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  • 1.
    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.

  • 2. 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.

  • 3.
    Persson, Gustav
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Temporal Modulation in Fluorescence Spectroscopy and Imaging for Biological Applications2009Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    This thesis explores the benefits of intensity modulation for the purpose of extending the range of applications of fluorescence spectroscopy and imaging in cellular and molecular biology and medicine.

    Long-lived transient states of fluorescent molecules can, because of their long lifetimes, be used to detect subtle changes in the microenvironment of the molecule. A method for determining the kinetic rates for transitions to and from such states by registration of changes in the average fluorescence intensity related to different modulation of the excitation source is introduced. It combines the detection sensitivity of fluorescence with the environmental sensitivity of the long-lived transient states and allows the use of slow detectors such as CCD cameras, making parallelization and wide-field imaging possible developments. An extension of this method, generating image contrast based on triplet state population using a standard laser scanning microscope, is also shown.

    A strategy to combine fluorescence correlation spectroscopy (FCS) with modulated excitation, in a way that allows extraction of correlation data for all correlation times, is presented. This enables the use of modulation to optimize measurement conditions with respect to photophysical properties of the dyes used. FCS with modulated excitation will probably prove useful in future studies involving multiple kinetic processes occurring in overlapping time ranges. One of the ideas from this project also constitutes a powerful method for generating artifact free correlation curves from data sets where sections have been removed. This is potentially very useful in biological studies where spikes in the measurements often cause problems.

    In the final project, cross-correlation and alternating excitation are combined in measurements on a pH-sensitive ratiometric dye to clearly distinguish the protonation–deprotonation dynamics from other processes. The presented approach makes the protonation related fluctuations manifest themselves as a very distinct anti-correlating component in the correlation curve. This enables robust data analysis using a simple model.

  • 4.
    Persson, Gustav
    KTH, School of Engineering Sciences (SCI), Applied Physics.
    Time-Varying Excitation in Fluorescence Spectroscopy for Biological Applications2007Licentiate thesis, comprehensive summary (Other scientific)
    Abstract [en]

    The focus of this thesis is to explore and use the benefits of time-varying excitation in fluorescence spectroscopy for studies of biomolecular dynamics. Two new techniques taking advantage of modulated excitation are presented. Also described are the first efforts in a project where single molecule FRET and multi-parameter fluorescence detection are used for characterization of the conformational dynamics of the retinoid X receptor (RXR).

    RXR is one of the most important proteins in the group of nuclear receptors. It is believed to be involved in many diseases and is hence most interesting as a potential drug target. Our study is at present at a very early stage and some sample issues are still to be resolved. However, single molecule measurements should give insights not attainable by previously applied ensemble methods and help explaining how RXR can regulate so many different processes.

    Long-lived transient states of fluorescent molecules can, because of their long lifetimes, be used to detect subtle changes in the microenvironment of the molecule. A method for determining the kinetic rates for transitions to and from such states by registration of changes in the average fluorescence intensity related to different modulation of the excitation source is introduced. It combines the sensitivity of fluorescence with the environmental sensitivity of the long-lived transient states and allows the use of slow detectors such as CCD cameras, making parallelization and imaging possible developments. The approach was experimentally verified by measurements of the triplet kinetics of rhodamine 6G (Rh6G) in aqueous solution and compared with fluorescence correlation spectroscopy (FCS). It should also be applicable to any other photoinduced transient states affecting the fluorescence intensity.

    A strategy to combine FCS with modulated excitation, in a way that allows extraction of correlation data for all correlation times, is presented. This enables the use of modulation to optimize the measurement conditions with respect to the photophysical properties of the dyes used. Measurements were made on Rh6G to verify the method. To illustrate its usefulness, it was applied to measurements of protonation kinetics of fluorescein at different pH. FCS with modulated excitation will most probably prove very useful in many future studies involving multiple kinetic processes occurring in overlapping time ranges.

