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Biomedical subjects

Michael Gösch

Publications and source records attributed to Michael Gösch.

11 recordsLinked to original sources
PubMed2006

Stochastic approach to data analysis in fluorescence correlation spectroscopy.

Fluorescence correlation spectroscopy (FCS) has emerged as a powerful technique for measuring low concentrations of fluorescent molecules and their diffusion constants. In FCS, the experimental data is conventionally fit using standard local search techniques, for example, the Marquardt-Levenberg (ML) algorithm. A prerequisite for these categories of algorithms is the sound knowledge of the behavior of fit parameters and in most cases good initial guesses for accurate fitting, otherwise leading to fitting artifacts. For known fit models and with user experience about the behavior of fit parameters, these local search algorithms work extremely well. However, for heterogeneous systems or where automated data analysis is a prerequisite, there is a need to apply a procedure, which treats FCS data fitting as a black box and generates reliable fit parameters with accuracy for the chosen model in hand. We present a computational approach to analyze FCS data by means of a stochastic algorithm for global search called PGSL, an acronym for Probabilistic Global Search Lausanne. This algorithm does not require any initial guesses and does the fitting in terms of searching for solutions by global sampling. It is flexible as well as computationally faster at the same time for multiparameter evaluations. We present the performance study of PGSL for two-component with triplet fits. The statistical study and the goodness of fit criterion for PGSL are also presented. The robustness of PGSL on noisy experimental data for parameter estimation is also verified. We further extend the scope of PGSL by a hybrid analysis wherein the output of PGSL is fed as initial guesses to ML. Reliability studies show that PGSL and the hybrid combination of both perform better than ML for various thresholds of the mean-squared error (MSE).

Computer Simulation↗
PubMed2006

Prism-based multicolor fluorescence correlation spectrometer.

We report the design and application of a prism-based detection system for fluorescence (cross) correlation spectroscopy. The system utilizes a single laser wavelength for the simultaneous excitation of several dyes of different emission spectra. Fluorescence light is spectrally separated with a prismatic setup, and wavelengths are selected by scanning a fiber-coupled avalanche photodiode across the image spots. Multicolor autocorrelations are demonstrated with standard and tandem dyes, and fluorescence cross-correlation measurements of biotinylated nanocontainers and streptavidin are presented. This spectrometer offers high optical stability and no focal volume mismatch for the multicolor detection of molecular dynamics and interactions, with single-molecule sensitivity.

Equipment Design↗
PubMed2006

Simultaneous multicolor fluorescence cross-correlation spectroscopy to detect higher order molecular interactions using single wavelength laser excitation.

Fluorescence cross-correlation spectroscopy is a powerful method for the study of molecular interactions and dynamics in solution and even in living cells. Usually, in the optical setup, either two laser beams have to be superimposed in their respective confocal volumes or two-photon excitation is used for a dual-color detection system. It has been shown recently that fluorescence cross correlation can be achieved with spectrally similar fluorophores using single wavelength excitation fluorescence cross-correlation spectroscopy (SW-FCCS). In this study, we show that SW-FCCS allows the simultaneous excitation of up to three fluorophores in which the cross correlation of their fluctuation signals is detected separately in three detection channels. The experimental and theoretical model to describe triple pairwise cross correlations incorporating cross talk and possible changes in emission characteristics such as quenching upon binding are outlined. The effectiveness of SW-FCCS to detect binding of three interacting partners is experimentally verified with a standard ligand-receptor model, biotin-streptavidin, where differently labeled biotin ligands and their binding to a third-color labeled streptavidin are studied. The cross-correlation amplitudes and their changes with stoichiometric binding are analyzed and the upper limits of dissociation constants are determined. Performed with appropriate negative controls, SW-FCCS can determine interaction patterns between ligands and receptors.

Biotin↗
PubMed2005

Ultrasensitive reporter protein detection in genetically engineered bacteria.

We demonstrate the use of laser-induced fluorescence confocal spectroscopy to measure analyte-stimulated enhanced green fluorescent protein (egfp) synthesis by genetically modified Escherichia coli bioreporter cells. Induction is measured in cell lysates and, since the spectroscopic focal volume is approximately the size of one bioreporter cell, also in individual live bacteria. This is, to our knowledge, the first ever proof-of-concept work utilizing instrumentation with single-molecule detection capability to monitor bioreporter response. Although we use arsenic inducible bioreporters here, the method is extensible to gfp/egfp bioreporters that are responsive to other substances.

Arsenites↗
PubMed2005

Fluorescence correlation spectroscopy of molecular motions and kinetics.

