Time-Varying Excitation in Fluorescence Spectroscopy for Biological Applications

Abstract: The focus of this thesis is to explore and use the benefits of time-varying excitation in fluorescence spectroscopy for studies of biomolecular dynamics. Two new techniques taking advantage of modulated excitation are presented. Also described are the first efforts in a project where single molecule FRET and multi-parameter fluorescence detection are used for characterization of the conformational dynamics of the retinoid X receptor (RXR).RXR is one of the most important proteins in the group of nuclear receptors. It is believed to be involved in many diseases and is hence most interesting as a potential drug target. Our study is at present at a very early stage and some sample issues are still to be resolved. However, single molecule measurements should give insights not attainable by previously applied ensemble methods and help explaining how RXR can regulate so many different processes.Long-lived transient states of fluorescent molecules can, because of their long lifetimes, be used to detect subtle changes in the microenvironment of the molecule. A method for determining the kinetic rates for transitions to and from such states by registration of changes in the average fluorescence intensity related to different modulation of the excitation source is introduced. It combines the sensitivity of fluorescence with the environmental sensitivity of the long-lived transient states and allows the use of slow detectors such as CCD cameras, making parallelization and imaging possible developments. The approach was experimentally verified by measurements of the triplet kinetics of rhodamine 6G (Rh6G) in aqueous solution and compared with fluorescence correlation spectroscopy (FCS). It should also be applicable to any other photoinduced transient states affecting the fluorescence intensity.A strategy to combine FCS with modulated excitation, in a way that allows extraction of correlation data for all correlation times, is presented. This enables the use of modulation to optimize the measurement conditions with respect to the photophysical properties of the dyes used. Measurements were made on Rh6G to verify the method. To illustrate its usefulness, it was applied to measurements of protonation kinetics of fluorescein at different pH. FCS with modulated excitation will most probably prove very useful in many future studies involving multiple kinetic processes occurring in overlapping time ranges.