Dissected spine structures reveal that the DARPP-32 signal rarely overlapped with the D1R signal. The D1R receptor is positioned in an "aggregated" manner primarily in the spine head and to some extent in the neck, while DARPP-32 forms several neighboring small nanoclusters spanning the whole spine structure. The DARPP-32 clusters have a mean size of 52 +/- 6 nm, which is close to the resolution limit of the microscope and corresponds to the physical size of a few individual phosphoprotein immunocomplexes. Dissection of synaptic proteins using superresolution microscopy gives possibilities to reveal in better detail biologically relevant information, as compared to diffraction-limited microscopy. In this work, the dissected postsynaptic topology of the DARPP-32 phosphoprotein provides strong evidence for a compartmentalized and confined distribution in dendritic spines. The protein topology and the relatively low copy number of phosphoprotein provides a conception of DARPP-32's possibilities to fine-tune the regulation of synaptic signaling, which should have an impact on the performance of the neuronal circuits in which it is expressed.

  • 11.
    Blom, Hans
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Rönnlund, Daniel
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Scott, Lena
    Spicarova, Zuzana
    Rantanen, Ville
    Widengren, Jerker
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Aperia, Anita
    Brismar, Hjalmar
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Nearest neighbor analysis of dopamine D1 receptors and Na plus -K plus -ATPases in dendritic spines dissected by STED microscopy2012In: Microscopy research and technique (Print), ISSN 1059-910X, E-ISSN 1097-0029, Vol. 75, no 2, p. 220-228Article in journal (Refereed)
    Abstract [en]

    Protein localization in dendritic spines is the focus of intense investigations within neuroscience. Applications of super-resolution microscopy to dissect nanoscale protein distributions, as shown in this work with dual-color STED, generate spatial correlation coefficients having quite small values. This means that colocalization analysis to some extent looses part of its correlative impact. In this study we thus introduced nearest neighbor analysis to quantify the spatial relations between two important proteins in neurons, the dopamine D1 receptor and Na+,K+-ATPase. The analysis gave new information on how dense the D1 receptor and Na+,K+-ATPase constituting nanoclusters are located both with respect to the homogenous (self to same) and the heterogeneous (same to other) topology. The STED dissected nanoscale topologies provide evidence for both a joint as well as a separated confinement of the D1 receptor and the Na+,K+-ATPase in the postsynaptic areas of dendritic spines. This confined topology may have implications for generation of local sodium gradients and for structural and functional interactions modulating slow synaptic transmission processes. Microsc. Res. Tech., 2011.

  • 12.
    Blom, Hans
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Widengren, Jerker
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    STED microscopy: towards broadened use and scope of applications2014In: Current opinion in chemical biology, ISSN 1367-5931, E-ISSN 1879-0402, Vol. 20, no 1, p. 127-133Article, review/survey (Refereed)
    Abstract [en]

    High resolution Stimulated Emission Depletion (STED) microscopy has been demonstrated for fundamental studies in cells, living tissue and organisms. Today, a major trend in the STED technique development is to make the instruments simpler and more user-friendly, without compromising performance. This has become possible by new low-cost, turn-key laser technology and by implementing specifically designed phase plates and polarization elements, extending and simplifying the shaping of the laser beam profiles. These simpler and cheaper realizations of STED are now becoming more broadly available. In parallel with the continuous development of sample preparation and fluorophore reporter molecules ultimately setting the limit of the image quality, contrast and resolution, we can thus expect a significant increase in the use of STED, in science as well as for clinical and drug development purposes.

  • 13.
    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.

  • 14.
    Brändén, Magnus
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Sandén, Tor
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Brzezinski, Peter
    Widengren, Jerker
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Localized Proton Microcircuits at the Biological Membrane-Water Interface2006In: PNAS, ISSN 0027-8424, Vol. 103, no 52, p. 19677-19770Article in journal (Refereed)
    Abstract [en]

    Cellular processes such as nerve conduction, energy metabolism, and import of nutrients into cells all depend on transport of ions across biological membranes through specialized membrane-spanning proteins. Understanding these processes at a molecular level requires mechanistic insights into the interaction between these proteins and the membrane itself. To explore the role of the membrane in ion translocation we used an approach based on fluorescence correlation spectroscopy. Specifically, we investigated exchange of protons between the water phase and the membrane surface, as well as diffusion of protons along membrane surfaces, at a single-molecule level. We show that the lipid head groups collectively act as a proton-collecting antenna, dramatically accelerating proton uptake from water to a membrane-anchored proton acceptor. Furthermore, the results show that proton transfer along the surface can be significantly faster than that between the lipid head groups and the surrounding water phase. Thus, ion translocation across membranes and between the different membrane protein components is a complex interplay between the proteins and the membrane itself, where the membrane acts as a proton-conducting link between membrane-spanning proton transporters

  • 15. Cebula, Marcus
    et al.
    Turan, Ilke Simsek
    Sjodin, Birgitta
    Thulasingam, Madhuranayaki
    Brock, Joseph
    Chmyrov, Volodymyr
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Widengren, Jerker
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Abe, Hiroshi
    Mannervik, Bengt
    Haeggstrom, Jesper Z.
    Rinaldo-Matthis, Agnes
    Akkaya, Engin U.
    Morgenstern, Ralf
    Catalytic Conversion of Lipophilic Substrates by Phase constrained Enzymes in the Aqueous or in the Membrane Phase2016In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 6, article id 38316Article in journal (Refereed)
    Abstract [en]

    Both soluble and membrane-bound enzymes can catalyze the conversion of lipophilic substrates. The precise substrate access path, with regard to phase, has however, until now relied on conjecture from enzyme structural data only (certainly giving credible and valuable hypotheses). Alternative methods have been missing. To obtain the first experimental evidence directly determining the access paths (of lipophilic substrates) to phase constrained enzymes we here describe the application of a BODIPY-derived substrate (PS1). Using this tool, which is not accessible to cytosolic enzymes in the presence of detergent and, by contrast, not accessible to membrane embedded enzymes in the absence of detergent, we demonstrate that cytosolic and microsomal glutathione transferases (GSTs), both catalyzing the activation of PS1, do so only within their respective phases. This approach can serve as a guideline to experimentally validate substrate access paths, a fundamental property of phase restricted enzymes. Examples of other enzyme classes with members in both phases are xenobiotic-metabolizing sulphotransferases/UDP-glucuronosyl transferases or epoxide hydrolases. Since specific GSTs have been suggested to contribute to tumor drug resistance, PS1 can also be utilized as a tool to discriminate between phase constrained members of these enzymes by analyzing samples in the absence and presence of Triton X-100.

