Two-Focus Fluorescence Correlation Spectroscopy: A New Tool for Accurate and Absolute Diffusion Measurements

Graphical Abstract

Absolute diffusion coefficients: Two-focus-FCS (2fFCS) is a powerful modification of fluorescence correlation spectroscopy. By using two overlapping foci with a well-defined distance (see figure), 2fFCS introduces an external length scale which allows for precise and absolute measurements of diffusion coefficients. This method is robust to optical and photophysical artefacts inherent to standard FCS.

Abstract

We present a new method to measure absolute diffusion coefficients at nanomolar concentrations with high precision. Based on a modified fluorescence correlation spectroscopy (FCS)-setup, this method is improved by introducing an external ruler for measuring the diffusion time by generating two laterally shifted and overlapping laser foci at a fixed and known distance. Data fitting is facilitated by a new two-parameter model to describe the molecule detection function (MDF). We present a recorded MDF and show the excellent agreement with the fitting model. We measure the diffusion coefficient of the red fluorescent dye Atto655 under various conditions and compare these values with a value achieved by gradient pulsed field NMR (GPF NMR). From these measurements we conclude, that the new measurement scheme is robust against optical and photophysical artefacts which are inherent to standard FCS. With two-focus-FCS, the diffusion coefficient of 4.26×10⁻⁶ cm² s⁻¹ for Atto655 in water at 25 °C compares well with the GPF NMR value of 4.28×10⁻⁶ cm² s⁻¹.