Claus B. Müller

Precise measurement of diffusion by multi-color dual-focus fluorescence correlation spectroscopy

Dual-focus fluorescence correlation spectroscopy is a method for precisely measuring the diffusion coefficient of fluorescing molecules close to the infinite dilution limit in a reference-free and absolute manner. We apply the method to determine the diffusion coefficients of three fluorescent dyes across the visible spectrum. These values can be used as absolute reference standards for fluorescence correlation spectroscopy. In particular, it is found that the diffusion coefficient of the widely used reference dye Rhodamine 6G is by 37% larger than the value used in most publications on fluorescence correlation spectroscopy over the last three decades.

The optics and performance of dual-focus fluorescence correlation spectroscopy.

Fluorescence correlation spectroscopy (FCS) is an important spectroscopic technique which can be used for measuring the diffusion and thus size of fluorescing molecules at pico- to nanomolar concentrations. Recently, we introduced an extension of conventional FCS, which is called dual-focus FCS (2fFCS) and allows absolute diffusion measurements with high precision and repeatability. It was shown experimentally that the method is robust against most optical and sample artefacts which are troubling conventional FCS measurements, and is furthermore able to yield absolute values of diffusion coefficients without referencing against known standards. However, a thorough theoretical treatment of the performance of 2fFCS is still missing. The present paper aims at filling this gap. Here, we have systematically studied the performance of 2fFCS with respect to the most important optical and photophysical factors such as cover slide thick-ness, refractive index of the sample, laser beam geometry, and optical satu-ration. We show that 2fFCS has indeed a superior performance when com-pared with conventional FCS, being mostly insensitive to most potential ab-errations when working under optimized conditions.

Study of Layer-by-Layer Films on Thermoresponsive Nanogels Using Temperature-Controlled Dual-Focus Fluorescence Correlation Spectroscopy

While a few studies have reported on the layer-by-layer (LbL) assembly of polyelectrolytes on soft and porous templates, none have really demonstrated direct proof that the layers are actually on the template. Thermoresponsive nanogels present challenges that render a quantitative proof of successful polyelectrolyte deposition extremely difficult. Additionally, the fate of the polyelectrolyte has never been investigated during the phase transition of the coated nanogel. Here, the auto- and cross-correlation functions of a labeled polyelectrolyte assembled via the LbL technique onto soft and porous thermoresponsive labeled nanogels using dual-focus fluorescence correlation spectroscopy (2f-FCS) are presented. Performing 2f-FCS as a function of temperature, hydrodynamic radii of nanogels coated with various numbers of layers are determined, which are found to be in excellent agreement with values obtained from dynamic light scattering. This study presents irrefutable quantitative evidence of successful LbL assembly on thermoresponsive nanogels and demonstrates that the layers are not stripped off during the phase transition of the nanogels. Forster Resonance Energy Transfer (FRET) detection also supports our findings.

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

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.

Calibrating differential interference contrast microscopy with dual-focus fluorescence correlation spectroscopy.

We present a novel calibration technique for determining the shear distance of a Nomarski Differential Interference Contrast prism, which is used in Differential Interference Contrast microscopy as well as for the recently developed dual-focus fluorescence correlation spectroscopy. In both applications, an exact knowledge of the shear distance induced by the Nomarski prism is important for a quantitative data evaluation. In Differential Interference Contrast microscopy, the shear distance determines the spatial resolution of imaging, in dual-focus fluorescence correlation spectroscopy, it represents the extrinsic length scale for determining diffusion coefficients. The presented calibration technique is itself based on a combination of fluorescence correlation spectroscopy and dynamic light scattering. The method is easy to implement and allows for determining the shear distance with nanometer accuracy.

Dual-Focus Fluorescence Correlation Spectroscopy of Colloidal Solutions : Influence of Particle Size

Fluorescence correlation spectroscopy (FCS) is a powerful technique for measuring diffusion coefficients of small fluorescent molecules at pico- to nanomolar concentrations. Recently, a modified version of FCS, dual-focus FCS (2fFCS), was introduced that significantly improves the reliability and accuracy of FCS measurements and allows for obtaining absolute values of diffusion coefficients without the need of referencing again a known standard. It was shown that 2fFCS gives excellent results for measuring the diffusion of small molecules. However, when measuring colloids or macromolecules, the size of these objects can no longer be neglected with respect to the excitation laser focus. Here, we analyze how 2fFCS data evaluation has to be modified for correctly taking into a count these finite size effects. We exemplify the new method of measuring the absolute size of polymeric particles with simple and complex fluorophore distributions.

Polymer-Stabilized Emulsions: Influence of Emulsion Components on Rheological Properties and Droplet Size

We investigated o/w-emulsions containing polymeric thickener C10–C30 acrylate (acrylate) and silicone-based emulsifier PEG-12 dimethicone (PEG-12) but without low molecular mass surfactants. Mechanical properties of emulsions were probed by oscillatory and continuous flow rheometry while droplet size was studied by flow particle image analysis (FPIA). By varying thickener and emulsifier content rheological properties and droplet size of emulsions changed significantly. Experimental results and a statistical analysis showed that the physical network, built up by acrylate in a concentration range from 0.1–%[wt.]1.0, was the dominating factor for rheological properties and increased moduli and viscosity of emulsions. The development of droplet diameters revealed that a systematic control of droplet parameters was possible by increasing the PEG-12 concentration from 0.0–%[wt.]5.0. In contrast, increasing acrylate concentration led to either large or small droplets. The influence of larger droplets in the emulsions was revealed when the arithmetic diameter and the Sauter diameter were compared and displayed huge differences. These differences resulted from a rather small amount of big droplets with diameters above 40 μm. An influence of oil droplets on emulsion elasticity was only observed for emulsions with low acrylate concentration (≤ 0.1 wt. %), because at higher concentrations the influence of oil droplets was superimposed by thickening properties of acrylate.