Monitoring dynamic systems with multiparameter fluorescence imaging |
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Authors: | Volodymyr Kudryavtsev Suren Felekyan Anna K Woźniak Marcelle König Carl Sandhagen Ralf Kühnemuth Claus A M Seidel Filipp Oesterhelt |
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Institution: | 1.Lehrstuhl für Molekulare Physikalische Chemie,Heinrich-Heine-Universit?t Düsseldorf, Universit?tsstra?e 1,Düsseldorf,Germany |
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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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Keywords: | Multiparameter fluorescence imaging Fluorescence correlation spectroscopy FCS Confocal microscopy Lifetime imaging Microtubules |
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