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In light harvesting complex II (LHCII) of higher plants and green algae, carotenoids (Cars) have an important function to quench chlorophyll (Chl) triplet states and therefore avoid the production of harmful singlet oxygen. The resulting Car triplet states lead to a non-linear self-quenching mechanism called singlet-triplet (S-T) annihilation that strongly depends on the excitation density. In this work we investigated the fluorescence decay kinetics of single immobilized LHCIIs at room temperature and found a two-exponential decay with a slow (3.5 ns) and a fast (35 ps) component. The relative amplitude fraction of the fast component increases with increasing excitation intensity, and the resulting decrease in the fluorescence quantum yield suggests annihilation effects. Modulation of the excitation pattern by means of an acousto-optic modulator (AOM) furthermore allowed us to resolve the time-dependent accumulation and decay rate (∼7 μs) of the quenching species. Inspired by singlet-singlet (S-S) annihilation studies, we developed a stochastic model and then successfully applied it to describe and explain all the experimentally observed steady-state and time-dependent kinetics. That allowed us to distinctively identify the quenching mechanism as S-T annihilation. Quantitative fitting resulted in a conclusive set of parameters validating our interpretation of the experimental results. The obtained stochastic model can be generalized to describe S-T annihilation in small molecular aggregates where the equilibration time of excitations is much faster than the annihilation-free singlet excited state lifetime.

Singlet–triplet annihilation in single LHCII complexes