Correlation Spectroscopy

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Theo Lasser - One of the best experts on this subject based on the ideXlab platform.

Rudolf Rigler - One of the best experts on this subject based on the ideXlab platform.

  • dynamic disorder in horseradish peroxidase observed with total internal reflection fluorescence Correlation Spectroscopy
    Optics Express, 2007
    Co-Authors: Kai Hassler, Rudolf Rigler, Hans Blom, Jerker Widengren, Per Rigler, Theo Lasser
    Abstract:

    This paper discusses the application of objective-type total internal reflection fluorescence Correlation Spectroscopy (TIR-FCS) to the study of the kinetics of immobilized horseradish peroxidase on a single molecule level. Objective-type TIR-FCS combines the advantages of FCS with TIRF microscopy in a way that allows for simultaneous ultra-sensitive spectroscopic measurements using a single-point detector and convenient localization of single molecules on a surface by means of parallel imaging.

  • high count rates with total internal reflection fluorescence Correlation Spectroscopy
    Biophysical Journal, 2005
    Co-Authors: Kai Hassler, Rudolf Rigler, Tiemo Anhut, Michael Gosch, Theo Lasser
    Abstract:

    We achieved photon count rates per molecule as high as with commonly used confocal fluorescence Correlation Spectroscopy instruments using a new total internal reflection fluorescence Correlation Spectroscopy system based on an epi-illumination configuration.

  • molecular interactions of peptides with phospholipid vesicle membranes as studied by fluorescence Correlation Spectroscopy
    Chemistry and Physics of Lipids, 2000
    Co-Authors: Aladdin Pramanik, Per Thyberg, Rudolf Rigler
    Abstract:

    Interactions of the peptides melittin and magainin with phospholipid vesicle membranes have been studied using fluorescence Correlation Spectroscopy. Molecular interactions of melittin and magainin with phospholipid membranes are performed in rhodamine-entrapped vesicles (REV) and in rhodamine-labelled phospholipid vesicles (RLV), which did not entrap free rhodamine inside. The results demonstrate that melittin makes channels into vesicle membranes since exposure of melittin to vesicles causes rhodamine release only from REV but not from RLV. It is obvious that rhodamine can not be released from RLV because the inside of RLV is free of dye molecules. In contrast, magainin breaks vesicles since addition of magainin to vesicles results in rhodamine release from both REV and RLV. As the inside of RLV is free of rhodamine, the appearance of rhodamine in solution confirms that these vesicles are broken into rhodamine-labelled phospholipid fragments after addition of magainin. This study is of pharmaceutical significance since it will provide insights that fluorescence Correlation Spectroscopy can be used as a rapid protocol to test incorporation and release of drugs by vesicles.

  • fluorescence Correlation Spectroscopy of enzymatic dna polymerization
    Biochemistry, 1998
    Co-Authors: Sofie Bjorling, Per Thyberg, Masataka Kinjo, Zeno Foldespapp, Ebba Hagman, Rudolf Rigler
    Abstract:

    We show that fluorescence Correlation Spectroscopy (FCS) can be used as a reliable, simple, and fast tool for detecting products of the polymerase chain reaction (PCR). By use of autoCorrelation experiments, it is demonstrated that fluorescent 217-bp DNA fragments can be detected at very low initial ss M13mp18(+) DNA and tetramethylrhodamine-4-dUTP concentrations and that these polymers are cleaved by the chosen restriction enzymes. A FCS calibration curve is presented, where the translational diffusion times of different size DNA fragments are plotted versus the number of base pairs they contain. At zero and very low template concentrations a large "background" species emerges, which is a reflection of the experimental conditions chosen and the extremely high sensitivity of FCS. The relative amount of nonspecific product formation is less than 1%. The ease by which a FCS measurement can be performed (a few minutes at most) also enables the technique to be used as an effective screening method.

  • dual color fluorescence cross Correlation Spectroscopy for multicomponent diffusional analysis in solution
    Biophysical Journal, 1997
    Co-Authors: Petra Schwille, Franzjosef Meyeralmes, Rudolf Rigler
    Abstract:

    The present paper describes a new experimental scheme for following diffusion and chemical reaction systems of fluorescently labeled molecules in the nanomolar concentration range by fluorescence Correlation analysis. In the dual-color fluorescence cross-Correlation Spectroscopy provided here, the concentration and diffusion characteristics of two fluorescent species in solution as well as their reaction product can be followed in parallel. By using two differently labeled reaction partners, the selectivity to investigate the temporal evolution of reaction product is significantly increased compared to ordinary one-color fluorescence autoCorrelation systems. Here we develop the theoretical and experimental basis for carrying out measurements in a confocal dual-beam fluorescence Correlation Spectroscopy setup and discuss conditions that are favorable for cross-Correlation analysis. The measurement principle is explained for carrying out DNA-DNA renaturation kinetics with two differently labeled complementary strands. The concentration of the reaction product can be directly determined from the cross-Correlation amplitude.

Maria Angela Franceschini - One of the best experts on this subject based on the ideXlab platform.

Petra Schwille - One of the best experts on this subject based on the ideXlab platform.

  • fluorescence lifetime Correlation Spectroscopy for precise concentration detection in vivo by background subtraction
    Clinical and Biomedical Spectroscopy (2009) paper 7368_1V, 2009
    Co-Authors: Maria Gartner, Thomas Ohrt, Jorg Mutze, Petra Schwille
    Abstract:

    In vivo studies of single molecule dynamics by means of Fluorescence Correlation Spectroscopy can suffer from high background. Fluorescence lifetime Correlation Spectroscopy provides a tool to distinguish between signal and unwanted contributions via lifetime separation. By studying the motion of the RNA-induced silencing complex (RISC) within two compartments of a human cell, the nucleus and the cytoplasm, we observed clear differences in concentration as well as mobility of the protein complex between those two locations. Especially in the nucleus, where the fluorescence signal is very weak, a correction for background is crucial to provide reliable results of the particle number. Utilizing the fluorescent lifetime of the different contributions, we show that it is possible to distinguish between the fluorescent signal and the autofluorescent background in vivo in a single measurement.

