Applications of two focus fluorescence correlation spectroscopy in colloid and polymer science

Thermal fluctuations in liquid samples can be used to calculate the hydrodynamic radii (Rh) of observed particles. Therefore, it is necessary to generate scattering contrast between the observed particles and the surrounding liquid phase. In static- and dynamic light scattering (SLS/DLS) experiments this contrast is usually created by the different refractive indices of sample and solvent, whereas in small angle neutron scattering (SANS) the differences in the scattering lengths of hydrogen and deuterium are responsible for the contrast. This thesis presents results obtained on colloidal systems by a new method, dual focus Fluorescence Correlation Spectroscopy (2fFCS), which permits investigations of comparable precision to established methods. 2fFCS is insensitive to many optical and photo-physical effects. An external length scale is introduced into the measurement by creating two overlapping excitation foci at a precisely known distance, thus allowing reference free and absolute measurements of diffusion coefficients (D) close to the infinite dilution limit. 2fFCS data evaluation is modified for taking into account correctly finite size effects in colloidal and macromolecular systems. Nevertheless, it is necessary to consider some instrumental and experimental issues to achieve the best results. The most important instrumental part is precise temperature control of the sample environment, which plays a key role, on account of the strong temperature dependence of the solvent viscosity. The presented sample cell for inverted microscopes has a temperature accuracy of +/- 0.05 K in a temperature range of 5 to 65°C. A tunable independent setting of heating- and cooling behavior is possible and allows the application to microscopes without any objective heating. Diffusion experiments performed in aqueous solutions demonstrate the excellent temperature stability and reproducibility of the device. Due to this, it is possible to carry out remote temperature measurements in microfluidic devices with higher accuracy than before. The most popular methods with micrometer spatial resolution use temperature dependent fluorescence lifetime or temperature dependent size of thermo responsive materials. Because 2fFCS is generally applicable under various conditions the possible field of applications is much broader with comparable or even better accuracy than with common methods. Another instrumental factor and necessary condition for quantitative data evaluation of 2fFCS experiments is exact knowledge of the shear distance induced by the Nomarski DIC prism, which represents the extrinsic length scale for determining D. The presented calibration technique based on a combination of FCS and DLS allows determination of shear distances with nanometer accuracy, and high precision measurements of diffusion of reference standard fluorescence dyes close to the regime of infinite dilution is possible. In particular, it is found that D of the reference dye Rhodamine 6G is by 37% larger than the value used in most publications on FCS! However, many investigations do not deal with systems at the limit of infinite dilution but with molecular diffusion in crowding environments and it is necessary to consider the differences between the local refractive index in a sample and the refractive index of the immersion liquid. The theory of 2fFCS enables the absolute determination of D without additional calibration; this is confirmed by investigations on a well known system of a fluorescently labeled 70 kDa dextrane in an unlabeled 70 kDa dextrane matrix. As an example for a special kind of molecular dynamics qualitative and quantitative investigations on Layer-by-Layer (LbL) assembly of polyelectrolyte (PE) multilayers on soft and porous temperature sensitive microgels are presented in this thesis. As PEs are able to interdigitate with each other during multilayer formation as well as with the microgel it was not posible so far to distinguish between PE adsorbed on or in the microgel. But a clear difference between free labeled PE and those that are bound to the microgel is possible; dual color correlation confirmed the presence of both PE's bound to the same particle, while fluorescence imaging provided the visual proof.

Grantor dc:publisher

Publikationsserver der RWTH Aachen University

Year dc:date

2008

Author dc:creator

• Müller, Claus Bernhard

Contributors dc:contributor

• Richtering, Walter

• info:eu-repo/classification/ddc/540
• Spektroskopie
• Optische Spektroskopie
• Laserinduzierte Fluoreszenz
• Fluoreszenz
• Fluoreszenz-Resonanz-Energie-Transfer
• Mikroskopie
• Konfokale Mikroskopie
• Kolloidchemie
• Kolloidphysik
• Polymere
• Chemie
• Zwei-Fokus
• Korrelationspektroskopie
• 2fFCS
• Two focus
• fluorescence
• correlation spectroscopy
• dual focus fluorescence correlation spectroscopy, 2fFCS

Statement dc:rights

• info:eu-repo/semantics/openAccess

Language dc:language

eng

Identifier

https://publications.rwth-aachen.de/search?p=id:%22RWTH-CONV-112792%22

OAI record oai:identifier

https://publications.rwth-aachen.de/oai2d?verb=GetRecord&metadataPrefix=oai_dc&identifier=oai%3Apublications.rwth-aachen.de%3A50239