Size-Dependent Fluorescence Kinetics Reveal Contributions of Intrinsic Quenching and Singlet-Triplet Annihilation during LHCII Aggregation

TL;DR

This study quantitatively separates intrinsic quenching from singlet-triplet annihilation in LHCII aggregation, revealing how these processes influence fluorescence decay and intensity as aggregate size varies, using combined FCS and TCSPC techniques.

Contribution

It introduces a size-dependent framework that distinguishes intrinsic quenching from annihilation effects in LHCII aggregation, advancing understanding of photophysical mechanisms.

Findings

Singlet-triplet annihilation dominates fluorescence quenching at moderate excitation intensities.

Fluorescence lifetime decreases semi-logarithmically with aggregate size.

Intrinsic quenching increases gradually with aggregate size.

Abstract

Aggregation of the main antenna complex of higher plants, Light-Harvesting Complex II (LHCII), is widely used as an in vitro model for energy-dependent quenching (qE), yet fluorescence reduction in aggregates is frequently interpreted without a quantitative separation of intrinsic quenching from excitation-induced annihilation. Here, we address this ambiguity by directly correlating aggregate size, concentration, steady-state fluorescence intensity, and decay kinetics during controlled, incremental aggregation of isolated LHCII. By combining fluorescence correlation spectroscopy (FCS) with TCSPC in a unified experimental framework, we monitored structural and photophysical changes in real time as detergent removal drives biphasic aggregation. We quantified the aggregate composition from the particle concentrations, enabling direct scaling of the absorption cross-section with aggregate size. The average fluorescence lifetime decreased semi-logarithmically with increases in hydrodynamic radius, whereas steady-state fluorescence intensities deviated strongly from this trend. Intensitydependent measurements and steady-state kinetic modeling reveal that singlet-triplet annihilation (STA) emerges at moderate excitation intensities and rapidly becomes the dominant contributor to fluorescence quenching, even for relatively small aggregates. In contrast, intrinsic quenching increases more gradually with aggregate size. By quantitatively disentangling intrinsic excitation quenching from annihilation processes, this work demonstrates that STA can govern the apparent photophysical response of aggregated LHCII across excitation regimes commonly considered non-annihilating. The size-dependent mechanistic framework presented here provides a basis for distinguishing intrinsic quenching from annihilation effects in aggregation-based studies of photosynthetic antenna complexes.