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  • 101.
    Widengren, Jerker
    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.
    Eggeling, Christian
    Max-Planck-Institut fu¨r Biophysikalische Chemie.
    Löfdahl, Per-Åke
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Seidel, Claus
    Heinrich-Heine Universität.
    Strategies to Improve Photostabilities in Ultrasensitive Fluorescence Spectroscopy2007In: Journal of Physical Chemistry A, ISSN 1089-5639, E-ISSN 1520-5215, Vol. 111, no 3, p. 429-440Article in journal (Refereed)
    Abstract [en]

    Given the particular importance of dye photostability for single-molecule and fluorescence fluctuation spectroscopy investigations, refined strategies were explored for how to chemically retard dye photobleaching. These strategies will be useful for fluorescence correlation spectroscopy (FCS), fluorescence-based confocal single-molecule detection (SMD) and related techniques. In particular, the effects on the addition of two main categories of antifading compounds, antioxidants (n-propyl gallate, nPG, ascorbic acid, AA) and triplet state quenchers (mercaptoethylamine, MEA, cyclo-octatetraene, COT), were investigated, and the relevant rate parameters involved were determined for the dye Rhodamine 6G. Addition of each of the compound categories resulted in significant improvements in the fluorescence brightness of the monitored fluorescent molecules in FCS measurements. For antioxidants, we identify the balance between reduction of photoionized fluorophores on the one hand and that of intact fluorophores on the other as an important guideline for what concentrations to be added for optimal fluorescence generation in FCS and SMD experiments. For nPG/AA, this optimal concentration was found to be in the lower micromolar range, which is considerably less than what has previously been suggested. Also, for MEA, which is a compound known as a triplet state quencher, it is eventually its antioxidative properties and the balance between reduction of fluorophore cation radicals and that of intact fluorophores that defines the optimal added concentration. Interestingly, in this optimal concentration range the triplet state quenching is still far from sufficient to fully minimize the triplet populations. We identify photoionization as the main mechanism of photobleaching within typical transit times of fluorescent molecules through the detection volume in a confocal FCS or SMD instrument (<1−20 ms), and demonstrate its generation via both one- and multistep excitation processes. Apart from reflecting a major pathway for photobleaching, our results also suggest the exploitation of the photoinduced ionization and the subsequent reduction by antioxidants for biomolecular monitoring purposes and as a possible switching mechanism with applications in high-resolution microscopy.

  • 102.
    Widengren, Jerker
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Kudryavtsev, V.
    Antonik, M.
    Berger, S.
    Gerken, M.
    Seidel, C. A. M.
    Single-molecule detection and identification of multiple species by multiparameter fluorescence detection2006In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 78, no 6, p. 2039-2050Article in journal (Refereed)
    Abstract [en]

    Two general strategies are introduced to identify and quantify single molecules in dilute solutions by employing a spectroscopic method for data registration and specific burst analysis, denoted multiparameter fluorescence detection (MFD). MFD uses pulsed excitation and time-correlated single-photon counting to simultaneously monitor the evolution of the eight-dimensional fluorescence information (fundamental anisotropy, fluorescence lifetime, fluorescence intensity, time, excitation spectrum, fluorescence spectrum, fluorescence quantum yield, distance between fluorophores) in real time and allows for selection of specific events for subsequent analysis. Using the multiple fluorescence dimensions, we demonstrate a dye labeling scheme of oligonucleotides, by which it is possible to identify and separate 16 different compounds in the mixture via their characteristic pattern by MFD. Such identification procedures and multiplex assays with single-molecule sensitivity may have a great impact on screening of species and events that do not lend themselves so easily to amplification, such as disease-specific proteins and their interactions.

