126,537 research outputs found

    Polymer dynamics, fluorescence correlation spectroscopy, and the limits of optical resolution

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    In recent years, fluorescence correlation spectroscopy has been increasingly applied for the study of polymer dynamics on the nanometer scale. The core idea is to extract, from a measured autocorrelation curve, an effective mean-square displacement function that contains information about the underlying conformational dynamics. The paper presents a fundamental study of the applicability of fluorescence correlation spectroscopy for the investigation of nanoscale conformational and diffusional dynamics. We find that fluorescence correlation spectroscopy cannot reliably elucidate processes on length scales much smaller than the resolution limit of the optics used and that its improper use can yield spurious results for the observed dynamics.Comment: 4 pages, 4 figures, accepted by Physical Review Letter

    Tracking-FCS: Fluorescence correlation spectroscopy of individual particles

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    We exploit recent advances in single-particle tracking to perform fluorescence correlation spectroscopy on individual fluorescent particles, in contrast to traditional methods that build up statistics over a sequence of many measurements. By rapidly scanning the focus of an excitation laser in a circular pattern, demodulating the measured fluorescence, and feeding these results back to a piezoelectric translation stage, we track the Brownian motion of fluorescent polymer microspheres in aqueous solution in the plane transverse to the laser axis. We discuss the estimation of particle diffusion statistics from closed-loop position measurements, and we present a generalized theory of fluorescence correlation spectroscopy for the case that the motion of a single fluorescent particle is actively tracked by a time-dependent laser intensity. We model the motion of a tracked particle using Ornstein-Uhlenbeck statistics, using a general theory that contains a umber of existing results as specific cases. We find good agreement between our theory and experimental results, and discuss possible future applications of these techniques to passive, single-shot, single-molecule fluorescence measurements with many orders of magnitude in time resolution

    Chloroplast and Cell Imaging at Submicron Resolution by Two-Photon Excitation

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    Novel, two-photon NIR excitation fluorescence correlation microspectroscopy tests and preliminary results were presented in this article with submicron resolution for concentrated suspensions of functional cells and chloroplast membranes. Related developments of these technique involve applications of Fluorescence Cross-Correlation Spectroscopy (FCCS) detection to monitoring: 
DNA- telomerase interactions, DNA hybridization kinetics, ligand-receptor interactions, and HIV-HBV testing. 
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    Intramolecular fluorescence correlation spectroscopy in a feedback tracking microscope

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    We derive the statistics of the signals generated by shape fluctuations of large molecules studied by feedback tracking microscopy. We account for the influence of intramolecular dynamics on the response of the tracking system, and derive a general expression for the fluorescence autocorrelation function that applies when those dynamics are linear. We show that tracking provides enhanced sensitivity to translational diffusion, molecular size, heterogeneity and long time-scale decays in comparison to traditional fluorescence correlation spectroscopy. We demonstrate our approach by using a three-dimensional tracking microscope to study genomic λ\lambda-phage DNA molecules with various fluorescence label configurations.Comment: 11 pages, 5 figures, supplemental info: http://minty.stanford.edu/papers/Publications/McHale10aSI.pd

    Localization-based fluorescence correlation spectroscopy

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    Two-Focus Fluorescence Correlation Spectroscopy

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    Fluorescence Correlation Spectroscopy (FCS) has been invented more than 30 years ago and experienced a renaissance after stable and affordable laser sources and low-noise single-photon detectors have become available. Its ability to measure diffusion coefficients at nanomolar concentrations of analyte made it a widely used tool in biophysics. However, in recent years it has been shown by many authors that aberrational (e.g. astigmatism) and photophysical effects (e.g. optical saturation) may influence the result of an FCS experiment dramatically, so that a precise and reliable estimation of the diffusion coefficient is no longer possible. In this thesis, we report on the development, implementation, and application of a new and robust modification of FCS that we termed two-focus FCS (2fFCS) and which fulfils two requirements: (i) It introduces an external ruler into the measurement by generating two overlapping laser foci of precisely known and fixed distance. (ii) These two foci and corresponding detection regions are generated in such a way that the corresponding molecule detection functions (MDFs) are sufficiently well described by a simple two-parameter model yielding accurate diffusion coefficients when applied to 2fFCS data analysis. Both these properties enable us to measure absolute values of the diffusion coefficient with an accuracy of a few percent. Moreover, it will turn out that the new technique is robust against refractive index mismatch, coverslide thickness deviations, and optical saturation effects, which so often trouble conventional FCS measurements. This thesis deals mainly with the introduction of the new measurement scheme, 2fFCS, but also presents several applications with far-reaching importance

