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.
Here, 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 turns 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. Additionally, we will
show data that indicates that with 2fFCS it is even possible to monitor conformational changes of a calcium bindig
protein affecting the hydrodynamic radius by as little as two Angstrom.
We report on our application of a new fluorescence-correlation spectroscopy technique, 2-focus FCS, for measuring the
hydrodynamic radius of molecules with sub- Ångstrøm precision. The method is applied of monitoring conformational
changes of proteins upon ion binding. In particular, we present measurements on Ca<sup>2+</sup>-binding of recoverin. Recoverin
belongs to the superfamily of EF-hand Ca<sup>2+</sup>-binding proteins and operates as a Ca<sup>2+</sup>-sensor in vertebrate photoreceptor cells, where it regulates the activity of rhodopsin kinase GRK1 in a Ca<sup>2+</sup>-dependent manner. The protein undergoes conformational changes upon Ca<sup>2+</sup>-changes that are reflected as changes in their hydrodynamic radius. By using 2fFCS
we were able to resolve hydrodynamic radius changes of ca. one Ångstrøm and used the Ca<sup>2+</sup> dependence of this radius
for recording binding curves in solution. We compare our results with those obtained by other techniques.
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 <sup>1, 2</sup>. 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).