  • 103. Widengren, Jerker
    et al.
    Schweinberger, E.
    Berger, S.
    Seidel, C. A. M.
    Two new concepts to measure fluorescence resonance energy transfer via fluorescence correlation spectroscopy: Theory and experimental realizations2001In: Journal of Physical Chemistry A, ISSN 1089-5639, E-ISSN 1520-5215, Vol. 105, no 28, p. 6851-6866Article in journal (Refereed)
    Abstract [en]

    In this study, we demonstrate two new concepts, using fluorescence correlation spectroscopy (FCS), to characterize fluorescence resonance energy transfer (FRET). The two approaches were tested experimentally by measuring a series of double-stranded DNA molecules, with different numbers of base-pairs separating the donor (Alexa488) and acceptor (Cy5) fluorophores. In the first approach, FRET efficiencies are determined from the detected acceptor fluorescence rate per molecule. Here, the unique possibility with FCS to determine the mean number of molecules within the detection volume is exploited, making a concentration calibration superfluous. The second approach takes advantage of FRET-dependent fluorescence fluctuations of photophysical origin, in particular fluctuations generated by trans-cis isomerization of the acceptor dye. The rate of interchange,between the trans and cis states is proportional to the excitation rate and can be conveniently measured by FCS. Under FRET-mediated excitation, this rate can be used as a direct measure of the FRET efficiency. The measured isomerization rate depends only on the fluctuations in the acceptor fluorescence, and is not affected by donor, fluorescence cross-talk, background, dye labeling efficiencies, or by the concentration of molecules under study. The measured FRET efficiencies are well in agreement with a structural model of DNA. Furthermore, additional structural information is obtained from simulations of the measured fraction of acceptor dyes being in a nonfluorescent cis conformation, from which differences in the position and orientation of the trans and cis form of the acceptor dye can be predicted.

  • 104. Widengren, Jerker
    et al.
    Schwille, P.
    Characterization of photoinduced isomerization and back-isomerization of the cyanine dye Cy5 by fluorescence correlation spectroscopy2000In: Journal of Physical Chemistry A, ISSN 1089-5639, E-ISSN 1520-5215, Vol. 104, no 27, p. 6416-6428Article in journal (Refereed)
    Abstract [en]

    Cy5 is one of a few commercially available dyes in the near-infrared wavelength range. In this study, the fluorescence fluctuations of Cy5 have been investigated under steady-state excitation conditions by fluorescence correlation spectroscopy (FCS). The fluctuations in fluorescence are compatible with and can be used to characterize the photoinduced isomerization and back-isomerization, as well as the transitions between the singlet and triplet states of the dye. By employing a simple kinetic model, the rate constants of these processes can be determined. The model was used over a broad range of experimental conditions, where the influence on the isomerization properties of solvent viscosity, polarity, and temperature, excitation intensity and wavelength, and the presence of different side groups was investigated. We propose FCS as a useful and simple complementary approach to study isomerization processes of cyanine dyes yielding information about the rates of both the photoinduced isomerization and the back-isomerization, as well as of the kinetic properties of the triplet states. Our data show that for most excitation conditions relevant for ultrasensitive fluorescence spectroscopy a photostationary equilibrium is established between the isomeric forms, where approximately 50% of the Cy5 dye molecules can be expected to be in their weakly fluorescent cis states. The fluorophores therefore lose about half of their fluorescence capacity. This is of relevance for the performance of the dye in all applications of fluorescence spectroscopy where a high sensitivity or a fast readout is required, such as in single-molecule detection experiments and in many applications of confocal laser scanning microscopy.

  • 105. Widengren, Jerker
    et al.
    Seidel, C. A. M.
    Manipulation and characterization of photo-induced transient states of Merocyanine 540 by fluorescence correlation spectroscopy2000In: Physical Chemistry, Chemical Physics - PCCP, ISSN 1463-9076, E-ISSN 1463-9084, Vol. 2, no 15, p. 3435-3441Article in journal (Refereed)
    Abstract [en]

    In this study, fluorescence correlation spectroscopy (FCS) is used to investigate the photo-induced transient states of Merocyanine 540 (MC540), a fluorescent agent for photodynamic therapy. Two relaxation processes are observed in the FCS measurements, which can be attributed to trans-cis isomerization and triplet state formation. Under the photostationary conditions present in the detection volume of the FCS measurements, the steady state populations of the photo-isomer and the triplet state, as well as their relaxation rates, can be determined. The population of the triplet states was noticeably reduced by light-induced deactivation at 515 nm excitation, and by simultaneous excitation at 647 nm the triplet state build-up could be almost eliminated with a concomitant increase in fluorescence intensity. By applying a simplified kinetic model for the measured fluorescence fluctuations it is possible to determine the rates for intersystem crossing, triplet state decay, as well as photo-induced isomerization and back-isomerization. In relation to other present techniques, FCS offers a relatively simple way to monitor photo-induced trans-cis isomerization, and cis-trans back-isomerization. For MC540, it is desirable to increase the triplet state formation at the expense of trans-cis isomerization in order to optimize the photodynamic action. FCS is well suited to monitor these processes on a microscopic scale, and thus to follow the local potency of MC540 as a photodynamic agent, at its site of action in target cells.