  • 5.
    Persson, Gustav
    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.
    Sandberg, AnnSofi
    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 cross-correlation spectroscopy of a pH-sensitive ratiometric dye for molecular proton exchange studies2009In: Physical Chemistry, Chemical Physics - PCCP, ISSN 1463-9076, E-ISSN 1463-9084, Vol. 11, no 21, p. 4410-4418Article in journal (Refereed)
    Abstract [en]

    Fluorescence fluctuation analysis of individual pH-sensitive fluorophores has recently proven to be a useful approach for biomolecular proton exchange studies. In this work, dual-color fluorescence cross-correlation spectroscopy (FCCS) is demonstrated on a ratiometric pH-sensitive dye, for which both the excitation and emission spectra shift as a function of pH. In the FCCS measurements, the fluorescence signal from the predominant emission wavelength range of the protonated form of the dye is cross-correlated with that of the deprotonated form. Two lasers are used alternatingly to excite predominantly the protonated and the deprotonated form of the dye. The alternating excitation modulation scheme is combined with detection gating, and is based on a recently developed concept that allows extraction of correlation data for all correlation times regardless of the chosen modulation period. The scheme can thus be applied without concern for the time-scales of the molecular dynamic processes to be studied. By this combined discrimination based on both excitation and emission, spectral cross-talk is dramatically reduced and a very distinct and unambiguous anticorrelation can be recorded in the correlation curves as a consequence of the proton exchange. The strong discrimination power makes the approach applicable also to ratiometric dyes with less pronounced spectral shifts. It should also be useful in combination with ratiometric dyes sensitive to other ambient conditions and ions, such as the biologically very important calcium ion.

  • 6.
    Persson, Gustav
    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.
    Thyberg, Per
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Widengren, Jerker
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Modulated or alternating excitation in fluorescence correlation spectroscopy2009In: SINGLE MOLECULE SPECTROSCOPY AND IMAGING / [ed] Enderlein J; Gryczynski ZK; Erdmann R, 2009, Vol. 7185Conference paper (Refereed)
    Abstract [en]

    We have previously shown that formation of triplet states and other photo-induced states can be controlled by modulating the excitation with pulse widths and periods in the range of the transition times of the involved states. However, modulating the excitation in fluorescence correlation spectroscopy (FCS) measurements normally destroys correlation information and induces ringing in the correlation curve. We have introduced and experimentally verified a method to retrieve the full correlation curves from FCS measurements with modulated excitation and arbitrarily low fraction of active excitation. Modulated excitation applied to FCS experiments was shown to suppress the triplet build-up more efficiently than reducing excitation power with continuous wave excitation. The usefulness of the method was demonstrated by measurements done on fluorescein at different pH, where suppression of the triplet significantly facilitates the analysis of the protonation kinetics. Using a fluorophore where the protonation-coupled fluorescence intensity fluctuations are due to spectral shifts, introduction of two-color alternating excitation and spectral cross-correlation can turn the protonation component of the correlation curve into an anti-correlation and further facilitate the distinction of this component from those of other processes.

  • 7.
    Persson, Gustav
    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.
    Sandén, Tor
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Widengren, Jerker
    KTH, School of Engineering Sciences (SCI), Applied Physics.
    Modulation Filtering Enables Removal of Spikes in Fluorescence Correlation Spectroscopy Measurements without Affecting the Temporal Information2009In: Journal of Physical Chemistry B, ISSN 1520-6106, E-ISSN 1520-5207, Vol. 113, no 25, p. 8752-8757Article in journal (Refereed)
    Abstract [en]

    The appearance of intensity spikes in measurements is a common problem in fluorescence correlation spectroscopy (FCS) studies of biological samples. In this work, we present a new method for generating artifact-free correlation curves from fluorescence traces that have undergone spike removal. This method preserves the temporal information throughout the measurement and properly represents the correlation between events separated by removed spikes. The method was validated using experimental data. The proposed algorithm is demonstrated herein to be generally applicable, but it is particularly powerful for cases where spikes occur frequently.

  • 8.
    Persson, Gustav
    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.
    Widengren, Jerker
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Modulated Fluorescence Correlation Spectroscopy with Complete Time Range Information2008In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 94, no 3, p. 977-985Article in journal (Refereed)
    Abstract [en]

    Two methods to combine fluorescence correlation spectroscopy (FCS) with modulated excitation, in a way that allows extraction of correlation data for all correlation times have been developed and experimentally verified. One method extracts distortion-free correlation data from measurements acquired with standard hardware correlators provided the fluorescence does not change systematically within the excitation pulses. This restriction does not apply to the second method, which, however, requires time-resolved acquisition of the fluorescence intensity. Modulation of the excitation in an FCS experiment is demonstrated to suppress triplet population buildup more efficiently than a corresponding reduction in continuous wave excitation intensity (shown for the dye rhodamine 6G in aqueous solution). Excitation modulation thus offers an additional means to optimize the FCS measurement conditions with respect to the photophysical properties of the dyes used. This possibility to suppress photoinduced states also provides a useful tool to distinguish additional processes occurring in the same time regime in the FCS measurements, as demonstrated here for the protonation kinetics of fluorescein at different pH. In general, the proposed concept opens for FCS measurements with a complete correlation timescale in a range of applications where a modulated excitation is either necessary or brings specific advantages.

