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During the last decades, fluorescence spectroscopy has emerged as a powerful tool in the fields of biophysics, biotechnology, biochemistry, cellular biology and the medical sciences. These techniques are highly sensitive, and allow us to study the structure and dynamics of (bio)molecular systems (Valeur 2001). A significant advantage of fluorescence techniques is that they can often be non-invasive and measurements can be performed in real time. In this thesis different advanced fluorescence methods will be used to study two important biological processes: (1) DNA dynamics and (2) plant photosynthesis. The first part aims at improving the smFRET technique for the analysis of DNA dynamics and other fast conformational changes. This improvement is made by combining and developing instrumentation and data evaluation tools. The second part is the continuous development of time-resolved fluorescence spectroscopy methods, as well their application in the field of photosynthesis to study ultrafast processes in thylakoid membranes and leaves. The two fluorescence techniques are technically and conceptually very different, but they are both designed for analysis of biomolecular systems. In this thesis, the techniques are applied to study energy transfer and dynamical changes in DNAs, thylakoid membranes and leaves. REFERENCE: VALEUR B 2001. Molecular Fluorescence: Principles and Applications. 1 ed: Wiley-VCH.

Chlorophyll fluorescence research began in the early 1930s with Kautsky (Kautsky and Hirsch, 1931) who was trained as a physical chemist and stumbled upon the phenomenon by accident. The main paradigm in those days was that the chlorophyll molecules alone bound CO2, absorbed photon energy and transferred that energy to the carbon atom, releasing oxygen to produce carbohydrates. When Kautsky presented his work in 1931 and stated that there was hardly any change in chlorophyll fluorescence yield when going from a CO2-containing atmosphere to a CO2-free atmosphere, it was believed that his equipment had failed. It was thought impossible, if he really was measuring chlorophyll fluorescence, that it would be unperturbed by the presence or absence of CO2. Although he was not a plant physiologist, Kautsky suggested that the light-dependent reactions and carbon fixation could be spatially separated processes in photosynthesis. It was not until the 1950s with the work of Rabinowitch (1951), Rabinowitch (1956) and Duysens (Duysens et al., 1961; Duysens and Sweers, 1963) that the idea of separate light and dark reactions was accepted.