  • 106.
    Widengren, Jerker
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Thyberg, Per
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    FCS cell surface measurements - Photophysical limitations and consequences on molecular ensembles with heterogenic mobilities2005In: Cytometry Part A, ISSN 1552-4922, Vol. 68A, no 2, p. 101-112Article in journal (Refereed)
    Abstract [en]

    Background: Fluorescence Correlation Spectroscopy is a powerful method to analyze densities and diffusive behavior of molecules in membranes, but effects of photodegradation can easily be overlooked. Method: Based on experimental photophysical parameters, calculations were performed to analyze the consequences of photobleaching in fluorescence correlation spectroscopy (FCS) cell surface experiments, covering a range of standard measurement conditions. Results: Cumulative effects of photobleaching can be prominent, although an absolute majority of the fluorescent molecules would pass the laser excitation beam without being photobleached. Given a distribution of molecules on a cell surface with different diffusive properties, the fraction of molecules that is actually analyzed depends strongly on the excitation intensities and measurement times, as well as on the size of the reservoir of freely diffusing molecules. Both the slower and the faster diffusing molecules can be disfavored. Conclusions: Apart from quantifying photobleaching effects, the calculations suggest that the effects can be used to extract additional information, for instance about the size of the reservoirs of free diffusion. By certain choices of measurement conditions, it may be possible to more specifically analyze certain species within a population, based on their different diffusive properties, different areas of free diffusion, or different kinetics of possible transient binding.

  • 107. Wigenius, Jens
    et al.
    Persson, Gustav
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Widengren, Jerker
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Inganäs, Olle
    Interactions Between a Luminescent Conjugated Oligoelectrolyte and Insulin During Early Phases of Amyloid Formation2011In: Macromolecular Bioscience, ISSN 1616-5187, E-ISSN 1616-5195, Vol. 11, no 8, p. 1120-1127Article in journal (Refereed)
    Abstract [en]

    Aggregates of misfolded proteins play an important role in diseases such as Alzheimer's. Here it is demonstrated how the anionic oligothiophene p-FTAA interacts with and influences pre-fibrillar protein assemblies during the earlier stages of in vitro fibrillation. Conjugated polythiophenes have previously been demonstrated to detect and discriminate between different types of protein aggregates and also introduce luminescent or conductive properties to these nanoscale fiber structures. Fluorescence spectroscopy, DLS, TEM and FCS are employed to follow the interplay between p-FTAA and insulin during in vitro fibrillation.

  • 108.
    Xu, Hao
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    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.
    Brismar, Hjalmar
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Fu, Ying
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Mechanisms of fluorescence decays of colloidal CdSe-CdS/ZnS quantum dots unraveled by time-resolved fluorescence measurement2015In: Physical Chemistry, Chemical Physics - PCCP, ISSN 1463-9076, E-ISSN 1463-9084, Vol. 17, no 41, p. 27588-27595Article in journal (Refereed)
    Abstract [en]

    By narrowing the detection bandpass and increasing the signal-to-noise ratio in measuring the time-resolved fluorescence decay spectrum of colloidal CdSe-CdS/ZnS quantum dots (QDs), we show that directly after the photoexcitation, the fluorescence decay spectrum is characterized by a single exponential decay, which represents the energy relaxation of the photogenerated exciton from its initial high-energy state to the ground exciton state. The fluorescence decay spectrum of long decay time is in the form of beta/t(2), where beta is the radiative recombination time of the ground-state exciton and t is the decay time. Our findings provide us with a direct and quantitative link between fluorescence decay measurement data and fundamental photophysics of QD exciton, thereby leading to a novel way of applying colloidal QDs to study microscopic, physical and chemical processes in many fields including biomedicine.