  • 16. Chen, Xingqi
    et al.
    Shi, Chengxi
    Yammine, Samer
    Göndör, Anita
    Rönnlund, Daniel
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Fernandez-Woodbridge, Alejandro
    Sumida, Noriyuki
    Widengren, Jerker
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Ohlsson, Rolf
    Chromatin in situ proximity (ChrISP): Single-cell analysis of chromatin proximities at a high resolution2014In: BioTechniques, ISSN 0736-6205, E-ISSN 1940-9818, Vol. 56, no 3, p. 117-124Article in journal (Refereed)
    Abstract [en]

    Current techniques for analyzing chromatin structures are hampered by either poor resolution at the individual cell level or the need for a large number of cells to obtain higher resolution. This is a major problem as it hampers our understanding of chromatin conformation in single cells and how these respond to environmental cues. Here we describe a new method, chromatin in situ proximity (ChrISP), which reproducibly scores for proximities between two different chromatin fibers in 3-D with a resolution of similar to 170 angstrom in single cells. The technique is based on the in situ proximity ligation assay (ISPLA), but ChrISP omits the rolling circle amplification step (RCA). Instead, the proximities between chromatin fibers are visualized by a fluorescent connector oligonucleotide DNA, here termed splinter, forming a circular DNA.with another circle-forming oligonucleotide, here termed backbone, upon ligation. In contrast to the regular ISPLA technique, our modification enables detection of chromatin fiber proximities independent of steric hindrances from nuclear structures. We use this method to identify higher order structures of individual chromosomes in relation to structural hallmarks of interphase nuclei and beyond the resolution of the light microscope.

  • 17.
    Chmyrov, Andriy
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Arden-Jacob, J.
    Zilles, A.
    Drexhage, K. -H
    Widengren, Jerker
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Characterization of new fluorescent labels for ultrahigh resolution microscopy2009In: Novel Techniques in Microscopy (NTM) 2009, Optical Society of America, 2009Conference paper (Refereed)
    Abstract [en]

    A set of modified dyes was investigated, of which several candidates combine prominent triplet state yield with reasonable photostability. They can be used to achieve ultrahigh optical resolution by photo-induced switching into dark (triplet) states.

  • 18.
    Chmyrov, Andriy
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Arden-Jacob, Jutta
    ATTO-TEC GmbH, Germany.
    Zilles, Alexander
    University of Siegen, Germany.
    Drexhage, Karl-Heinz
    University of Siegen, Germany.
    Widengren, Jerker
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Characterization of new fluorescent labels for ultra-high resolution microscopy2008In: Photochemical and Photobiological Sciences, ISSN 1474-905X, E-ISSN 1474-9092, Vol. 7, no 11, p. 1378-1385Article in journal (Refereed)
    Abstract [en]

    Photo-induced switching of dyes into dark, long-lived states, such as a triplet state, has recently gained increasing interest, as a means to achieve ultra-high optical resolution. Additionally, these long lived states are often highly environment-sensitive and their photodynamics can thus offer additional independent fluorescence-based information. However, although providing a useful mechanism for photo-induced switching, the triplet state often appears as a precursor state for photobleaching, which potentially can limit its usefulness. In this work, a set of rhodamine and pyronin dyes, modified by substitution of heavy atoms and nitrogen within or close to the central xanthene unit of the dyes, were investigated with respect to their triplet state dynamics and photostabilities, under conditions relevant for ultra-high resolution microscopy. Out of the dyes investigated, in particular the rhodamine and pyronin dyes with a sulfur atom replacing the central oxygen atom in the xanthene unit were found to meet the requirements for ultra-high resolution microscopy, combining a prominent triplet state yield with reasonable photostability.

  • 19.
    Chmyrov, Andriy
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Sandén, Tor
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Widengren, Jerker
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Iodide as a Fluorescence Quencher and Promoter-Mechanisms and Possible Implications2010In: Journal of Physical Chemistry B, ISSN 1520-6106, E-ISSN 1520-5207, Vol. 114, no 34, p. 11282-11291Article in journal (Refereed)
    Abstract [en]

    In this work, fluorescence correlation spectroscopy (FCS) was used to investigate the effects of potassium iodide (KI) on the electronic-state population kinetics of a range of organic dyes in the visible wavelength range. Apart from a heavy atom effect promoting intersystem crossing to the triplet states in all dyes, KI was also found to enhance the triplet-state decay rate by a charge-coupled deactivation. This deactivation was only found for dyes with excitation maximum in the blue range, not for those with excitation maxima at wavelengths in the green range or longer. Consequently, under excitation conditions sufficient for triplet state formation, KI can promote the triplet state buildup of one dye and reduce it for another, red-shifted dye. This anticorrelated, spectrally separable response of two different dyes to the presence of one and the same agent may provide a useful readout for biomolecular interaction and microenvironmental monitoring studies. In contrast to the typical notion of KI as a fluorescence quencher, the FCS measurements also revealed that when added in micromolar concentrations KI can act as an antioxidant, promoting the recovery of photo-oxidized fluorophores. However, in millimolar concentrations KI also reduces intact, fluorescently viable fluorophores to a considerable extent. In aqueous solutions, for the dye Rhodamine Green, an optimal concentration of KI of approximately 5 mM can be defined at which the fluorescence signal is maximized. This concentration is not high enough to allow full triplet state quenching. Therefore, as a fluorescence enhancement agent, it is primarily the antioxidative properties of KI that play a role.

  • 20.
    Chmyrov, Andriy
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Sandén, Tor
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Widengren, Jerker
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Iodide as a Triplet State Promoter and Quencher –Mechanisms and Possible ImplicationsManuscript (preprint) (Other academic)
    Abstract [en]

    In this work, Fluorescence Correlation Spectroscopy(FCS) was used to investigate the effects of potassium iodide(KI) on the electronic state population kinetics of arange of organic dyes in the visible wavelength range. Apartfrom a heavy atom effect promoting intersystem crossing tothe triplet states in all dyes, KI was also found to enhancethe triplet state decay by a charge-coupled deactivation.This deactivation was only found for dyes with excitationmaximum in the blue range, not for those with excitationmaxima at wavelengths in the green range or longer. Consequently,under excitation conditions sufficient for tripletstate formation, KI can promote the triplet state build-up ofone dye and reduce it for another, red-shifted dye. The anticorrelated,spectrally separable responses of two dyes to thepresence of one and the same agent are likely to provide auseful readout for biomolecular interaction and micro-environmentalmonitoring studies. In contrast to the typicalnotion of KI as a fluorescence quencher, the FCS measurementsalso revealed that when added in micromolar concentrationsKI can act as an anti-oxidant, promoting the recoveryof photo-oxidized fluorophores. However, in millimolarconcentrations KI also reduces intact, fluorescently viablefluorophores to a considerable extent. In aqueous solutions,an optimal concentration of KI of approximately 5 mM canbe defined at which the fluorescence signal is maximized.This concentration is not high enough to allow full tripletstate quenching. Therefore, as a fluorescence enhancementagent, it is primarily the anti-oxidative properties of KI thatplay a role.