  • 9.
    Sanden, Tor
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    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.
    Transient state microscopy: a new tool for biomolecular imaging2009In: MULTIPHOTON MICROSCOPY IN THE BIOMEDICAL SCIENCES IX / [ed] Periasamy A, So PTC, Bellingham, WA: SPIE-INT SOC OPTICAL ENGINEERING , 2009, Vol. 7183Conference paper (Refereed)
    Abstract [en]

    Photoinduced transient dark states are exhibited by practically all common fluorophores. However, their information content has to date only been sparsely exploited due to methodological constraints. Here, a new concept is presented and verified that can monitor and image these states via their photodynamic fingerprints. It unites the environmental sensitivity of these states with the sensitivity of fluorescence-based detection. For demonstration, triplet state images of liposomes in different environments were generated, showing how local environmental differences can be resolved, not clearly distinguishable via other fluorescence parameters. The concept can provide several new, useful and independent fluorescence-based parameters in biomolecular imaging.

  • 10.
    Sandén, Tor
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Persson, Gustav
    KTH, School of Engineering Sciences (SCI), Applied Physics.
    Thyberg, Per
    KTH, School of Engineering Sciences (SCI), Applied Physics.
    Blom, Hans
    KTH, School of Engineering Sciences (SCI), Applied Physics.
    Widengren, Jerker
    KTH, School of Engineering Sciences (SCI), Applied Physics.
    Monitoring Kinetics of Highly Environment Sensitive States of Fluorescent Molecules by Modulated Excitation and Time-Averaged Fluorescence Intensity Recording2007In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 79, no 9, p. 3330-3341Article in journal (Refereed)
    Abstract [en]

    In this work, a concept is described for how the kinetics of photoinduced, transient, long-lived, nonfluorescent or weakly fluorescent states of fluorophore marker molecules can be extracted from the time-averaged fluorescence by using time-modulated excitation. The concept exploits the characteristic variation of the population of these states with the modulation parameters of the excitation and thereby circumvents the need for time resolution in the fluorescence detection. It combines the single-molecule sensitivity of fluorescence detection with the remarkable environmental responsiveness obtainable from long-lived transient states, yet does not in itself impose any constraints on the concentration or the fluorescence brightness of the sample molecules that can be measured. Modulation of the excitation can be performed by variation of the intensity of a stationary excitation beam in time or by repeated translations of a CW excitation beam with respect to the sample. As a first experimental verification of the approach, we have shown how the triplet-state parameters of the fluorophore rhodamine 6G in different aqueous enviroments can be extracted. We demonstrate that the concept is fully compatible with low time-resolution detection by a CCD camera. The concept opens for automated transient-state monitoring or imaging on a massively parallel scale and for high-throughput biomolecular screening as well as for more fundamental biomolecular studies. The concept should also be applicable to the monitoring of a range of other photoinduced nonfluorescent or weakly fluorescent transient states, from which subtle changes in the immediate microenvironment of the fluorophore marker molecules can be detected

  • 11.
    Sandén, Tor
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    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.
    Transient State Imaging for Microenvironmental Monitoring by Laser Scanning Microscopy2008In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 80, no 24, p. 9589-9596Article in journal (Refereed)
    Abstract [en]

    Photoinduced transient dark states are exhibited by practically all common fluorophores. These relatively long-lived states are very sensitive to the local environment and thus highly attractive for microenvironmental imaging purposes. However, because of methodological constraints, their sensitivity has to date been very sparsely exploited. Here, a concept based on spatio-temporal modulation of the excitation intensity is presented that can image these states via their photodynamic fingerprints. With the use of a standard laser scanning microscope, it unites the outstanding environmental sensitivity of the transient state parameters with the high sensitivity of the fluorescence readout and is easily implemented. For demonstration, triplet state images of liposomes with different internal environments were generated. These images provide an example of bow local environmental differences can be resolved, which are not clearly distinguishable via other fluorescence parameters. Having minor instrumental and sample constraints the concept can be foreseen to provide several new, useful, and independent fluorescence-based parameters in biomolecular imaging.

  • 12. 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.

1 - 12 of 12
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