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Monitoring dynamic systems with multiparameter fluorescence imaging

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Abstract

A new general strategy based on the use of multiparameter fluorescence detection (MFD) to register and quantitatively analyse fluorescence images is introduced. Multiparameter fluorescence imaging (MFDi) uses pulsed excitation, time-correlated single-photon counting and a special pixel clock to simultaneously monitor the changes in 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. The three spatial coordinates are also stored. The most statistically efficient techniques known from single-molecule spectroscopy are used to estimate fluorescence parameters of interest for all pixels, not just for the regions of interest. Their statistical significance is judged from a stack of two-dimensional histograms. In this way, specific pixels can be selected for subsequent pixel-based subensemble analysis in order to improve the statistical accuracy of the parameters estimated. MFDi avoids the need for sequential measurements, because the registered data allow one to perform many analysis techniques, such as fluorescence-intensity distribution analysis (FIDA) and fluorescence correlation spectroscopy (FCS), in an off-line mode. The limitations of FCS for counting molecules and monitoring dynamics are discussed. To demonstrate the ability of our technique, we analysed two systems: (i) interactions of the fluorescent dye Rhodamine 110 inside and outside of a glutathione sepharose bead, and (ii) microtubule dynamics in live yeast cells of Schizosaccharomyces pombe using a fusion protein of Green Fluorescent Protein (GFP) with Minichromosome Altered Loss Protein 3 (Mal3), which is involved in the dynamic cycle of polymerising and depolymerising microtubules.

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Acknowledgements

We thank Ursula Fleig and Christoph Beuter for their generous support. They provided the cells and prepared these cell samples for imaging. We thank Stanislav Kalinin for helping us to find appropriate and statistically stable parameters to describe noisy FCS curves. We thank Stefan Marawske for helping us to perform live cell measurements. We thank Alexander Gaiduk for helping us to establish MFD imaging. CS gratefully acknowledges financial support from the BMBF Biofuture grant 0311865 and the SFB 590, Heinrich-Heine-University Düsseldorf.

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  1. Lehrstuhl für Molekulare Physikalische Chemie, Heinrich-Heine-Universität Düsseldorf, Universitätsstraße 1, 40225, Düsseldorf, Germany

    Volodymyr Kudryavtsev, Suren Felekyan, Anna K. Woźniak, Marcelle König, Carl Sandhagen, Ralf Kühnemuth, Claus A. M. Seidel & Filipp Oesterhelt

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  1. Volodymyr Kudryavtsev
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  2. Suren Felekyan
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  3. Anna K. Woźniak
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  4. Marcelle König
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Correspondence to Claus A. M. Seidel or Filipp Oesterhelt.

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Kudryavtsev, V., Felekyan, S., Woźniak, A.K. et al. Monitoring dynamic systems with multiparameter fluorescence imaging. Anal Bioanal Chem 387, 71–82 (2007). https://doi.org/10.1007/s00216-006-0917-0

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  • DOI: https://doi.org/10.1007/s00216-006-0917-0

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