Changes in spatial distribution of fluorescence lifetimes in individual aggregates of LHCII and chloroplasts revealed by two-photon excitation time-resolved fluorescence microscopy

Two-photon excitation, fluorescence lifetime imaging microscopy (FLIM) was used to investigate the heterogeneity, connectivity and fluorescence quenching mechanisms in aggregates of light-harvesting chlorophyll a/b pigment protein complexes of photosystem II from green plants (LHCII) and chloroplasts. Fluorescence decays of individual LHCII aggregates and chloroplasts were recorded with two-photon excitation microscopes equipped with time-gated detection module or with a time-correlated single photon counting. Microscopy images show the presence of large heterogeneity in fluorescence lifetimes not only for different LHCII aggregates but also within a single aggregate. Different structural/functional units were resolved and their excited states dynamics were investigated individually. We found that the lifetime distribution of a single aggregate is sensitive to the concentration of quenchers contained in the system. Singlet-triplet annihilation affected the distribution of fluorescence lifetimes. For LHCII aggregates, higher repetition rates of pulse excitation preferentially quenched domains with initially shorter fluorescence lifetimes (500-700 ps) and domains with long lifetimes (>1 ns) were not affected. This verified our previous prediction from singlet-singlet annihilation investigations that shorter fluorescence lifetimes originate from larger domains in LHCII aggregates. We found that singlet-singlet annihilation has a strong effect in time-resolved fluorescence microscopy of connective systems. High connectivity domains could be found inside individual LHCII aggregate. In this work, we showed that time-resolved fluorescence microscopy renders new information about excited state dynamics in heterogeneous photosynthetic systems. Spatially resolved fluorescence decays give us a lifetime distribution, which provides the information about changes in the ensemble of domains, that is not available in the macroscopic time-resolved measurements.