  • fluorescence Correlation Spectroscopy and fluorescence cross Correlation Spectroscopy reveal the cytoplasmic origination of loaded nuclear risc in vivo in human cells
    Nucleic Acids Research, 2008
    Co-Authors: Thomas Ohrt, Jorg Mutze, Wolfgang Staroske, Lasse Weinmann, Julia Hock, Karin Crell, Gunter Meister, Petra Schwille
    Abstract:

    Studies of RNA interference (RNAi) provide evidence that in addition to the well-characterized cytoplasmic mechanisms, nuclear mechanisms also exist. The mechanism by which the nuclear RNA-induced silencing complex (RISC) is formed in mammalian cells, as well as the relationship between the RNA silencing pathways in nuclear and cytoplasmic compartments is still unknown. Here we show by applying fluorescence Correlation and cross-Correlation Spectroscopy (FCS/FCCS) in vivo that two distinct RISC exist: a large ∼3 MDa complex in the cytoplasm and a 20-fold smaller complex of ∼158 kDa in the nucleus. We further show that nuclear RISC, consisting only of Ago2 and a short RNA, is loaded in the cytoplasm and imported into the nucleus. The loaded RISC accumulates in the nucleus depending on the presence of a target, based on an miRNA-like interaction with impaired cleavage of the cognate RNA. Together, these results suggest a new RISC shuttling mechanism between nucleus and cytoplasm ensuring concomitant gene regulation by small RNAs in both compartments.

  • fluorescence Correlation Spectroscopy novel variations of an established technique
    Annual Review of Biophysics and Biomolecular Structure, 2007
    Co-Authors: Elke Haustein, Petra Schwille
    Abstract:

    Fluorescence Correlation Spectroscopy (FCS) is one of the major biophysical techniques used for unraveling molecular interactions in vitro and in vivo. It allows minimally invasive study of dynamic processes in biological specimens with extremely high temporal and spatial resolution. By recording and correlating the fluorescence fluctuations of single labeled molecules through the exciting laser beam, FCS gives information on molecular mobility and photophysical and photochemical reactions. By using dual-color fluorescence cross-Correlation, highly specific binding studies can be performed. These have been extended to four reaction partners accessible by multicolor applications. Alternative detection schemes shift accessible time frames to slower processes (e.g., scanning FCS) or higher concentrations (e.g., TIR-FCS). Despite its long tradition, FCS is by no means dated. Rather, it has proven to be a highly versatile technique that can easily be adapted to solve specific biological questions, and it continues to find exciting applications in biology and medicine.

  • real time enzyme kinetics monitored by dual color fluorescence cross Correlation Spectroscopy
    Proceedings of the National Academy of Sciences of the United States of America, 1998
    Co-Authors: Ulrich Kettling, Petra Schwille, Andre Koltermann, Manfred Eigen
    Abstract:

    A method for sensitively monitoring enzyme kinetics and activities by using dual-color fluorescence cross-Correlation Spectroscopy is described. This universal method enables the development of highly sensitive and precise assays for real-time kinetic analyses of any catalyzed cleavage or addition reaction, where a chemical linkage is formed or cleaved through an enzyme's action between two fluorophores that can be discriminated spectrally. In this work, a homogeneous assay with restriction endonuclease EcoRI and a 66-bp double-stranded DNA containing the GAATTC recognition site and fluorophores at each 5' end is described. The enzyme activity can be quantified down to the low picomolar range (>1.6 pM) where the rate constants are linearly dependent on the enzyme concentrations over two orders of magnitude. Furthermore, the reactions were monitored on-line at various initial substrate concentrations in the nanomolar range, and the reaction rates were clearly represented by the Michaelis-Menten equation with a KM of 14 +/- 1 nM and a kcat of 4.6 +/- 0.2 min-1. In addition to kinetic studies and activity determinations, it is proposed that enzyme assays based on the dual-color fluorescence cross-Correlation Spectroscopy will be very useful for high-throughput screening and evolutionary biotechnology.

  • dual color fluorescence cross Correlation Spectroscopy for multicomponent diffusional analysis in solution
    Biophysical Journal, 1997
    Co-Authors: Petra Schwille, Franzjosef Meyeralmes, Rudolf Rigler
    Abstract:

    The present paper describes a new experimental scheme for following diffusion and chemical reaction systems of fluorescently labeled molecules in the nanomolar concentration range by fluorescence Correlation analysis. In the dual-color fluorescence cross-Correlation Spectroscopy provided here, the concentration and diffusion characteristics of two fluorescent species in solution as well as their reaction product can be followed in parallel. By using two differently labeled reaction partners, the selectivity to investigate the temporal evolution of reaction product is significantly increased compared to ordinary one-color fluorescence autoCorrelation systems. Here we develop the theoretical and experimental basis for carrying out measurements in a confocal dual-beam fluorescence Correlation Spectroscopy setup and discuss conditions that are favorable for cross-Correlation analysis. The measurement principle is explained for carrying out DNA-DNA renaturation kinetics with two differently labeled complementary strands. The concentration of the reaction product can be directly determined from the cross-Correlation amplitude.

Stefan A Carp - One of the best experts on this subject based on the ideXlab platform.

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