Fluorescence lifetime imaging microscopy on individual chloroplasts in intact leaves

Fluorescence lifetime imaging microscopy (FLIM) provides the possibility to perform picosecond fluorescence measurements at a microscopic level. We started to explore the possibilities to perform FLIM measurements on individual chloroplasts within a leaf, with the aim to monitor the response of the chloroplast to changes of the environmental conditions. This was done on a fluorescence lifetime imaging microscope with 2-photon excitation (840 nm, 100 fs pulses). We performed FLIM on Arabidopsis thaliana leaves where either ferricyanide or DCMU was added by vacuum infiltration to keep the reaction centers of photosystem II open or closed, respectively. The results were compared with the results of macroscopic time-correlated single photon counting measurements on chloroplasts. The kinetics show good agreement, demonstrating that FLIM can indeed be used to study individual chloroplasts in leaves. As a next step we performed singlet-singlet annihilation measurements on individual chloroplasts. The fluorescence kinetics appears to depend strongly on the laser intensity. The results can be related to domain sizes of connected pigments and thus provide information on the organization within the membrane in planta. This will provide an extra tool to study the response of chloroplasts to changes in their direct environment.