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.

TRANSPARENT TESTA GLABRA1 and GLABRA1 Compete for Binding to GLABRA3 in Arabidopsis

“…The fluorescence lifetime of CFP was determined and analyzed pixel-wise in merged images to increase photon numbers for analysis using the software tools AnI-3SF and Margarita (Software Package for Multiparameter Fluorescence Spectroscopy, Full Correlation, and Multiparameter Fluorescence Imaging [http://www.mpc.hhu.de/software.html] for Multiparameter Fluorescence Image Spectroscopy; Kudryavtsev et al, 2007;Weidtkamp-Peters et al, 2009). In fluorescence lifetime microscopy with high spatial resolution and low excitation power to prevent photobleaching, the number of photons per pixel is exceptionally low, ranging from 100 to 2,000 photons per pixel.…”

Single-molecule spectroscopy of fluorescent proteins

“…Other properties addressed by single-molecule multiparameter detection are the polarization of the emitted light and the fluorescence lifetime [123,124].…”

Transient State Monitoring by Total Internal Reflection Fluorescence Microscopy

“…To increase information content, multiplexing by simultaneous registration of several independent parameters has evolved as an important means in wide-field and confocal fluorescence microscopy, 1 for readouts of DNA and protein microarrays, 2 as well as in single molecule spectroscopy 3 and imaging. 4 Attention has been addressed to photophysical properties determining the fluorescence saturation properties and photostabilities of the fluorophore marker molecules. Long-lived, photo-induced states, such as triplet, photo-isomerised, and photo-oxidized states are often found to reduce the signal in fluorescence microscopy, 5 and can act as precursor states of photobleaching.…”