  • 21.
    Chmyrov, Andriy
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Sandén, Tor
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Widengren, Jerker
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Recovery of Photoinduced Reversible Dark States Utilized for Molecular Diffusion Measurements2010In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 82, no 24, p. 9998-10005Article in journal (Refereed)
    Abstract [en]

    For a spatially restricted excitation volume, the effective modulation of the excitation in time is influenced by the passage times of the molecules through the excitation volume. By applying an additional time-modulated excitation, the buildup of photoinduced reversible dark states in fluorescent molecules can be made to vary significantly with their passage times through the excitation volume. The variations in the dark state populations are reflected by the time-averaged fluorescence intensity, which thus can be used to characterize the mobilities of the molecules. The concept was experimentally verified by measuring the fluorescence response of freely diffusing cyanine fluorophores (Cy5), undergoingtrans-cis isomerization when subject to time-modulated excitation in a focused laser beam. From the fluorescence response, and by applying a simple photodynamic model, the transition times of the Cy5 molecules could be well reproduced when applying different laminar flow speeds through the detection volume. The presented approach puts no constraints on sample concentration, no requirements for high time resolution or sensitivity in the detection, nor requires a high fluorescence brightness of the characterized molecules. This can make the concept useful for a broad range of biomolecular mobility studies.

  • 22.
    Chmyrov, Volodymyr
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Spielmann, Thiemo
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Hevekerl, Heike
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Widengren, Jerker
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Trans-Cis isomerization of lipophilic dyes probing membrane microviscosity in biological membranes and in live cells2015In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 87, no 11, p. 5690-5697Article in journal (Refereed)
    Abstract [en]

    Membrane environment and fluidity can modulate the dynamics and interactions of membrane proteins and can thereby strongly influence the function of cells and organisms in general. In this work, we demonstrate that trans-cis isomerization of lipophilic dyes is a useful parameter to monitor packaging and fluidity of biomembranes. Fluorescence fluctuations, generated by trans-cis isomerization of the thiocarbocyanine dye Merocyanine 540 (MC540), were first analyzed by fluorescence correlation spectroscopy (FCS) in different alcohol solutions. Similar isomerization kinetics of MC540 in lipid vesicles could then also be monitored, and the influence of lipid polarity, membrane curvature, and cholesterol content was investigated. While no influence of membrane curvature and lipid polarity could be observed, a clear decrease in the isomerization rates could be observed with increasing cholesterol contents in the vesicle membranes. Finally, procedures to spatially map photoinduced and thermal isomerization rates on live cells by transient state (TRAST) imaging were established. On the basis of these procedures, MC540 isomerization was studied on live MCF7 cells, and TRAST images of the cells at different temperatures were found to reliably detect differences in the isomerization parameters. Our studies indicate that trans-cis isomerization is a useful parameter for probing membrane dynamics and that the TRAST imaging technique can provide spatial maps of photoinduced isomerization as well as both photoinduced and thermal back-isomerization, resolving differences in local membrane microviscosity in live cells.

  • 23.
    Chmyrov, Volodymyr
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Spielmann, Thiemo
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Hevekerl, Heike
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Widengren, Jerker
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Trans-cis isomerization of lipophilic dyes provides a measure of membrane microviscosity in biological membranes and in live cellsManuscript (preprint) (Other academic)
  • 24. Doerr, Denis
    et al.
    Sandrin, Deborah
    Kalinin, Stanislav
    Kuehnemuth, Ralf
    Overmann, Sebastian
    Pfiffi, Daniela
    Schaper, Klaus
    Seidel, Claus A. M.
    Mueller, Thomas J. J.
    Chmyrov, Andriy
    Widengren, Jerker
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Bier, Brigitte A.
    Maximizing the Fluorescence Signal and Photostability of Fluorophores by Quenching Dark-States2014In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 106, no 2, p. 196A-196AArticle in journal (Other academic)
  • 25.
    Du, Zhixue
    et al.
    Shanghai Jiao Tong Univ, Sch Chem & Chem Engn, State Key Lab Met Matrix Composites, 800 Dongchuan Rd, Shanghai 200240, Peoples R China..
    Yu, Jing
    Shanghai Jiao Tong Univ, Sch Life Sci & Biotechnol, State Key Lab Microbial Metab, 800 Dongchuan Rd, Shanghai 200240, Peoples R China..
    Li, Fucai
    Shanghai Jiao Tong Univ, Sch Chem & Chem Engn, State Key Lab Met Matrix Composites, 800 Dongchuan Rd, Shanghai 200240, Peoples R China..
    Deng, Liyun
    Shanghai Jiao Tong Univ, Sch Chem & Chem Engn, State Key Lab Met Matrix Composites, 800 Dongchuan Rd, Shanghai 200240, Peoples R China..
    Wu, Fang
    Shanghai Jiao Tong Univ, Shanghai Ctr Syst Biomed, Minist Educ, Key Lab Syst Biomed, 800 Dongchuan Rd, Shanghai 200240, Peoples R China..
    Huang, Xiangyi
    Shanghai Jiao Tong Univ, Sch Chem & Chem Engn, State Key Lab Met Matrix Composites, 800 Dongchuan Rd, Shanghai 200240, Peoples R China..
    Bergstrand, Jan
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Widengren, Jerker
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Dong, Chaoqing
    Shanghai Jiao Tong Univ, Sch Chem & Chem Engn, State Key Lab Met Matrix Composites, 800 Dongchuan Rd, Shanghai 200240, Peoples R China..
    Ren, Jicun
    Shanghai Jiao Tong Univ, Sch Chem & Chem Engn, State Key Lab Met Matrix Composites, 800 Dongchuan Rd, Shanghai 200240, Peoples R China..
    In Situ Monitoring of p53 Protein and MDM2 Protein Interaction in Single Living Cells Using Single-Molecule Fluorescence Spectroscopy2018In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 90, no 10, p. 6144-6151Article in journal (Refereed)
    Abstract [en]

    Protein-protein interactions play a central role in signal transduction, transcription regulations, enzymatic activity, and protein synthesis. The p53 protein is a key transcription factor, and its activity is precisely regulated by the p53-MDM2 interaction. Although the p53-MDM2 interaction has been studied, it is still not clear how p53 structures and external factors influence the p53-MDM2 interaction in living cells. Here, we developed a direct method for monitoring the p53-MDM2 interaction in single living cells using single-molecule fluorescence cross-correlation spectroscopy with a microfluidic chip. First, we labeled p53 and MDM2 proteins with enhanced green fluorescent protein (EGFP) and mCherry, respectively, using lentivirus infection. We then designed various mutants covering the three main domains of p53 (tetramerization, transactivation, and DNA binding domains) and systematically studied effects of p53 protein primary, secondary, and quaternary structures on p53 MDM2 binding affinity in single living cells. We found that p53 dimers and tetramers can bind to MDM2, that the binding affinity of p53 tetramers is higher than that of p53 dimers, and that the affinity is closely correlated to the helicity of the p53 transactivation domain. The hot-spot mutation R175H in the DNA-binding domain reduced the binding of p53 to MDM2. Finally, we studied effects of inhibitors on p53-MDM2 interactions and dissociation dynamics of pS3-MDM2 complexes in single living cells. We found that inhibitors Nutlin 3 alpha and MI773 efficiently inhibited the pS3-MDM2 interaction, but RITA did not work in living cells. This study provides a direct way for quantifying the relationship between protein structure and protein protein interactions and evaluation of inhibitors in living cells.