  • 109.
    Xu, Lei
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Braun, Laura J.
    Karolinska Inst, Dept Microbiol Tumor & Cell Biol, Stockholm, Sweden..
    Rönnlund, Daniel
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Widengren, Jerker
    KTH, School of Engineering Sciences (SCI), Applied Physics, Quantum and Biophotonics.
    Aspenstrom, Pontus
    Karolinska Inst, Dept Microbiol Tumor & Cell Biol, Stockholm, Sweden..
    Gad, Annica K. B.
    Univ Madeira, CQM, Campus Penteada, P-9020105 Funchal, Portugal..
    Nanoscale localization of proteins within focal adhesions indicates discrete functional assemblies with selective force-dependence2018In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 285, no 9, p. 1635-1652Article in journal (Refereed)
    Abstract [en]

    Focal adhesions (FAs) are subcellular regions at the micrometer scale that link the cell to the surrounding microenvironment and control vital cell functions. However, the spatial architecture of FAs remains unclear at the nanometer scale. We used two-color and three-color super-resolution stimulated emission depletion microscopy to determine the spatial distributions and co-localization of endogenous FA components in fibroblasts. Our data indicate that adhesion proteins inside, but not outside, FAs are organized into nanometer size units of multi-protein assemblies. The loss of contractile force reduced the nanoscale co-localization between different types of proteins, while it increased this co-localization between markers of the same type. This suggests that actomyosin-dependent force exerts a nonrandom, specific, control of the localization of adhesion proteins within cell-matrix adhesions. These observations are consistent with the possibility that proteins in cell-matrix adhesions are assembled in nanoscale particles, and that force regulates the localization of the proteins therein in a protein-specific manner. This detailed knowledge of how the organization of FA components at the nanometer scale is linked to the capacity of the cells to generate contractile forces expands our understanding of cell adhesion in health and disease.

  • 110.
    Xu, Lei
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Braun, Laura
    Karolinska Institutet, Department of Microbiology, Tumor and Cell Biology.
    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.
    Aspenström, Pontus
    Karolinska Institutet, Department of Microbiology, Tumor and Cell Biology.
    Gad, Annica
    Karolinska Institutet, Department of Microbiology, Tumor and Cell Biology.
    Nanoscale analysis of cell-matrix adhesionsManuscript (preprint) (Other academic)
    Abstract [en]

    Cell adhesion to the extracellular matrix is required for physiological processes, such as morphogenesis and wound healing. The cell adheres to the extracellular matrix via focal adhesions, which are considered to be cell adhesion organelles that govern cell function. However, the spatial architecture and organization of focal adhesions at a nanometer scale remain unclear. Therefore, we compared the spatial distribution of focal adhesion components within and outside of focal adhesions, using STED microscopy with resolution of 40-50nm. Our results are consistent with the concept that at the nanometer scale, adhesion proteins within but not outside of focal adhesions are composed by nanoscale protein clusters that attach to the extracellular matrix.

  • 111. Xu, Lei
    et al.
    Ojemyr, Linda Nasvik
    Bergstrand, Jan
    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.
    Protonation Dynamics on Lipid Nanodiscs: Influence of the Membrane Surface Area and External Buffers2016In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 110, no 9, p. 1993-2003Article in journal (Refereed)
    Abstract [en]

