Monitoring dynamic systems with multiparameter fluorescence imaging

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