  • 26. Eggeling, C.
    et al.
    Widengren, Jerker
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Brand, L.
    Schaffer, J.
    Felekyan, S.
    Seidel, C. A. M.
    Analysis of photobleaching in single-molecule multicolor excitation and forster resonance energy transfer measurement2006In: Journal of Physical Chemistry A, ISSN 1089-5639, E-ISSN 1520-5215, Vol. 110, no 9, p. 2979-2995Article in journal (Refereed)
    Abstract [en]

    we investigated the influence of photobleaching in fluorescence experiments applying multicolor laser as well as Forster resonance energy transfer (FRET) mediated excitation using several red-emitting dyes frequently used in multicolor experiments or as FRET acceptors. The chosen dyes (cyanine 5 (Cy5), MR121, Alexa660, Alexa680, Atto647N, Atto655) have chemically distinct chromophore systems and can be excited at 650 nm. Several fluorescence analysis techniques have been applied to detect photobleaching and to disclose the underlying photophysics, all of which are based on single-molecule detection: (1) fluorescence correlation spectroscopy (FCS) of bulk solutions, (2) fluorescence cross-correlation of single-molecule trajectories, and (3) multiparameter fluorescence detection (MFD) of single-molecule events. The maximum achievable fluorescence signals as well as the survival times of the red dyes were markedly reduced under additional laser irradiation in the range of 500 nm. Particularly at excitation levels at or close to saturation, the 500 nm irradiation effectively induced transitions to higher excited electronic states on already excited dye molecules, leading to a pronounced bleaching reactivity. A theoretical model for the observed laser irradiance dependence of the fluorescence brightness of a Cy5 FRET acceptor dye has been developed introducing the full description of the underlying photophysics. The model takes into account acceptor as well as donor photobleaching from higher excited electronic states, population of triplet states, and energy transfer to both the ground and excited states of the acceptor dye. Also, photoinduced reverse intersystem crossing via higher excited triplet states is included, which was found to be very efficient for Cy5 attached to DNA. Comparing continuous wave (cw) and pulsed donor excitation, a strong enhancement of acceptor photobleaching by a factor of 5 was observed for the latter. Thus, in the case of fluorescence experiments utilizing multicolor pulsed laser excitation, the application of the appropriate timing of synchronized green and red laser pulses in an alternating excitation mode can circumvent excessive photobleaching. Moreover, important new single-molecule analysis diagnosis tools are presented: (1) For the case of excessive acceptor photobleaching, cross-correlation analysis of single-molecule trajectories of the fluorescence signal detected in the donor and acceptor detection channels and vice versa shows an anticorrelated exponential decay and growth, respectively. (2) The time difference, T-g - T-r of the mean observation times of all photons detected for the donor and acceptor detection channels within a single-molecule fluorescence burst allows one to identify and exclude molecules with an event of acceptor photobleaching. The presented single-molecule analysis methods can be constrained to, for example, FRET-active subpopulations, reducing bias from FRET-inactive molecules. The observations made are of strong relevance for and demand a careful choice of laser action in multicolor and FRET experiments, in particular when performed at or close to saturation.

  • 27. Fridberger, A.
    et al.
    Widengren, Jerker
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    de Monvel, J. B.
    Measuring hearing organ vibration patterns with confocal microscopy and optical flow2004In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 86, no 1, p. 535-543Article in journal (Refereed)
    Abstract [en]

    A new method for visualizing vibrating structures is described. The system provides a means to capture very fast repeating events by relatively minor modi. cations to a standard confocal microscope. An acousto-optic modulator was inserted in the beam path, generating brief pulses of laser light. Images were formed by summing consecutive frames until every pixel of the resulting image had been exposed to a laser pulse. Images were analyzed using a new method for optical flow computation; it was validated through introducing artificial displacements in confocal images. Displacements in the range of 0.8 to 4 pixels were measured with 5% error or better. The lower limit for reliable motion detection was 20% of the pixel size. These methods were used for investigating the motion pattern of the vibrating hearing organ. In contrast to standard theory, we show that the organ of Corti possesses several degrees of freedom during sound-evoked vibration. Outer hair cells showed motion indicative of deformation. After acoustic overstimulation, supporting cells contracted. This slowly developing structural change was visualized during simultaneous intense sound stimulation and its speed measured with the optical flow technique.

  • 28. Gad, Annica K. B.
    et al.
    Rönnlund, Daniel
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Spaar, Alexander
    Savchenko, Andrii A.
    Petranyi, Gabor
    Blom, Hans
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Szekely, Laszlo
    Widengren, Jerker
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Aspenström, Pontus
    Rho GTPases link cellular contractile force to the density and distribution of nanoscale adhesions2012In: The FASEB Journal, ISSN 0892-6638, E-ISSN 1530-6860, Vol. 26, no 6, p. 2374-2382Article in journal (Refereed)
    Abstract [en]

    The ability of cells to adhere and to exert contractile forces governs their capacity to move within an organism. The cytoskeletal regulators of the Rho GTPase proteins are involved in control of the contractile forces of cells. To elucidate the basis of cell migration, we analyzed contractile forces and nanoscale adhesion-related particles in single cells expressing constitutively active variants of Rho GTPases by using traction-force microscopy and ultra-high-resolution stimulated emission depletion microscopy, respectively. RhoAV14 induced large increases in the contractile forces of single cells, with Rac1L61 and RhoDV26 having more moderate effects. The RhoAV14- and RhoDV26-induced forces showed similar spatial distributions and were accompanied by reduced or unaltered cell spreading. In contrast, the Rac1L61-induced force had different, scattered, force distributions that were linked to increased cell spreading. All three of these Rho GTPase activities caused a loss of thick stress fibers and focal adhesions and a more homogenous distribution of nanoscale adhesion-related particles over the ventral surface of the cells. Interestingly, only RhoAV14 increased the density of these particles. Our data suggest a Rac1-specific mode for cells to generate contractile forces. Importantly, increased density and a more homogenous distribution of these small adhesion-related particles promote cellular contractile forces.-Gad, A. K. B., Ronnlund, D., Spaar, A., Savchenko, A. A., Petranyi, G., Blom, H., Szekely, L., Widengren, J., Aspenstrom, P. Rho GTPases link cellular contractile force to the density and distribution of nanoscale adhesions.

