Förster Resonance Energy Transfer (FRET) between single molecule donor (D) and acceptor (A) is well understood from fundamental perspective and is widely applied in biology, biotechnology, medical diagnostics and bio-imaging. However, the reliability of molecular FRET measurements can be affected by numerous artefacts which eventually hamper quantitative and reliable analysis, mostly due to issues with the donor and acceptor molecules. Lanthanide doped upconverting nanoparticles (UCNPs) have demonstrated their suitability as alternative donor species. Nevertheless, while they solved most disadvantageous features of organic donor molecules, such as photo-bleaching, spectral cross-excitation and emission bleed-through, the fundamental understanding and practical realizations of bio-assays with UCNP donors remain challenging. Among others, the actual donor ions in individual donor UCNPs are the numerous activator ions randomly distributed in the nanoparticle at various distances to acceptors anchored on the nanoparticle surface. Further, the power dependent, complex energy transfer upconversion and energy migration between sensitizing and activating lanthanide ions within UCNPs complicate the decay based analysis of D-A interaction. In this work, the assessment of designed virtual core-shell nanoparticle (VNP) models led us to the new designs of UCNPs, such as …@Er, Yb@Er, Yb@YbEr, which were experimentally evaluated as donor nanoparticles and compared to the simulations. Moreover, the specific properties of lanthanide-based upconversion motivated us to analyze not only steady-state luminescence and luminescence decay responses of both the UNCP donor and the sensitized acceptor, but also the effects of their luminescence rise kinetics upon RET was discussed in newly proposed disparity measurements. The presented studies help to understand the role of energy-transfer and energy migration between lanthanide ion dopants (due to their concentration and spatial distribution) and how the architecture of core-shell UCNPs affects their performance as FRET donors to organic acceptor dyes.

A.P.-W. and A.M.K. contributed equally to this work. S.L. and T.S. acknowledge Business Finland for the financial support. A.K. acknowledges financial support from the National Science Centre Poland (Grant No 2021/41/N/ST5/02753). Calculations have been carried out using resources provided by Wroclaw Centre for Networking and Supercomputing (https://wcss.pl), grant No. 529.