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  • 1.
    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.

  • 2.
    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.

  • 3.
    Chmyrov, Andriy
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Photo-induced dark states influorescence spectroscopy – investigations & applications2010Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    This thesis focuses on investigations of transient dark states of fluorescentmolecules using spectroscopic techniques. The main purpose is to show andconvince the reader that transient dark states are not always a nuisance, butalso represent an additional source of information. Several studies with fluorescencecorrelation spectroscopy were performed, all related to non-fluorescentstates such as triplet state or isomerized states.Photobleaching is one of the main problems in virtually all of the fluorescencetechniques. In this thesis, mechanisms that retard photobleaching arecharacterized. Several compounds, antioxidants and triplet state quenchers,which decrease photobleaching, are studied, and guidelines for achieving optimalfluorescence brightness using these compounds are presented.Triplet state quenching by several compounds was studied. Detailed investigationsof the fluorescence quencher potassium iodide demonstratedthat for some of fluorophores, except of quenching, there is fluorescence enhancementmechanism present. In agreement with the first publication inthis thesis, antioxidative properties were found to play an important role inthe fluorescence enhancement. Quenching of the triplet state is proposedas a tool for monitoring diffusion mediated reactions over a wide range offrequencies.Specially designed fluorophores combining high triplet yields with reasonablefluorescence brightness and photostability were characterized forpossible applications in novel super-resolution imaging techniques based onfluorescence photoswitching. Except of benefits for imaging techniques, photoinducedswitching to non-fluorescent states could be used for monitoringmolecular diffusion, which was also demonstrated in this thesis.Studies of the triplet state kinetics of fluorophores close to dielectric interfaceswere performed using fluorescence spectroscopy. The analysis of thetriplet state kinetic can provide information about the local microenvironmentand electrostatic interactions near dielectric interfaces.

  • 4.
    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.

  • 5.
    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.

  • 6.
    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.

  • 7.
    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.

  • 8.
    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.

  • 9.
    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.

  • 10.
    Schaper, Klaus
    et al.
    Univ Dusseldorf, Dept Chem, D-40225 Dusseldorf, Germany..
    Bier, Brigitte
    Univ Dusseldorf, Dept Chem, D-40225 Dusseldorf, Germany..
    Taureg, Patrick
    Univ Dusseldorf, Dept Chem, D-40225 Dusseldorf, Germany..
    von Dahlen, Steffen
    Pfiffi, Daniela
    Seidel, Claus A. M.
    Chmyrov, Andriy
    KTH.
    Widengren, Jerker
    KTH, School of Engineering Sciences (SCI), Applied Physics, Quantum and Biophotonics.
    PHYS 624-Strategies to improve signal strength and photostability of fluorophors in single molecule spectroscopy2007In: Abstract of Papers of the American Chemical Society, ISSN 0065-7727, Vol. 234Article in journal (Other academic)
  • 11.
    Strömqvist, Johan
    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.
    Johansson, Sofia
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Andersson, August
    Department of Biochemistry and Biophysics, Stockholm University.
    Mäler, Lena
    Department of Biochemistry and Biophysics, Stockholm University.
    Widengren, Jerker
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Nitroxide Spin-Label Quenching of Fluorophore’s Triplet Stateas a Tool for Studying Diffusion Mediated Reactions in LipidMembranesManuscript (preprint) (Other (popular science, discussion, etc.))
    Abstract [en]

    In this work, we introduce an approach to study bimolecularinteractions in model lipid bilayers and biologicalmembranes, which exploits the influence of membrane-associatedElectron Spin Resonance (ESR) labels on the fluorescencesignal of likewise membrane-bound fluorophoremarkers. It is shown how one can exploit the high detectionsensitivity of the fluorescence signal without loosing the abilityto follow low-frequency molecular interactions, takingplace on a time scale well beyond that of the fluorescencelifetimes. The approach utilizes triplet state monitoring byFluorescence Correlation Spectroscopy (FCS), whereby thefluctuations in a strong fluorescence signal is used to characterizetransition rates to and from the lowest triplet stateof the fluorophores, which take place on a time scale 3 to 6orders of magnitude slower than the fluorescence lifetimesof the fluorophores. FCS measurements were performed onthe dye Lissamine Rhodamine B (LRB) in aqueous solutionsand bound to a lipid in a liposome, and in the presence of differentlocal concentrations of the ESR label TEMPO. Bothin the aqueous solution and in the lipid membrane measurements,the measured relative changes in the singlet-triplettransitions rates were found to well reflect the collisionalfrequencies between the LRB and TEMPO molecules. Theproposed approach, allowing low-frequency interactionsto be monitored with a bright fluorescence signal offers abroad applicability, both in terms of read-out means, typesof molecular interactions that can be followed, and in whatenvironment these interactions can be measured. From thispoint of view, it can prove useful for a broad category ofmolecular interaction studies.

  • 12.
    Strömqvist, Johan
    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.
    Johansson, Sofia
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Andersson, August
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Mäler, Lena
    Widengren, Jerker
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Quenching of Triplet State Fluorophores for Studying Diffusion-Mediated Reactions in Lipid Membranes2010In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 99, no 11, p. 3821-3830Article in journal (Refereed)
    Abstract [en]

    An approach to study bimolecular interactions in model lipid bilayers and biological membranes is introduced, exploiting the influence of membrane associated electron spin resonance labels on the triplet state kinetics of membrane bound fluorophores Singlet triplet state transitions within the dye Lissamine Rhodamine B (LRB) were studied when free in aqueous solutions, with LRB bound to a lipid in a liposome and in the presence of different local concentrations of the electron spin resonance label TEMPO By monitoring the triplet state kinetics via variations in the fluorescence signal, in this study using fluorescence correlation spectroscopy a strong fluorescence signal can be combined with the ability to monitor low frequency molecular interactions at timescales much longer than the fluorescence lifetimes Both in solution and in membranes the measured relative changes in the singlet triplet transitions rates were found to well reflect the expected collisional frequencies between the LRB and TEMPO molecules These collisional rates could also be monitored at local TEMPO concentrations where practically no quenching of the excited state of the fluorophores can be detected The proposed strategy is broadly applicable in terms of possible read out means types of molecular interactions that can be followed, and in what environments these interactions can be measured

  • 13.
    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.

1 - 13 of 13
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