Biomedical subjects

Ralf Kühnemuth

Publications and source records attributed to Ralf Kühnemuth.

3 recordsLinked to original sources

PubMed2006

Monitoring dynamic systems with multiparameter fluorescence imaging.

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.

Algorithms↗

PubMed2005

Fluorescence correlation spectroscopy, a tool to investigate supramolecular dynamics: inclusion complexes of pyronines with cyclodextrin.

The control of supramolecular systems requires a thorough understanding of their dynamics on a molecular level. We present fluorescence correlation spectroscopy (FCS) as a powerful spectroscopic tool to study supramolecular dynamics with single molecule sensitivity. The formation of a supramolecular complex between beta-cyclodextrin (beta-CD) as host and pyronines Y (PY) and B (PB) as guests is studied by FCS. Global target analysis of full correlation curves with a newly derived theoretical model yields in a single experiment the fluorescence lifetimes and the diffusion coefficients of free and complexed guests and the rate constants describing the complexation dynamics. These data give insight into the recently published surprising fact that the association equilibrium constant of beta-CD with PY is much lower than that with the much bulkier guest PB. FCS shows that the stability of the complexes is dictated by the dissociation and not by the association process. The association rate constants are very similar for both guests and among the highest reported for this type of systems, although much lower than the diffusion-controlled collision rate constant. A two-step model including the formation of an encounter complex allows one to identify the unimolecular inclusion reaction as the rate-limiting step. Simulations indicate that this step may be controlled by geometrical and orientational requirements. These depend on critical molecular dimensions which are only weakly affected by the different alkyl substituents of PY and PB. Diffusion coefficients of PY and PB, of their complexes, and of rhodamine 110 are given and compared to those of similar molecules.

Coloring Agents↗

PubMed2005

Optical characteristics of atomic force microscopy tips for single-molecule fluorescence applications.

Knowledge of the optical properties of atomic force microscopy (AFM) tips is relevant for the combination of optical and force spectroscopy. The luminescence properties of five commercial AFM tips were characterized using a combination of multiparameter fluorescence detection (MFD) and scanning confocal techniques. These include three Si3N4 tips, one silicon tip, and one high-density carbon (HDC) tip grown on top of a silicon tip. Time-decay histograms of the signal were analyzed to determine the strength of scatter, constant background, and fluorescence in the observed signal. Intensity and anisotropy images with optical resolution down to the diffraction limit were generated. The optical signal recorded from the apex of the Si3N4 tips ranged from 0.7 to 1.9 times the count rates from single Rhodamine 110 molecules under similar illumination conditions. The signal is predominantly composed of scatter and background (>85%), plus a small fluorescence component with lifetimes between 1 and 3 ns. The intensity of the recorded signal fell with increasing distance from the apex, and by 300 nm the signals fell below single-molecule levels for all Si3N4 cantilevers. Silicon cantilevers demonstrated very low count rates relative to single-molecule measurements under all conditions, and virtually no fluorescence. The high-density carbon tips also demonstrated low count rates, but the signal contained a short lifetime fluorescence component (0.7 ns). The intensity of the signals from each of the tips was geometry dependent, demonstrating the highest intensities at the edges and corners. Likewise, the anisotropy of all tip signals was observed to be geometry dependent, with the dependence varying on a case-by-case basis. The implications for using confocal illumination instead of total internal reflection are discussed.

Anisotropy↗