The foundations for fluorescence correlation spectroscopy (FCS) were already laid in the early 1970s, but this technique did not become widely used until single-molecule detection was established almost 20 years later with the use of diffraction-limited confocal volume element. The analysis of molecular noise from the GHz- to the Hz-region facilitates measurements over a large dynamic range covering photophysics, conformational transitions and interactions as well as transport properties of fluorescent biomolecules. From the Poissonian nature of the noise spectrum the absolute number of molecules is obtainable. Originally used for the analysis of molecular interactions in solutions, the strength of FCS lies also in its applicability to molecular processes at either the surface or interior of single cells. Examples for the analysis of surface kinetics including on and off rates of ligand-receptor interactions will be given. The possibility of obtaining this type of information by FCS will be of particular interest for cell-based drug screening.

Kinetics↗
PubMed2003

Gene expression analysis using single molecule detection.

Recent developments of single molecule detection techniques and in particular the introduction of fluorescence correlation spectroscopy (FCS) led to a number of important applications in biological research. We present a unique approach for the gene expression analysis using dual-color cross-correlation. The expression assay is based on gene-specific hybridization of two dye-labeled DNA probes to a selected target gene. The counting of the dual-labeled molecules within the solution allows the quantification of the expressed gene copies in absolute numbers. As detection and analysis by FCS can be performed at the level of single molecules, there is no need for any type of amplification. We describe the gene expression assay and present data demonstrating the capacity of this novel technology. In order to prove the gene specificity, we performed experiments with gene-depleted total cDNA. The biological application was demonstrated by quantifying selected high, medium and low abundant genes in cDNA prepared from HL-60 cells.

Actins↗
PubMed2002

Parallel flow measurements in microstructures by use of a multifocal 4 x 1 diffractive optical fan-out element.

We have developed a multifocal optical fluorescence correlation spectroscopy system for parallel flow analyses. Multifocal excitation was made possible through a 4 x 1 diffractive optical fan-out element, which produces uniform intensity in all four foci. Autocorrelation flow analyses inside a 20 microm x 20 microm square microchannel, with the 4 x 1 fan-out foci perpendicular to the flow direction, made it possible to monitor different flows in all four foci simultaneously. We were able to perform cross-correlation flow analyses by turning the microstructure, thereby having all four foci parallel to the direction of flow. Transport effects of the diffusion as a function of flow and distance could then also be studied.

Fluorescent Dyes↗
PubMedDate not supplied

Parallel single molecule detection with a fully integrated single-photon 2x2 CMOS detector array.

We present parallel single molecule detection (SMD) and fluorescence correlation spectroscopy (FCS) experiments with a fully integrated complementary metal oxide semiconductor (CMOS) single-photon 2x2 detector array. Multifocal excitation is achieved with a diffractive optical element (DOE). Special emphasis is placed on parallelization of the total system. The performance of the novel single-photon CMOS detector is investigated and compared to a state-of-the-art single-photon detecting module [having an actively quenched avalanche photodiode (APD)] by measurements on free diffusing molecules at different concentrations. Despite the order of magnitude lower detection efficiency of the CMOS detector compared to the state-of-the-art single-photon detecting module, we achieve single molecule sensitivity and reliably determine molecule concentrations. In addition, the CMOS detector performance for the determination of the fraction of slowly diffusing molecules in a primer solution (two-component analysis) is demonstrated. The potential of this new technique for high-throughput confocal-detection-based systems is discussed.

Biopolymers↗
PubMedDate not supplied

Parallel dual-color fluorescence cross-correlation spectroscopy using diffractive optical elements.

Dual-color cross-correlation spectroscopy allows the detection and quantification of labeled biomolecules at ultra-low concentrations, whereby the sensitivity of the assay correlates with the measurement time. We now describe a parallel multifocal dual-color spectroscopic configuration employing multiple avalanche photodiodes and hardware correlators. Cross-correlation curves are obtained from several dual-color excitation foci simultaneously. Multifocal dual-color excitation is achieved by splitting each of two laser beams (488 and 633 nm) into four sub-beams with the help of two 2x2 fan-out diffractive optical elements (DOEs), and subsequent superposition of the two sets of four foci. The fluorescence emission from double-labeled biomolecules is detected by two 2x2 fiber arrays.

Equipment Design↗
PubMedDate not supplied

Dual-color total internal reflection fluorescence cross-correlation spectroscopy.

We present the development and first application of a novel dual-color total internal reflection (TIR) fluorescence system for single-molecule coincidence analysis and fluorescence cross-correlation spectroscopy (FCCS). As a performance analysis, we measured a synthetic DNA-binding assay, demonstrating this dual-color TIR-FCCS approach to be a suitable method for measuring coincidence assays such as biochemical binding, fusion, or signal transduction at solid/liquid interfaces. Due to the very high numerical aperture of the epi-illumination configuration, our setup provides a very high fluorescence collection efficiency resulting in a two- to three-fold increase in molecular brightness compared to conventional confocal FCCS. Further improvements have been achieved through global analysis of the spectroscopic data.

DNA↗