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  • 1.
    Krmpot, Aleksandar J.
    et al.
    Karolinska Inst, CMM, Dept Clin Neurosci CNS, S-17176 Stockholm, Sweden.;Univ Belgrade, Inst Phys Belgrade, Belgrade 11080, Serbia..
    Nikolic, Stanko N.
    Karolinska Inst, CMM, Dept Clin Neurosci CNS, S-17176 Stockholm, Sweden.;Univ Belgrade, Inst Phys Belgrade, Belgrade 11080, Serbia..
    Oasa, Sho
    Karolinska Inst, CMM, Dept Clin Neurosci CNS, S-17176 Stockholm, Sweden..
    Papadopoulos, Dimitrios K.
    Max Planck Inst Mol Cell Biol & Genet, D-01307 Dresden, Germany.;Univ Edinburgh, Inst Genet & Mol Med, MRC Human Genet Unit, Edinburgh EH4 2XU, Midlothian, Scotland..
    Vitali, Marco
    Sicoya GmbH, D-12489 Berlin, Germany..
    Oura, Makoto
    Hokkaido Univ, Fac Adv Life Sci, Lab Mol Cell Dynam, Sapporo, Hokkaido 0010021, Japan..
    Mikuni, Shintaro
    Hokkaido Univ, Fac Adv Life Sci, Lab Mol Cell Dynam, Sapporo, Hokkaido 0010021, Japan..
    Thyberg, Per
    KTH, School of Engineering Sciences (SCI), Applied Physics, Quantum and Biophotonics.
    Tisa, Simone
    MPD, I-39100 Bolzano, Italy..
    Kinjo, Masataka
    Hokkaido Univ, Fac Adv Life Sci, Lab Mol Cell Dynam, Sapporo, Hokkaido 0010021, Japan..
    Nilsson, Lennart
    Karolinska Inst, Dept Biosci & Nutr, S-14183 Huddinge, Sweden..
    Terenius, Lars
    Karolinska Inst, CMM, Dept Clin Neurosci CNS, S-17176 Stockholm, Sweden..
    Rigler, Rudolf
    Karolinska Inst, CMM, Dept Clin Neurosci CNS, S-17176 Stockholm, Sweden.;Karolinska Inst, Dept Med Biochem & Biophys MBB, S-17177 Stockholm, Sweden..
    Vukojevic, Vladana
    Karolinska Inst, CMM, Dept Clin Neurosci CNS, S-17176 Stockholm, Sweden..
    Functional Fluorescence Microscopy Imaging: Quantitative Scanning-Free Confocal Fluorescence Microscopy for the Characterization of Fast Dynamic Processes in Live Cells2019In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 91, no 17, p. 11129-11137Article in journal (Refereed)
    Abstract [en]

    Functional fluorescence microscopy imaging (fFMI), a time-resolved (21 mu s/frame) confocal fluorescence microscopy imaging technique without scanning, is developed for quantitative characterization of fast reaction-transport processes in solution and in live cells. The method is based on massively parallel fluorescence correlation spectroscopy (FCS). Simultaneous excitation of fluorescent molecules in multiple spots in the focal plane is achieved using a diffractive optical element (DOE). Fluorescence from the DOE-generated 1024 illuminated spots is detected in a confocal arrangement by a matching matrix detector comprising 32 x 32 single-photon avalanche photodiodes (SPADs). Software for data acquisition and fast auto- and cross-correlation analysis by parallel signal processing using a graphic processing unit (GPU) allows temporal autocorrelation across all pixels in the image frame in 4 s and cross-correlation between first- and second-order neighbor pixels in 45 s. We present here this quantitative, time-resolved imaging method with single-molecule sensitivity and demonstrate its usefulness for mapping in live cell location-specific differences in the concentration and translational diffusion of molecules in different subcellular compartments. In particular, we show that molecules without a specific biological function, e.g., the enhanced green fluorescent protein (eGFP), exhibit uniform diffusion. In contrast, molecules that perform specialized biological functions and bind specifically to their molecular targets show location-specific differences in their concentration and diffusion, exemplified here for two transcription factor molecules, the glucocorticoid receptor (GR) before and after nuclear translocation and the Sex combs reduced (Scr) transcription factor in the salivary gland of Drosophila ex vivo.

  • 2. Krmpot, Aleksandar J.
    et al.
    Nikolic, Stanko N.
    Vitali, Marco
    Papadopoulos, Dimitrios K.
    Oasa, Sho
    Thyberg, Per
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Tisa, Simone
    Kinjo, Masataka
    Nilsson, Lennart
    Gehring, Walter J.
    Terenius, Lars
    Rigler, Rudolf
    Vukojevic, Vladana
    Quantitative confocal fluorescence microscopy of dynamic processes by multifocal fluorescence correlation spectroscopy2015In: ADVANCED MICROSCOPY TECHNIQUES IV; AND NEUROPHOTONICS II, 2015, Vol. 9536, article id 95360OConference paper (Refereed)
    Abstract [en]

    Quantitative confocal fluorescence microscopy imaging without scanning is developed for the study of fast dynamical processes. The method relies on the use of massively parallel Fluorescence Correlation Spectroscopy (mpFCS). Simultaneous excitation of fluorescent molecules across the specimen is achieved by passing a single laser beam through a Diffractive Optical Element (DOE) to generate a quadratic illumination matrix of 32x32 light sources. Fluorescence from 1024 illuminated spots is detected in a confocal arrangement by a matching matrix detector consisting of the same number of single-photon avalanche photodiodes (SPADs). Software was developed for data acquisition and fast auto- and cross-correlation analysis by parallel signal processing using a Graphic Processing Unit (GPU). Instrumental performance was assessed using a conventional single-beam FCS instrument as a reference. Versatility of the approach for application in biomedical research was evaluated using ex vivo salivary glands from Drosophila third instar larvae expressing a fluorescently-tagged transcription factor Sex Combs Reduced (Scr) and live PC12 cells stably expressing the fluorescently tagged mu-opioid receptor (MOPeGFP). We show that quantitative mapping of local concentration and mobility of transcription factor molecules across the specimen can be achieved using this approach, which paves the way for future quantitative characterization of dynamical reaction-diffusion landscapes across live cells/tissue with a sub-millisecond temporal resolution (presently 21 mu s/frame) and single-molecule sensitivity.

  • 3.
    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.

  • 4.
    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.

  • 5.
    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.

  • 6.
    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

  • 7.
    Strömqvist, Johan
    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.
    Diffusion-mediated reaction rates in biological membranes: theory, simulations and its manifestation in triplet statequenching studies in liposomesManuscript (preprint) (Other academic)
  • 8.
    Wennmalm, Stefan
    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.
    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.
    Inverse-Fluorescence Correlation Spectroscopy2009In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 81, no 22, p. 9209-9215Article in journal (Refereed)
    Abstract [en]

    An alternative version of fluorescence correlation spectroscopy is presented, where the signal from a medium surrounding the particles of interest is analyzed, as opposed to a signal from the particles themselves. Ibis allows for analysis of unlabeled particles and potentially of biomolecules. Here, the concept together with principal experiments on polystyrene beads of 100, 200, 400, and 800 nm diameter in an aqueous solution of alexa 488-fluorophores are presented. The use of photo detectors allowing higher photon fluxes, or of reduced detection volumes, should enable analysis of significantly smaller particles or even biomolecules.

  • 9.
    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.

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