%0 Journal Article
%A Casillas Ruibio, Alejandro
%A Hamraoui, Khouloud
%A Méndez González, Diego
%A Laurenti, Marco
%A Rubio Retama, Benito Jorge
%A Gómez Calderón, Óscar
%A Melle Hernández, Sonia
%T Influence of excitation pulse duration on the efficiency of upconversion nanoparticle-based FRET
%D 2025
%@ 2040-3364
%U https://hdl.handle.net/20.500.14352/131487
%X Accurate and reliable quantification of Förster Resonance Energy Transfer (FRET) is essential for the development of sensitive upconverting nanoparticle (UCNP)-based biosensors. While lifetime-based FRET measurements are generally considered robust, excitation conditions can significantly bias observed efficiencies. Here, we investigate how excitation pulse width and power influence lifetime-derived FRET efficiency in core–shell β-NaYF4:Yb0.2@NaYF4:Yb0.2,Er0.02 UCNPs functionalized with Cy3 dyes. Time-resolved upconversion luminescence (UCL) measurements reveal that apparent FRET efficiencies decrease with increasing excitation pulse duration and power. These variations stem from excitation-induced changes in the UCL lifetime, arising from the complex dynamics that accompany the upconversion emission process. A dynamic rate equation model reproduces the experimental trends, confirming that excitation parameters alter emissive state kinetics and thus bias lifetime-based FRET measurements. Our findings identify excitation conditions as a hidden variable in UCNP-FRET experiments and underscore the need for standardized measurement protocols.
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