Heike Hevekerl

Abstract

Fluorescence spectroscopy and imaging are wide-spread tools in life science. The main read-out parameters are still fluorescence intensity and wavelength, but given the benefits of multi-parameter characterization there are also good reasons to consider additional fluorescence-based read-out parameters. A major focus of this thesis is to extend the use of transient, non-fluorescent states as additional parameters for biomolecular studies. To-date, such states (including mainly triplet states, isomerized states and photo-ionized states) have been exploited to a very limited extent for this purpose. Their use has been limited because they show very weak, or no luminescence at all, and absorption measurements require relatively complex instrumentation which are typically not applicable for studies under biologically relevant conditions. Moreover, the long lifetime of these transient states make any readout signal very sensitive to changes in the micro-environment, e.g. presence of small amounts of quenchers, like oxygen. Those transient states can be accessed by fluorescence correlation spectroscopy (FCS) and the newly developed transient state (TRAST) monitoring technique. In this thesis, FCS and TRAST have been applied to demonstrate the use of transient state monitoring for biomolecular studies.
    In Paper I, we demonstrated that due to the low brightness requirements of TRAST, also autofluorescent molecules like tryptophan can be studied, making external labeling of molecules redundant. The photo-physical transient states of tryptophan and tryptophan-containing proteins could be analyzed and were found to provide information about protein conformational states and about the influence of pH and buffers on single tryptophan molecules. In Paper II investigations of the transient states of the oligothiophene p-FTAA with FCS as well as with dynamic light scattering and spectrofluorimetry revealed a pH dependent aggregation behavior and a very efficient fluorescence quenching by oxygen could be identified and analyzed. In Paper III, FCS and TRAST were used to monitor the isomerization kinetics of Merocyanine 540 incorporated in lipid membranes. Because isomerization of cyanine dyes strongly depends on the viscosity of the local environment, the isomerization kinetics could be used to characterize membrane fluidity in artificial lipid vesicles and in cellular membranes. In Paper IV, a new approach was developed, based on a combination of TRAST and FCS to determine the stoichiometry of a fluorescently labeled sample. Finally, in Paper V, FCS and TRAST were employed to demonstrate that triplet states of fluorophores can provide a useful readout for Förster Resonance Energy Transfer (FRET) reflecting intra- or intermolecular distances between two fluorophores. The sensitivity of the triplet state made it possible to monitor distances larger than 10 nm, which is often stated as the upper limit of FRET interactions.
    Taken together, the studies presented in this thesis show that there is a wealth of information that can be revealed by studying long-lived transient states. Both FCS and TRAST combine a sensitive readout via the fluorescence signal with the sensitivity of the long-lived transient states monitored via the fluorescence changes. It can therefore be predicted that these approaches will find additional applications in the future.