  • 29.
    Gräslund, Astrid
    et al.
    Stockholm University, dept Biochem and Biophys.
    Rigler, RudolfEPFL, Lausanne.Widengren, JerkerKTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Single Molecule Spectroscopy in Chemistry, Physics and Biology: Nobel symposium2010Collection (editor) (Refereed)
  • 30. Hassler, Kai
    et al.
    Rigler, Per
    Blom, Hans
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Rigler, Rudolf
    Widengren, Jerker
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Lasser, Theo
    Dynamic disorder in horseradish peroxidase observed with total internal reflection fluorescence correlation spectroscopy2007In: Optics Express, ISSN 1094-4087, E-ISSN 1094-4087, Vol. 15, no 9, p. 5366-5375Article in journal (Refereed)
    Abstract [en]

    This paper discusses the application of objective-type total internal reflection fluorescence correlation spectroscopy (TIR-FCS) to the study of the kinetics of immobilized horseradish peroxidase on a single molecule level. Objective-type TIR-FCS combines the advantages of FCS with TIRF microscopy in a way that allows for simultaneous ultra-sensitive spectroscopic measurements using a single-point detector and convenient localization of single molecules on a surface by means of parallel imaging.

  • 31.
    Hevekerl, Heike
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Spielmann, Thiemo
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Chmyrov, Andriy
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Widengren, Jerker
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Forster Resonance Energy Transfer beyond 10 nm: Exploiting the Triplet State Kinetics of Organic Fluorophores2011In: Journal of Physical Chemistry B, ISSN 1520-6106, E-ISSN 1520-5207, Vol. 115, no 45, p. 13360-13370Article in journal (Refereed)
    Abstract [en]

    Inter- or intramolecular distances of biomolecules can be studied by Forster resonance energy transfer (FRET). For most FRET methods, the observable range of distances is limited to 1-10 nm, and the labeling efficiency has to be controlled carefully to obtain accurate distance determinations, especially for intensity-based methods. In this study, we exploit the triplet state of the acceptor fluorophore as a FRET readout using fluorescence correlation spectroscopy and transient state monitoring. The influence of donor fluorescence leaking into the acceptor channel is minimized by a novel suppression algorithm for spectral bleed-through, thereby tolerating a high excess (up to 100-fold) of donor-only labeled samples. The suppression algorithm and the high sensitivity of the triplet state to small changes in the fluorophore excitation rate make it possible to extend the observable range of FRET efficiencies by up to 50% in the presence of large donor-only populations. Given this increased range of FRET efficiencies, its compatibility with organic fluorophores, and the low requirements on the labeling efficiency and instrumentation, we foresee that this approach will be attractive for in vitro and in vivo FRET-based spectroscopy and imaging.

  • 32.
    Hevekerl, Heike
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Tornmalm, Johan
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Widengren, Jerker
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Fluorescence-based characterization of non-fluorescent transient states of tryptophan - prospects for protein conformation and interaction studies2016In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 6, article id 35052Article in journal (Refereed)
    Abstract [en]

    Tryptophan fluorescence is extensively used for label-free protein characterization. Here, we show that by analyzing how the average tryptophan fluorescence intensity varies with excitation modulation, kinetics of tryptophan dark transient states can be determined in a simple, robust and reliable manner. Thereby, highly environment-, protein conformation- and interaction-sensitive information can be recorded, inaccessible via traditional protein fluorescence readouts. For verification, tryptophan transient state kinetics were determined under different environmental conditions, and compared to literature data. Conformational changes in a spider silk protein were monitored via the triplet state kinetics of its tryptophan residues, reflecting their exposure to an air-saturated aqueous solution. Moreover, tryptophan fluorescence anti-bunching was discovered, reflecting local pH and buffer conditions, previously observed only by ultrasensitive measurements in highly fluorescent photo-acids. Taken together, the presented approach, broadly applicable under biologically relevant conditions, has the potential to become a standard biophysical approach for protein conformation, interaction and microenvironment studies.

  • 33.
    Hevekerl, Heike
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Widengren, Jerker
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Characterization of non-fluorescent transient states of tryptophan by fluorescence relaxation: Influence of buffers, additives and prospects for protein studiesManuscript (preprint) (Other academic)
  • 34.
    Hevekerl, Heike
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Widengren, Jerker
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Determination of molecular stoichiometry without reference samples by analysing fluorescence blinking with and without excitation synchronizationManuscript (preprint) (Other academic)
    Abstract [en]

    Stoichiometry of molecular complexes plays a crucial role in biology. Moreover, for quantitative fluorescence studies, it is often useful to know the number of fluorophores labeled onto the molecules studied. In this work, we propose an approach to determine the number of independent fluorescence emitters on fluorescent molecules based on fluorescence blinking caused by photo-induced triplet state formation, photo-isomerization or charge transfer. The fluorescence blinking is measured under two different excitation regimes, on the same setup, and in one and the same sample. By comparing the fluorescence fluctuations under continuous excitation using Fluorescence Correlation Spectroscopy (FCS), when all the fluorophores are blinking independently of each other, with those occurring under square-pulsed excitation using Transient State (TRAST) spectroscopy, when all fluorophores are blinking in a synchronized manner, the number of fluorophores per molecule can be determined. No calibration sample is needed and the approach is independent of experimental conditions and of the specific environment of the molecules under study.

    The approach was experimentally validated by labeling double stranded DNA (dsDNA) with different concentrations of the intercalating dye YOYO-1 Iodide. The sample was then measured consecutively by TRAST and FCS and the number of fluorophores per molecule was calculated. The determined numbers were found to agree well with the number of fluorophores per dsDNA, as determined from FCS measurements using additional calibration samples.

