PARENT SESSION
Posters P8B Supermolecular organization of the photosynthetic apparatus. Abstracts (592-611)

Fluorescence quenching and exciton-exciton annihilation effects in individual chloroplasts investigated by fluorescence lifetime imaging microscopy. Richard Cisek^*,1, Jeff Squier², Juerg Aus der Au², Virginijus Barzda¹, ¹ Department of Physics, Ontario, Canada² Department of Physics, Colorado, USA

ABSTRACT- Chloroplast fluorescence quenching heterogeneity was investigated by time-resolved laser scanning fluorescence microscopy. Measurements were taken in vivo by employing multi-photon excitation, and fluorescence was detected using time correlated single photon counting. Three exponential fits to fluorescence decays were used to analyze fluorescence lifetime images. The shortest fluorescence lifetime was observed to be less than 100ps, which is characteristic to the effect of exciton-exciton annihilation previously observed in fluorescence lifetime imaging of aggregates of photosystem II light-harvesting antenna (V. Barzda et. al. (2001) Biophysical Journal, V.80, p.2409-2421). The second exponential decay with lifetimes between 350 and 750ps was attributed to quenched fluorescence in the light-harvesting antenna. The longest decay component fell into the range between 2000 and 3500ps, which is characteristic to unquenched light-harvesting antenna. Lifetime heterogeneity between chloroplasts in a single cell and inside chloroplasts was observed. Regions with high fluorescence yield, long fluorescence lifetime, as well as small amounts of annihilation and quenching were observed. These regions can most probably be ascribed to thylakoids with loosely associated light-harvesting antenna. We also observed high fluorescing regions with high annihilation, high amplitude of fluorescence quenching, while the long fluorescence component was small. Thus, these regions possess high connectivity of light-harvesting antenna and high concentrations of quenchers in thylakoid membranes. The investigation shows that adaptation of light-harvesting to changing environmental light conditions is heterogeneous and varies at the subcellular as well as at the subchloroplast levels. For the first time to our knowledge, we employed exciton-exciton annihilation effects in multi-photon excitation microscopy. We explored this technique to map structures that contain high connectivity domains. Annihilation microscopy allows us to resolve the structural/functional units in chloroplasts in vivo, and gives the opportunity to further investigate the excited state dynamics of these individual units.

KEY WORDS: light-harvesting antenna, excitation energy transfer, non-photochemical quenching, fluorescence excitation microscopy