[论文解读] Size-Dependent Fluorescence Kinetics Reveal Contributions of Intrinsic Quenching and Singlet-Triplet Annihilation during LHCII Aggregation

Francois Conradie, Bertus van Heerden+2 more|arXiv (Cornell University)|Mar 12, 2026

Photosynthetic Processes and Mechanisms被引用 0

一句话总结

本论文利用同时的荧光相关光谱 (FCS) 与时间相关单光子计数 (TCSPC)，将 LHCII 聚集体大小与荧光强度及衰减动力学相关联，以在去表面活性剂驱动的聚集过程中区分本征猝灭与单-三重态湮灭 (STA)。

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.

研究动机与目标

• Quantify how aggregation size influences fluorescence yield and lifetimes in LHCII.
• Disentangle intrinsic excitation quenching from annihilation processes during aggregation.
• Link aggregate structure (size, concentration) to photophysical pathways in a unified framework.

提出的方法

• Combine fluorescence correlation spectroscopy (FCS) with time-correlated single-photon counting (TCSPC) to monitor intensity, lifetime, particle number, and hydrodynamic radius during detergent-driven aggregation.
• Quantify aggregate composition from particle concentrations and scale absorption cross-section with aggregate size.
• Use biexponential fits to resolve lifetime components and model quenching with competing rates (radiative, internal conversion, intersystem crossing, and quenching).
• Perform intensity-dependent TCSPC measurements to distinguish aggregation effects from excitation-intensity effects.
• Correlate FCS-derived N and diffusion times to compute M, the average number of trimers per aggregate, and analyze fractal growth dynamics.

实验结果

研究问题

• RQ1 How does LHCII aggregate size affect fluorescence intensity and lifetime?
• RQ2 Can intrinsic quenching and singlet–triplet annihilation (STA) be quantitatively disentangled during aggregation?
• RQ3 What is the relationship between aggregate size, absorption cross-section, and quenching efficiency?
• RQ4 How does excitation intensity influence the relative contributions of aggregation-induced quenching versus STA?
• RQ5 What are the structural growth dynamics and fractal characteristics of LHCII aggregation under controlled detergent removal?

主要发现

• STA emerges at moderate excitation and rapidly becomes the dominant quenching pathway, even for relatively small aggregates.
• Intrinsic quenching increases more gradually with aggregate size, separate from STA.
• Two fluorescence lifetime components are observed (approximately 0.35 ns and 3.6 ns) with amplitudes indicating aggregation-related quenching and unquenched states, respectively.
• The average lifetime decreases semi-logarithmically with increasing hydrodynamic radius, while steady-state intensity declines nonlinearly due to changing effective fluorescence yield.
• Aggregation proceeds in biphasic kinetics with a transition around M ≈ 10–20; fractal analysis yields d_f = 1.6 ± 0.1, consistent with diffusion-limited cluster aggregation patterns.
• A method to partition the effective absorption cross-section among aggregates shows that the ensemble-averaged cross-section scales with the number of trimers per aggregate, M.