  • 35.
    Hevekerl, Heike
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Widengren, Jerker
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Determination of molecular stoichiometry without reference samples by analyzing fluorescence blinking with and without excitation synchronization2015In: METHODS AND APPLICATIONS IN FLUORESCENCE, ISSN 2050-6120, Vol. 3, no 2, article id 025001Article in journal (Refereed)
    Abstract [en]

    Stoichiometry of molecular complexes plays a crucial role in biology. Moreover, for quantitative fluorescence studies, it is often useful to know the number of fluorophores labeled onto the molecules studied. In this work, we propose an approach to determine the number of independent fluorescence emitters on fluorescent molecules based on fluorescence blinking caused by photo-induced triplet state formation, photo-isomerization or charge transfer. The fluorescence blinking is measured under two different excitation regimes, on the same setup, and in one and the same sample. By comparing the fluorescence fluctuations under continuous excitation using Fluorescence Correlation Spectroscopy (FCS), when all the fluorophores are blinking independently of each other, with those occurring under square-pulsed excitation using Transient State (TRAST) spectroscopy, when all fluorophores are blinking in a synchronized manner, the number of fluorophores per molecule can be determined. No calibration sample is needed and the approach is independent of experimental conditions and of the specific environment of the molecules under study. The approach was experimentally validated by labeling double stranded DNA (dsDNA) with different concentrations of the intercalating dye YOYO-1 Iodide. The sample was then measured consecutively by TRAST and FCS and the number of fluorophores per molecule was calculated. The determined numbers were found to agree well with the number of fluorophores per dsDNA, as determined from FCS measurements using additional calibration samples.

  • 36.
    Hevekerl, Heike
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Wigenius, Jens
    Persson, Gustav
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Inganäs, Olle
    Widengren, Jerker
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Dark states in ionic oligothiophene bioprobes-evidence from fluorescence correlation spectroscopy and dynamic light scattering2014In: Journal of Physical Chemistry B, ISSN 1520-6106, E-ISSN 1520-5207, Vol. 118, no 22, p. 5924--5933Article in journal (Refereed)
    Abstract [en]

    Luminescent conjugated polyelectrolytes (LCPs) can upon interaction with biological macromolecules change their luminescent properties, and thereby serve as conformation- and interaction-sensitive biomolecular probes. However, to exploit this in a more quantitative manner, there is a need to better understand the photophysical processes involved. We report studies of the conjugated pentameric oligothiophene, derivative p-FTAA, which changes optical properties with different p-FTAA concentrations in aqueous buffers, and in a pH and oxygen saturation dependent manner. Using dynamic light scattering, luminescence spectroscopy and fluorescence correlation spectroscopy, we find evidence for a monomer dimer equilibrium, for the formation of large clusters of p-FTAA in aqueous environment, and can couple aggregation to changed emission properties of oligothiophenes. In addition, we observe the presence of at least two dark transient states, one presumably being a triplet state. Oxygen was found to statically quench the p-FTAA fluorescence but also to promote molecular fluorescence by quenching dark transient states of the p-FTAA molecules. Taken together, this study provides knowledge of fluorescence and photophysical features essential for applying p-FTAA and other oligothiophene derivatives for diagnostic purposes, including detection and staining of amyloid aggregates.

  • 37.
    Huang, Bingru
    et al.
    South China Normal Univ, South China Acad Adv Optoelect, Ctr Opt & Electromagnet Res, Guangdong Prov Key Lab Opt Informat Mat & Technol, Guangzhou 510006, Guangdong, Peoples R China.
    Bergstrand, Jan
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Duan, Sai
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Theoretical Chemistry and Biology.
    Zhan, Qiuqiang
    South China Normal Univ, South China Acad Adv Optoelect, Ctr Opt & Electromagnet Res, Guangdong Prov Key Lab Opt Informat Mat & Technol, Guangzhou 510006, Guangdong, Peoples R China.
    Widengren, Jerker
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Ågren, Hans
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Theoretical Chemistry and Biology.
    Liu, Haichun
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Theoretical Chemistry and Biology.
    Overtone Vibrational Transition-Induced Lanthanide Excited-State Quenching in Yb3+/Er3+-Doped Upconversion Nanocrystals2018In: ACS Nano, ISSN 1936-0851, E-ISSN 1936-086X, Vol. 12, p. 10572-10575Article in journal (Refereed)
  • 38. Iovino, Federico
    et al.
    Engelen-Lee, Joo-Yeon
    Brouwer, Matthijs
    van de Beek, Diederik
    van der Ende, Arie
    Seron, Merche Valls
    Mellroth, Peter
    Muschiol, Sandra
    Bergstrand, Jan
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Widengren, Jerker
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Henriques-Normark, Birgitta
    pIgR and PEC AM-1 bind to pneumococcal adhesins RrgA and PspC mediating bacterial brain invasion2017In: Journal of Experimental Medicine, ISSN 0022-1007, E-ISSN 1540-9538, Vol. 214, no 6, p. 1619-1630Article in journal (Refereed)
    Abstract [en]

    Streptococcus pneumoniae is the main cause of bacterial meningitis, a life-threating disease with a high case fatality rate despite treatment with antibiotics. Pneumococci cause meningitis by invading the blood and penetrating the blood-brain barrier (BBB). Using stimulated emission depletion (STED) super-resolution microscopy of brain biopsies from patients who died of pneumococcal meningitis, we observe that pneumococci colocalize with the two BBB endothelial receptors: polymeric immunoglobulin receptor (pIgR) and platelet endothelial cell adhesion molecule (PECAM-1). We show that the major adhesin of the pneumococcal pilus-1, RrgA, binds both receptors, whereas the choline binding protein PspC binds, but to a lower extent, only pIgR. Using a bacteremia-derived meningitis model and mutant mice, as well as antibodies against the two receptors, we prevent pneumococcal entry into the brain and meningitis development. By adding antibodies to antibiotic (ceftriaxone)-treated mice, we further reduce the bacterial burden in the brain. Our data suggest that inhibition of pIgR and PECAM-1 has the potential to prevent pneumococcal meningitis.

  • 39. Kronqvist, Nina
    et al.
    Otikovs, Martins
    Chmyrov, Volodymyr
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Chen, Gefei
    Andersson, Marlene
    Nordling, Kerstin
    Landreh, Michael
    Sarr, Médoune
    Jörnvall, Hans
    Wennmalm, Stefan
    Widengren, Jerker
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Meng, Qing
    Rising, Anna
    Otzen, Daniel Erik Rik
    Knight, Stefan
    Jaudzems, Kristaps
    Johansson, Jan Ove
    Sequential pH-driven dimerization and stabilization of the N-terminal domain enables rapid spider silk formation2014In: Nature Communications, ISSN 2041-1723, E-ISSN 2041-1723, Vol. 5, p. 3254-Article in journal (Refereed)
    Abstract [en]

    The mechanisms controlling the conversion of spider silk proteins into insoluble fibres, which happens in a fraction of a second and in a defined region of the silk glands, are still unresolved. The N-terminal domain changes conformation and forms a homodimer when pH is lowered from 7 to 6; however, the molecular details still remain to be determined. Here we investigate site-directed mutants of the N-terminal domain from Euprosthenops australis major ampullate spidroin 1 and find that the charged residues D40, R60 and K65 mediate intersubunit electrostatic interactions. Protonation of E79 and E119 is required for structural conversions of the subunits into a dimer conformation, and subsequent protonation of E84 around pH 5.7 leads to the formation of a fully stable dimer. These residues are highly conserved, indicating that the now proposed three-step mechanism prevents premature aggregation of spidroins and enables fast formation of spider silk fibres in general.

