Monitoring dynamic systems with multiparameter fluorescence imaging

• Published in: Analytical and bioanalytical chemistry Vol. 387; no. 1; pp. 71 - 82
• Main Authors: Kudryavtsev, Volodymyr, Felekyan, Suren, Woźniak, Anna K, König, Marcelle, Sandhagen, Carl, Kühnemuth, Ralf, Seidel, Claus A. M, Oesterhelt, Filipp
• Format: Journal Article
• Language: English
• Published: Germany Berlin/Heidelberg : Springer-Verlag 2007; Springer Nature B.V
• Subjects: Algorithms; Anisotropy; Chemical compounds; Confocal microscopy; Diffusion; Dynamical systems; Excitation spectra; FCS; Fluorescence; Fluorescence correlation spectroscopy; Fluorescence Polarization; Fluorescence spectroscopy; Fluorescent dyes; Fluorescent indicators; Fluorophores; Fusion protein; Glutathione; Glutathione - chemistry; Green fluorescent protein; Green Fluorescent Proteins - genetics; Green Fluorescent Proteins - metabolism; Histograms; Image Processing, Computer-Assisted - methods; Laser Scanning Cytometry - methods; Lifetime imaging; Microscopy, Fluorescence - methods; Microtubule-Associated Proteins - genetics; Microtubule-Associated Proteins - metabolism; Microtubules; Microtubules - metabolism; Monitoring; Multiparameter fluorescence imaging; Parameter estimation; Pixels; Proteins; Rhodamine; Rhodamines - chemistry; Schizosaccharomyces - genetics; Schizosaccharomyces - metabolism; Schizosaccharomyces pombe Proteins - genetics; Schizosaccharomyces pombe Proteins - metabolism; Sepharose - chemistry; Spectrometry, Fluorescence - methods; Spectroscopy; Spectrum analysis; Statistical analysis; Statistics; Time correlation functions; Yeasts
• ISSN: 1618-2642; 1618-2650
• DOI: 10.1007/s00216-006-0917-0

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.