Measuring precise diffusion coefficients with two-focus fluorescence correlation spectroscopy

Thomas Dertinger; Ingo Gregor; Iris von der Hocht; Rainer Erdmann; Benedikt Krämer; Felix Koberling; Rudolf Hartmann; Jörg Enderlein

doi:10.1117/12.651053

27 February 2006 Measuring precise diffusion coefficients with two-focus fluorescence correlation spectroscopy

Thomas Dertinger, Ingo Gregor, Iris von der Hocht, Rainer Erdmann, Benedikt Krämer, Felix Koberling, Rudolf Hartmann, Jörg Enderlein

Author Affiliations +

Proceedings Volume 6092, Ultrasensitive and Single-Molecule Detection Technologies; 609203 (2006) https://doi.org/10.1117/12.651053
Event: SPIE BiOS, 2006, San Jose, California, United States

Abstract

We present a new method for precisely measuring diffusion coefficients of fluorescent molecules at nanomolar concentrations. The method is based on a modified Fluorescence Correlation Spectroscopy (FCS)-setup which is robust against many artifacts that are inherent to standard FCS ^{1, 2}. The core idea of the new method is the introduction of an external ruler by generating two laterally shifted and overlapping laser foci at a fixed and known distance. Data fitting is facilitated by ab initio calculations of resulting correlation curves and subsequent affine transformation of these curves to match the measured auto- and cross-correlation functions. The affine transformation coefficient along the time axis then directly yields the correct diffusion coefficient. This method is not relying on the rather inexact assumption of a 3D Gaussian shaped detection volume. We measured the diffusion coefficient of the red fluorescent dye Atto-655 (Atto-Tec GmbH) in water and compared the obtained value with results from Gradient Pulsed Field NMR (GPF-NMR).

Citation Download Citation

Thomas Dertinger, Ingo Gregor, Iris von der Hocht, Rainer Erdmann, Benedikt Krämer, Felix Koberling, Rudolf Hartmann, and Jörg Enderlein "Measuring precise diffusion coefficients with two-focus fluorescence correlation spectroscopy", Proc. SPIE 6092, Ultrasensitive and Single-Molecule Detection Technologies, 609203 (27 February 2006); https://doi.org/10.1117/12.651053

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