  • 40. Leutenegger, Marcel
    et al.
    Blom, Hans
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Widengren, Jerker
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Eggeling, Christian
    Goesch, Michael
    Leitgeb, Rainer A.
    Lasser, Theo
    Dual-color total internal reflection fluorescence cross-correlation spectroscopy2006In: Journal of Biomedical Optics, ISSN 1083-3668, E-ISSN 1560-2281, Vol. 11, no 4Article in journal (Refereed)
    Abstract [en]

    We present the development and first application of a novel dual-color total internal reflection (TIR) fluorescence system for single-molecule coincidence analysis and fluorescence cross-correlation spectroscopy (FCCS). As a performance analysis, we measured a synthetic DNA-binding assay, demonstrating this dual-color TIR-FCCS approach to be a suitable method for measuring coincidence assays such as biochemical binding, fusion, or signal transduction at solid/liquid interfaces. Due to the very high numerical aperture of the epi-illumination configuration, our setup provides a very high fluorescence collection efficiency resulting in a two- to three- fold increase in molecular brightness compared to conventional confocal FCCS. Further improvements have been achieved through global analysis of the spectroscopic data.

  • 41. Lin, Hongzhen
    et al.
    Tian, Yuxi
    Zapadka, Karolina
    Persson, Gustav
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Thomsson, Daniel
    Mirzov, Oleg
    Larsson, Per-Olof
    Widengren, Jerker
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Scheblykin, Ivan G.
    Fate of Excitations in Conjugated Polymers: Single-Molecule Spectroscopy Reveals Nonemissive "Dark" Regions in MEH-PPV Individual Chains2009In: Nano letters (Print), ISSN 1530-6984, E-ISSN 1530-6992, Vol. 9, no 12, p. 4456-4461Article in journal (Refereed)
    Abstract [en]

    Single chains of the conjugated polymer MEH-PPV (poly(2-methoxy-5-(2'-ethylhexyloxy)-1,4-phenylenevinylene)) were studied with wide-field fluorescence microscopy (dispersion in inert polymer matrices) and with fluorescence correlation spectroscopy (chloroform solution). The fluorescence yield of individual molecules in matrices was found to be 1-2 orders of magnitude lower than that in solution and it decreased substantially with increasing chain length. It suggests that isolation of MEH-PPV molecules in polymer matrices creates favorable conditions for photogeneration of nonemissive primary excited states.

  • 42.
    Linda, Öjemyr
    et al.
    Stockholm University, dept Biochem and Biophys.
    Sandén, Tor
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Widengren, Jerker
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Brzezinski, Peter
    Stockholm University, dept Biochem and Biophys.
    Lateral Proton Transfer between the Membrane and a Membrane Protein2009In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 48, no 10, p. 2173-2179Article in journal (Refereed)
    Abstract [en]

    Proton transport across biological membranes is a key step of the energy conservation machinery in living organisms, and it has been proposed that the membrane itself plays an important role in this process. In the present study we have investigated the effect of incorporation of a proton transporter, cytochrome c oxidase, into a membrane on the protonation kinetics of a fluorescent pH-sensitive probe attached at the surface of the protein. The results show that proton transfer to the probe was slightly accelerated upon attachment at the protein surface (similar to 7 x 10(10) s(-1) M-1, compared to the expected value of (1-2) x 10(10) s(-1) M-1), which is presumably due to the presence of acidic/His groups in the vicinity. Upon incorporation of the protein into small unilamellar phospholipid vesicles the rate increased by more than a factor of 400 to similar to 3 x 10(13) s(-1) M-1, which indicates that the protein-attached probe is in rapid protonic contact with the membrane surface. The results indicate that. the membrane acts to accelerate proton uptake by the membrane-bound proton transporter.

  • 43. Lomnytska, Marta
    et al.
    Pinto, Rui
    Becker, Susanne
    Engstrom, Ulla
    Gustafsson, Sonja
    Bjorklund, Christina
    Templin, Markus
    Bergstrand, Jan
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Xu, Lei
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Widengren, Jerker
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Epstein, Elisabeth
    Franzen, Bo
    Auer, Gert
    Platelet protein biomarker panel for ovarian cancer diagnosis2018In: BIOMARKER RESEARCH, ISSN 2050-7771, Vol. 6, article id 2Article in journal (Refereed)
    Abstract [en]

    Background: Platelets support cancer growth and spread making platelet proteins candidates in the search for biomarkers. Methods: Two-dimensional (2D) gel electrophoresis, Partial Least Squares Discriminant Analysis (PLS-DA), Western blot, DigiWest. Results: PLS-DA of platelet protein expression in 2D gels suggested differences between the International Federation of Gynaecology and Obstetrics (FIGO) stages III-IV of ovarian cancer, compared to benign adnexal lesions with a sensitivity of 96% and a specificity of 88%. A PLS-DA-based model correctly predicted 7 out of 8 cases of FIGO stages I-II of ovarian cancer after verification by western blot. Receiver-operator curve (ROC) analysis indicated a sensitivity of 83% and specificity of 76% at cut-off >0.5 (area under the curve (AUC) = 0.831, p < 0.0001) for detecting these cases. Validation on an independent set of samples by DigiWest with PLS-DA differentiated benign adnexal lesions and ovarian cancer, FIGO stages III-IV, with a sensitivity of 70% and a specificity of 83%. Conclusion: We identified a group of platelet protein biomarker candidates that can quantify the differential expression between ovarian cancer cases as compared to benign adnexal lesions.

  • 44. Luu, Thuy Thanh
    et al.
    Vignesh, Sunitha
    Wagner, Arnika Kathleen
    Chrobok, Michael
    Schlums, Heinrich
    Tornmalm, Johan
    KTH, School of Engineering Sciences (SCI), Applied Physics.
    Kadri, Nadir
    Meinke, Stephan
    Widengren, Jerker
    KTH, School of Engineering Sciences (SCI), Applied Physics.
    Bryceson, Yenan
    Alici, Evren
    Johannsson, Sofia
    Hoglund, Petter
    Reactive oxygen species are linked with general phosphorylation status and functional education of natural killer cells2017In: Scandinavian Journal of Immunology, ISSN 0300-9475, E-ISSN 1365-3083, Vol. 86, no 4, p. 295-295Article in journal (Other academic)
  • 45. Margittai, M.
    et al.
    Widengren, Jerker
    Schweinberger, E.
    Schroder, G. F.
    Felekyan, S.
    Haustein, E.
    Konig, M.
    Fasshauer, D.
    Grubmuller, H.
    Jahn, R.
    Seidel, C. A. M.
    Single-molecule fluorescence resonance energy transfer reveals a dynamic equilibrium between closed and open conformations of syntaxin 12003In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 100, no 26, p. 15516-15521Article in journal (Refereed)
    Abstract [en]

