title: Monitoring dynamic systems with multiparameter fluorescence imaging.
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.
authors.affiliation: Lehrstuhl für Molekulare Physikalische Chemie, Heinrich-Heine-Universität Düsseldorf, Universitätsstrasse 1, 40225, Düsseldorf, Germany.
authors.forename: Volodymyr
authors.initials: V
authors.lastname: Kudryavtsev
authors.forename: Suren
authors.initials: S
authors.lastname: Felekyan
authors.forename: Anna K
authors.initials: AK
authors.lastname: Woźniak
authors.forename: Marcelle
authors.initials: M
authors.lastname: König
authors.forename: Carl
authors.initials: C
authors.lastname: Sandhagen
authors.forename: Ralf
authors.initials: R
authors.lastname: Kühnemuth
authors.forename: Claus A M
authors.initials: CA
authors.lastname: Seidel
authors.forename: Filipp
authors.initials: F
authors.lastname: Oesterhelt
mesh_terms: D000465:Algorithms; D004058:Diffusion; D005454:Fluorescence Polarization; D005978:Glutathione / Q000737:chemistry; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D007091:Image Processing, Computer-Assisted / Q000379:methods*; D047569:Laser Scanning Cytometry / Q000379:methods*; D008856:Microscopy, Fluorescence / Q000379:methods; D008869:Microtubule-Associated Proteins / Q000235:genetics / Q000378:metabolism; D008870:Microtubules / Q000378:metabolism; D012235:Rhodamines / Q000737:chemistry; D012568:Schizosaccharomyces / Q000235:genetics / Q000378:metabolism; D029702:Schizosaccharomyces pombe Proteins / Q000235:genetics / Q000378:metabolism; D012685:Sepharose / Q000737:chemistry; D013050:Spectrometry, Fluorescence / Q000379:methods
chemical_list: C108851:Mal3 protein, S pombe; D008869:Microtubule-Associated Proteins; D012235:Rhodamines; D029702:Schizosaccharomyces pombe Proteins; C049025:rhodamine 110; D049452:Green Fluorescent Proteins; D012685:Sepharose; D005978:Glutathione