    Fluorescence Correlation Spectroscopy analysis of segmental dynamics in Actin filaments

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    We adapt Fluorescence Correlation spectroscopy (FCS) formalism to the studies of the dynamics of semi-flexible polymers and derive expressions relating FCS correlation function to the longitudinal and transverse mean square displacements of polymer segments. We use the derived expressions to measure the dynamics of actin filaments in two experimental situations: filaments labeled at distinct positions and homogeneously labeled filaments. Both approaches give consistent results and allow to measure the temporal dependence of the segmental mean-square displacement (MSD) over almost five decades in time, from ~0.04ms to 2s. These noninvasive measurements allow for a detailed quantitative comparison of the experimental data to the current theories of semi-flexible polymer dynamics. Good quantitative agreement is found between the experimental results and theories explicitly accounting for the hydrodynamic interactions between polymer segments

    Plasmonic antennas and zero mode waveguides to enhance single molecule fluorescence detection and fluorescence correlation spectroscopy towards physiological concentrations

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    Single-molecule approaches to biology offer a powerful new vision to elucidate the mechanisms that underpin the functioning of living cells. However, conventional optical single molecule spectroscopy techniques such as F\"orster fluorescence resonance energy transfer (FRET) or fluorescence correlation spectroscopy (FCS) are limited by diffraction to the nanomolar concentration range, far below the physiological micromolar concentration range where most biological reaction occur. To breach the diffraction limit, zero mode waveguides and plasmonic antennas exploit the surface plasmon resonances to confine and enhance light down to the nanometre scale. The ability of plasmonics to achieve extreme light concentration unlocks an enormous potential to enhance fluorescence detection, FRET and FCS. Single molecule spectroscopy techniques greatly benefit from zero mode waveguides and plasmonic antennas to enter a new dimension of molecular concentration reaching physiological conditions. The application of nano-optics to biological problems with FRET and FCS is an emerging and exciting field, and is promising to reveal new insights on biological functions and dynamics.Comment: WIREs Nanomed Nanobiotechnol 201

    Molecular Sizing using Fluorescence Correlation Spectroscopy

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    Die Größe ist eine grundlegende Eigenschaft eines jeden Objektes, speziell von Molekülen. Sie ist direkt verbunden mit fundamentalen Phänomenen, wie z.B. der Diffusion, und zwar unabhängig von anderen Eigenschaften. Die Größe von Molekülen kann sich ändern, wenn sie mit anderen Molekülen wechselwirken (z. B. wenn sie Ionen binden), oder wenn die Temperatur, der pH-Wert oder die chemische Zusammensetzung des umgebenden Mediums sich ändert. Somit kann die Größe ein sehr sensitiver Reporter für den Zustand eines Moleküls sein. Daher findet die Größenbestimmung von Molekülen breite Anwendung in der Physik, der Chemie und der Biologie. In den meisten Fällen erfordern diese Anwendungen eine Größenbestimmung mit einer Genauigkeit von wenigen Angström. In dieser Arbeit untersuche ich die Größe von Molekülen bei pico- und nanomolaren Konzentrationen mittels hoch-genauer Fluoreszenz-Korrelations-Spektroskopie (FCS). Eine spezielle Abwandlung dieser Methode, die zwei-Focus FCS (2fFCS) erlaubt die Bestimmung des absoluten Diffusionskoeffizienten und somit auch der absoluten Größe eines Moleküls. Die Genauigkeit dieser Methodik wird unter anderen anhand weit verbreiteter globulärer Proteine bestimmt. Weiterhin wird die gemessene quantitative Beziehung zwischen dem molekularen Gewicht und dem gemessenen Diffusionskoeffizienten diskutiert. Ausgehend von der Fluoreszenz-Korrelations-Spektroskopie habe ich eine neue Methode entwickelt, die Rotations-Diffusions-Konstanten von Makromolekülen bestimmt. Diese Methode ist geeignet, um Rotations-Diffusions-Konstanten zwischen zehn und einhundert Nanosekunden zu bestimmen, also einem Bereich in dem Fluoreszenz-Anisotropie-Messungen nicht gut angewendet werden können. Mittels des gemessenen Rotations-Diffusions-Koeffizienten wurden die hydrodynamischen Radien einiger weitverbreiteter, globulärer Proteine bestimmt. Die erhaltenen Werte werden mit den Resultaten aus den Diffusionsmessungen verglichen
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