    Lipid membrane surfaces can act as proton-collecting antennae, accelerating proton uptake by membrane-bound proton transporters. We investigated this phenomenon in lipid nanodiscs (NDs) at equilibrium on a local scale, analyzing fluorescence fluctuations of individual pH-sensitive fluorophores at the membrane surface by fluorescence correlation spectroscopy (FCS). The protonation rate of the fluorophores was similar to 100-fold higher when located at 9- and 12-nm diameter NDs, compared to when in solution, indicating that the proton-collecting antenna effect is maximal already for a membrane area of similar to 60 nm(2). Fluorophore-labeled cytochrome c oxidase displayed a similar increase when reconstituted in 12 nm NDs, but not in 9 nm NDs, i.e., an acceleration of the protonation rate at the surface of cytochrome c oxidase is found when the lipid area surrounding the protein is larger than 80 nm(2), but not when below 30 nm(2). We also investigated the effect of external buffers on the fluorophore proton exchange rates at the ND membrane-water interfaces. With increasing buffer concentrations, the proton exchange rates were found to first decrease and then, at millimolar buffer concentrations, to increase. Monte Carlo simulations, based on a simple kinetic model of the proton exchange at the membrane-water interface, and using rate parameter values determined in our FCS experiments, could reconstruct both the observed membrane-size and the external buffer dependence. The FCS data in combination with the simulations indicate that the local proton diffusion coefficient along a membrane is similar to 100 times slower than that observed over submillimeter distances by proton-pulse experiments (D-s similar to 10(-5)cm(2)/s), and support recent theoretical studies showing that proton diffusion along membrane surfaces is time- and length-scale dependent.

  • 112.
    Xu, Lei
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Rönnlund, Daniel
    KTH, School of Engineering Sciences (SCI), Applied Physics.
    Aspenstrom, Pontus
    Braun, Laura J.
    Gad, Annica K. B.
    Widengren, Jerker
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Resolution, target density and labeling effects in colocalization studies - suppression of false positives by nanoscopy and modified algorithms2016In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 283, no 5, p. 882-898Article in journal (Refereed)
    Abstract [en]

    Colocalization analyses of fluorescence images are extensively used to quantify molecular interactions in cells. In recent years, fluorescence nanoscopy has approached resolutions close to molecular dimensions. However, the extent to which image resolution influences different colocalization estimates has not been systematically investigated. In this work, we applied simulations and resolution-tunable stimulated emission depletion microscopy to evaluate how the resolution, molecular density and label size of targeted molecules influence estimates of the most commonly used colocalization algorithms (Pearson correlation coefficient, Manders' M1 and M2 coefficients), as well as estimates by the image cross-correlation spectroscopy method. We investigated the practically measureable extents of colocalization for stimulated emission depletion microscopy with positive and negative control samples with an aim to identifying the strengths and weaknesses of nanoscopic techniques for colocalization studies. At a typical optical resolution of a confocal microscope (200-300 nm), our results indicate that the extent of colocalization is typically overestimated by the tested algorithms, especially at high molecular densities. Only minor effects of this kind were observed at higher resolutions (< 60 nm). By contrast, underestimation of colocalization may occur if the resolution is close to the size of the label/affinity molecules themselves. To suppress false positives at confocal resolutions and high molecular densities, we introduce a statistical variant of Costes' threshold searching algorithm, used in combination with correlation-based methods like the Pearson coefficient and the image cross-correlation spectroscopy approach, to set intensity thresholds separating background noise from signals.

  • 113.
    Xu, Lei
    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.
    Gad, Annica
    Karolinska Institute, Department of Microbiology, Tumor and Cell Biology .
    Braun, Laura
    Karolinska Institute, Department of Microbiology, Tumor and Cell Biology .
    Aspenström, Pontus
    Karolinska Institute, Department of Microbiology, Tumor and Cell Biology .
    Widengren, Jerker
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Effects of resolution, target density and labeling on co-localization estimates – nanoscopy and modified algorithms to suppress false positivesManuscript (preprint) (Other academic)
    Abstract [en]

    Co-localization analysis of fluorescence images is an important and extensively used tool to quantify molecular interactions in cells. In recent years, fluorescence nanoscopy techniques have made great progress and approach resolutions close to molecular dimensions. Although a strong increase in image resolution evidently influences different co-localization estimates it has to date not been systematically evaluated to what extent co-localization analyses can benefit from such techniques. . In this work, we apply simulations and resolution-tunable stimulated emission depletion (STED) microscopy to evaluate how resolution, molecular density and label size for the targeted molecules, as well as intensity variation and signal-to-noise ratio influence the estimates of the most commonly used co-localization algorithms (Pearson correlation coefficient, Manders M1 & M2 coefficients, and the image cross correlation spectroscopy (ICCS) method). We investigated the practically measureable extents of co-localization when using STED microscopy with positive and negative control samples and discussed the strengths and weaknesses of using nanoscopic techniques for co-localization studies. At a typical optical resolution of a confocal microscope (200-300 nm) our results indicate that the extent of co-localization is typically over-estimated by the tested algorithms, especially at high molecular densities. Only minor effects of this kind were observed at higher resolutions (< 60 nm). Apart from higher resolution, we introduced as an additional remedy a statistical variant of Costes threshold searching algorithm to set intensity thresholds separating background noise from signals. By this variant, combined with correlation-based methods like the Pearson coefficient and the ICCS approach, false positives at confocal resolutions and high molecular densities were found to be strongly suppressed.