    Protein conformational transitions form the molecular basis of many cellular processes, such as signal transduction and membrane traffic. However, in many cases, little is known about their structural dynamics. Here we have used dynamic single-molecule fluorescence to study at high time resolution, conformational transitions of syntaxin 1, a soluble N-ethylmaleimide-sensitive factor attachment protein receptors protein essential for exocytotic membrane fusion. Sets of syntaxin double mutants were randomly labeled with a mix of donor and acceptor dye and their fluorescence resonance energy transfer was measured. For each set, all fluorescence information was recorded simultaneously with high time resolution, providing detailed information on distances and dynamics that were used to create structural models. We found that free syntaxin switches between an inactive closed and an active open configuration with a relaxation time of 0.8 ms, explaining why regulatory proteins are needed to arrest the protein in one conformational state.

  • 46. Mellroth, Peter
    et al.
    Daniels, Robert
    Eberhardt, Alice
    Rönnlund, Daniel
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Blom, Hans
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Widengren, Jerker
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Normark, Staffan
    Henriques-Normark, Birgitta
    LytA, Major Autolysin of Streptococcus pneumoniae, Requires Access to Nascent Peptidoglycan2012In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 287, no 14, p. 11018-11029Article in journal (Refereed)
    Abstract [en]

    Background: The regulation of cell wall hydrolysis by the pneumococcal autolysin LytA is poorly understood. Results: The cell wall is susceptible to extracellular LytA only during the stationary phase or after cell wall synthesis inhibition. Conclusion: LytA is regulated on the substrate level, where peptidoglycan modifications likely prevent LytA hydrolysis. Significance: The control of amidases is essential for bacterial survival, cell-wall synthesis, and division.

  • 47. Mocsar, Gabor
    et al.
    Volko, Julianna
    Rönnlund, Daniel
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Widengren, Jerker
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Nagy, Peter
    Szollosi, Janos
    Toth, Katalin
    Goldman, Carolyn K.
    Damjanovich, Sandor
    Waldmann, Thomas A.
    Bodnar, Andrea
    Vamosi, Gyorgy
    MHC I Expression Regulates Co-clustering and Mobility of Interleukin-2 and-15 Receptors in T Cells2016In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 111, no 1, p. 100-112Article in journal (Refereed)
    Abstract [en]

    MHC glycoproteins form supramolecular clusters with interleukin-2 and -15 receptors in lipid rafts of T cells. The role of highly expressed MHC I in maintaining these clusters is unknown. We knocked down MHC I in FT7.10 human T cells, and studied protein clustering at two hierarchic levels: molecular aggregations and mobility by Forster resonance energy transfer and fluorescence correlation spectroscopy; and segregation into larger domains or superclusters by superresolution stimulated emission depletion microscopy. Fluorescence correlation spectroscopy-based molecular brightness analysis revealed that the studied molecules diffused as tight aggregates of several proteins of a kind. Knockdown reduced the number of MHC I containing molecular aggregates and their average MHC I content, and decreased the heteroassociation of MHC I with IL-2R alpha/IL-15R alpha. The mobility of not only MHC I but also that of IL-2R alpha/IL-15R alpha increased, corroborating the general size decrease of tight aggregates. A multifaceted analysis of stimulated emission depletion images revealed that the diameter of MHC I superclusters diminished from 400-600 to 200-300 nm, whereas those of IL-2R alpha/IL-15R alpha hardly changed. MHC I and IL-2R alpha/IL-15R alpha colocalized with GM1 ganglioside-rich lipid rafts, but MHC I clusters retracted to smaller subsets of GM1-and IL-2R alpha/IL-15R alpha-rich areas upon knockdown. Our results prove that changes in expression level may significantly alter the organization and mobility of interacting membrane proteins.

  • 48.
    Mücksch, Jonas
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Spielmann, Thiemo
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Sisamakis, Evangelos
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Widengren, Jerker
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Transient state imaging of live cells using single plane illumination and arbitrary duty cycle excitation pulse trains2015In: Journal of Biophotonics, ISSN 1864-063X, E-ISSN 1864-0648, Vol. 8, no 5, p. 392-400Article in journal (Refereed)
    Abstract [en]

    We demonstrate the applicability of Single Plane Illumination Microscopy to Transient State Imaging (TRAST), offering sensitive microenvironmental information together with optical sectioning and reduced overall excitation light exposure of the specimen. The concept is verified by showing that transition rates can be determined accurately for free dye in solution and that fluorophore transition rates can be resolved pixel-wise in live cells. Furthermore, we derive a new theoretical framework for analyzing TRAST data acquired with arbitrary duty cycle pulse trains. By this analysis it is possible to reduce the overall measurement time and thereby enhance the frame rates in TRAST imaging.

  • 49.
    Nordgren, Niklas
    et al.
    KTH, School of Chemical Science and Engineering (CHE).
    Rathje, Li-Sophie
    Karolinska Inst, Dept Microbiol Tumor & Cell Biol, Stockholm, Sweden..
    Pettersson, Torbjörn
    KTH, School of Chemical Science and Engineering (CHE).
    Rönnlund, Daniel
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Widengren, Jerker
    KTH, School of Chemical Science and Engineering (CHE).
    Aspenstrom, Pontus
    Karolinska Inst, Dept Microbiol Tumor & Cell Biol, Stockholm, Sweden..
    Gad, Annica
    Karolinska Inst, Dept Microbiol Tumor & Cell Biol, Stockholm, Sweden..
    Oncogene induced stiffening of living cells2015In: Abstracts of Papers of the American Chemical Society, ISSN 0065-7727, Vol. 249Article in journal (Other academic)
  • 50.
    Ojemyr, Linda Nasvik
    et al.
    Stockholm Univ, Dept Biochem & Biophys, Arrhenius Labs Nat Sci, S-10691 Stockholm, Sweden..
    Sanden, Tor
    Royal Inst Technol, Dept Appl Phys, Stockholm, Sweden..
    Widengren, Jerker
    KTH, School of Engineering Sciences (SCI), Applied Physics, Quantum and Biophotonics.
    Brzezinski, Peter
    Stockholm Univ, Dept Biochem & Biophys, Arrhenius Labs Nat Sci, S-10691 Stockholm, Sweden..
    Membrane-facilitated proton transfer to the surface of a membrane-spanning proton transporter2010In: Biochimica et Biophysica Acta - Bioenergetics, ISSN 0005-2728, E-ISSN 1879-2650, Vol. 1797, p. 98-98Article in journal (Other academic)
123 1 - 50 of 119
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