Studying interfacial processes using nanosecond pump-probe brewster angle reflectometry and fluorescence spectroscopy
Siebenhofer, Dipl.-lng. Bernhard
Date of Issue2014
In this thesis, optical methods are employed to study interfacial processes, like conformational changes in monolayers, transient kinetics in proteins, as well as pH changes and membrane potential generation by integral membrane proteins in biomimetic lipid bilayers. Brewster angle microscopy has been developed as a tool to image monolayers and thin films. It is an ideal method to follow conformational changes at interfaces. In this work the technique of Brewster angle reflectometry is simplified by replacing the goniometer usually employed for these measurements with a cylindrical lens. This novel approach allows single shot measurements of Brewster's angle and its vicinity. Using two synchronized nanosecond pulsed lasers in pump-probe configuration it is possible to measure the kinetics of photoinduced conformational changes by altering the delay between pump and probe pulses. By using the tunable output of an optical parametric oscillator it is even possible to combine transient kinetics from different wavelengths, thus effectively measuring transient spectra. Model compounds used in this work are amphiphilic spiropyran, spirooxazine and benzophenone. Qualitative measurements on organic photochromic dyes show that the setup is feasible for conversion kinetics measurements in monolayers on water or spin-coated thin films on solid substrates. The nanosecond time resolution is only limited by the temporal convolution of the optical parametric oscillator or laser probe pulse and the pump laser pulse. Another optical method, time-resolved fluorescence spectroscopy is used to track changes in the surrounding of biomimetic membranes, like pH changes and membrane potential generation by the proton pumping of cytochrome c oxidase from Pamcoccus denitrificans. For this approach, Proteo-lipobeads are presented as a novel technique to reconstitute proteins in lipid membranes in a controlled orientation. These beads consist of a central particle, to which solubilized his-tagged membrane proteins are attached with the help of ion-functionalized linkers. During in situ dialysis a biomimetic membrane self-assembles between the protein scaffold.