Casillas Ruibio, AlejandroHamraoui, KhouloudMéndez González, DiegoLaurenti, MarcoRubio Retama, Benito JorgeGómez Calderón, ÓscarMelle Hernández, Sonia2026-02-042026-02-042025Casillas-Rubio, Alejandro, et al. «Influence of Excitation Pulse Duration on the Efficiency of Upconversion Nanoparticle-Based FRET». Nanoscale, vol. 17, n.o 31, 2025, pp. 18173-84. DOI.org (Crossref), https://doi.org/10.1039/D5NR01779C.2040-336410.1039/D5NR01779Chttps://hdl.handle.net/20.500.14352/1314872024 Acuerdos transformativos CRUE. This work was supported by Ministerio de Ciencia e Innovación (PID2023-150645OA-I00, PID2021-122806OB-I00, PID2021-123318OB-I00 and TED2021-132317B-I00), Fundación Alonso (2024/0041), and Comunidad de Madrid (P2022/ BMD-7403 RENIM-CM). A. C. R. thanks UCM-Santander for a predoctoral contract (CT15/23). Datos de investigación en: https://hdl.handle.net/20.500.14352/120840Accurate 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.engAttribution 4.0 Internationalhttp://creativecommons.org/licenses/by/4.0/journal article2040-3372https://doi.org/10.1039/D5NR01779Chttps://hdl.handle.net/20.500.14352/120840open accessÓpticaNanopartículasÓptica (Física)2209 Óptica