Dual-focus fluorescence correlation spectroscopy: a robust tool for studying molecular crowding

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Abstract
Conventional single-focus fluorescence correlation spectroscopy (FCS) is often used for studying molecular diffusion in crowded environments. However, these measurements usually deal with concentrations of the crowding agent far beyond the overlap-concentration, resulting in a crowding effect which slows down the diffusion coefficient by several orders of magnitude. In the present paper, we would like to study the transition range from free diffusion to crowding. Therefore, high accuracy of the determination of the diffusion coefficient is needed. In the majority of cases, the local refractive index in a sample is different from the refractive index of the immersion medium of the used objective. To achieve a high accuracy during experiments it is necessary to account for the refractive index mismatch in single-focus FCS calculations. In this work, we study theoretically and experimentally the influence of the refractive index mismatch on performance of single-focus FCS as well as the recently developed dual-focus FCS (2fFCS). By looking at the transition from free tracer diffusion to crowding it is shown that, in contrast to conventional FCS, 2fFCS allows measuring absolute values of the diffusion coefficient and its change in the range of half an order of magnitude. Even under conditions of strong refractive index mismatch between sample and immersion medium, without the need of additional calibration. This is demonstrated on a system of fluorescently labeled 70 kDa dextrane in an unlabeled 70 kDa dextrane matrix. Therefore, 2fFCS is a perfect tool for investigating molecular dynamics in crowded environments.