  • 114.
    Xu, Lei
    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.
    Gad, Annica K. B.
    Braun, Laura J.
    Aspenström, Pontus
    Widengren, Jerker
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Effects of resolution, target density and labeling on co-localization estimates: nanoscopy and modified algorithms to suppress falseManuscript (preprint) (Other academic)
  • 115.
    Xu, Lei
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Öjemyr, Linda
    Stockholm University, Department of Biochemistry and Biophysics.
    Brzezinski, Peter
    Stockholm University, Department of Biochemistry and Biophysics.
    Widengren, Jerker
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    The membrane proton collecting antenna effect studied by Fluorescence Correlation Spectroscopy in a lipid-nanodisc model system – influence of the membrane area and external buffersManuscript (preprint) (Other academic)
    Abstract [en]

    Lipid membranes can act as proton collecting antennae, thereby significantly accelerating the protonation uptake of proteins responsible for proton transport across biological membranes. This uptake is a crucial step in the energy conversion within all living cells. In this study, we investigated the membrane-size dependence of the proton collecting antenna effect in a lipid nanodisc membrane model system of different sizes, 9nm and 12nm in diameter, with and without incorporation of a proton transporter, cytochrome c oxidase (CytcO). We also investigated how the proton exchange at the membrane-water interface is influenced by buffer molecules in the bulk solution. The proton exchange was monitored via fluorescence fluctuations of individual pH-sensitive fluorophores attached to membranes or to CytcO incorporated into the nanodiscs using fluorescence correlation spectroscopy. Our data confirm the significance of the membrane-water interface for accelerating proton uptake, show that this acceleration depends on the size of the membrane area surrounding the dyes, and indicate that the proton collection antenna can be in operation over a planar membrane-water interface in the range of 10nm in diameter, or possible larger. The buffer dependence for membrane-bound protonatable compounds was found to strongly deviate from the linear dependence, previously observed in both purely three-dimensional and two-dimensional systems. This, more complex, buffer concentration dependence can be explained by considering that also the proton exchange between the membrane surface itself and the bulk is influenced at the different buffer concentrations. Taken together, our findings reveal important biologically relevant aspects for how proton exchange at and across biological membranes are mediated and motivate further studies to better understand how these mechanisms mediate the proton exchange in more complex environments, as experienced in a living cells.

  • 116. Zentis, Peter D.
    et al.
    Frohnapfel, Manuel
    Rantanen, Ville
    Weidtkamp-Peters, Stefanie
    Felekyan, Suren
    Kuehnemuth, Ralf
    Sisamakis, Evangelos
    Xu, Lei
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Perols, Anna
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Arden-Jacob, Jutta
    Zilles, Alexander
    Oberlaender, Martina
    Fritzsche, Britta
    Nygren, Per-Åke
    Eriksson Karlström, Amelie
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Hautaniemi, Sampsa
    Drexhage, Karl-Heinz
    Auer, Gert
    Habermann, Jens K.
    Widengren, Jerker
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Seidel, Claus A. M.
    Cancer Diagnostics by Multiparameter Fluorescence Image Spectroscopy: A Bioinformatic Classifier Trained on Cultured Immunostained Cells2013In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 104, no 2, p. 342A-342AArticle in journal (Other academic)
  • 117. Zhang, B.
    et al.
    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.
    Vakifahmetoglu-Norberg, H.
    Norberg, E.
    Characterization of the role of the Malate dehydrogenases to lung tumor cell survival2017In: Journal of Cancer, ISSN 1837-9664, E-ISSN 1837-9664, Vol. 8, no 11, article id 19373Article in journal (Refereed)
    Abstract [en]

