Measuring precise diffusion coefficients with two-focus fluorescence correlation spectroscopy

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 FCS1,2. 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).