    Cellular compartmentalization of biochemical processes in eukaryotic cells is critical for many functions including shuttling of reducing equivalents across membranes. Although coordination of metabolic flux between different organelles is vital for cell physiology, its impact on tumor cell survival is not well understood. By using an integrative approach, we have dissected the role of the key metabolic enzymes Malate dehydrogenases (MDH1 and MDH2) to the survival of Nonsmall Cell Lung Carcinomas. Here, we report that while both the MDH1 (cytosolic) and the MDH2 (mitochondrial) enzymes display elevated levels in patients compared to normal counterparts, only high expression of MDH1 is associated with poor prognosis. We further show that the MDH1 enzymatic activity is significantly higher in NSCLC cells than that of MDH2. Accordingly, genetic depletion of MDH1 leads to significantly higher toxicity than depletion of MDH2. These findings provide molecular insights into the metabolic characteristics of the malate isoenzymes and mark MDH1 as a potential therapeutic target in these tumors.

  • 118.
    Zhang, Fuguo
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Chemistry, Organic chemistry.
    Cong, Jiayan
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Chemistry, Applied Physical Chemistry.
    Li, Yuanyuan
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Fibre- and Polymer Technology. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Centres, Wallenberg Wood Science Center.
    Bergstrand, Jan
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Theoretical Chemistry and Biology.
    Liu, Haichun
    KTH, School of Engineering Sciences (SCI), Applied Physics.
    Cai, Bin
    State Key Laboratory of Fine Chemicals, Dalian University of Technology (DUT).
    Hajian, Alireza
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Fibre- and Polymer Technology. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Centres, Wallenberg Wood Science Center.
    Yao, Zhaoyang
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Chemistry, Organic chemistry.
    Wang, Linqin
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Chemistry, Organic chemistry.
    Hao, Yan
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Chemistry, Applied Physical Chemistry.
    Yang, Xichuan
    State Key Laboratory of Fine Chemicals, Dalian University of Technology (DUT).
    Gardner, James M.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Chemistry, Applied Physical Chemistry.
    Ågren, Hans
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Theoretical Chemistry and Biology.
    Widengren, Jerker
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Theoretical Chemistry and Biology. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Centres, Wallenberg Wood Science Center.
    Kloo, Lars
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Chemistry, Applied Physical Chemistry.
    Sun, Licheng
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Chemistry. State Key Laboratory of Fine Chemicals, Dalian University of Technology (DUT).
    A facile route to grain morphology controllable perovskite thin films towards highly efficient perovskite solar cells2018In: Nano Energy, ISSN 2211-2855, E-ISSN 2211-3282, Vol. 53, p. 405-414Article in journal (Refereed)
    Abstract [en]

    Perovskite photovoltaics have recently attracted extensive attention due to their unprecedented high power conversion efficiencies (PCEs) in combination with primitive manufacturing conditions. However, the inherent polycrystalline nature of perovskite films renders an exceptional density of structural defects, especially at the grain boundaries (GBs) and film surfaces, representing a key challenge that impedes the further performance improvement of perovskite solar cells (PSCs) and large solar module ambitions towards commercialization. Here, a novel strategy is presented utilizing a simple ethylammonium chloride (EACl) additive in combination with a facile solvent bathing approach to achieve high quality methyammonium lead iodide (MAPbI3) films. Well-oriented, micron-sized grains were observed, which contribute to an extended carrier lifetime and reduced trap density. Further investigations unraveled the distinctively prominent effects of EACl in modulating the perovskite film quality. The EACl was found to promote the perovskite grain growing without undergoing the formation of intermediate phases. Moreover, the EACl was also revealed to deplete at relative low temperature to enhance the film quality without compromising the beneficial bandgap for solar cell applications. This new strategy boosts the power conversion efficiency (PCE) to 20.9% and 19.0% for devices with effective areas of 0.126 cm2 and 1.020 cm2, respectively, with negligible current hysteresis and enhanced stability. Besides, perovskite films with a size of 10 × 10 cm2, and an assembled 16 cm2(5 × 5 cm2 module) perovskite solar module with a PCE of over 11% were constructed.

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