Fig. 2: Nonlocal excitation limits multicolor single-particle imaging.

a A schematic (not to scale) and SE image of a dense layer of NaDyF₄ LNPs and sparse NaHoF₄ LNPs on Si. b SE and CL images of NaHoF₄ LNPs at distances, D, from the NaDyF₄ layer. c CL brightness of single NaHoF₄ LNPs in Ho³⁺ and Dy³⁺ channels as a function of D. At least five LNPs 15–35 nm in diameter were imaged at each D. d Electron trajectory simulations of energy absorbed by a layer of NaDyF₄ LNPs upon excitation of a 20 nm-diameter NaHoF₄ LNP as a function of D. e–i CL brightness of single nanoparticles in the Ho³⁺ channel at distances, D, from a dense layer of NaHoF₄ LNPs. The subpanels show nanoparticle and substrate types. Three nanoparticles were imaged at each D. Diameters of sparse nanoparticles were 15–35 nm in (e, g, h, i) and 40 nm in (f). j SE and CL images of a NaGdF₄ LNP at D = 20 µm from the NaHoF₄ layer on Pt:Pd substrate. Note the shadow behind the LNP (right side). k Sample geometry for imaging NaHoF₄ LNPs on a 200-nm-thick Si₃N₄ window, with a dense layer of NaDyF₄ LNPs on the other side. Simulated electron trajectories for a beam energy of 5 keV are also shown. The top surface was coated with a 5-nm-thick layer of 80:20 Pt:Pd (not shown) to reduce charging artifacts. l SE and m CL images of NaHoF₄ LNPs for the geometry in (k). n Composite image of Ho³⁺ and Dy³⁺ channels from (m). o Cross-sectional profile of the LNP marked in (n). Scale bars: a 200 nm, b, j 20 nm, l–n 50 nm. Pixel intensity scaling across filters: b Ho³⁺: 400–3000 photons s⁻¹ (all D); Dy³⁺: 2000–11,000 (D = 20 µm); 400–4000 (D = 65 µm); 400–1000 (D = 90 µm); 100–1080 (D = 120 µm); 100–1080 photons s⁻¹ (D = 180 µm). j Ho³⁺: 70,000–110,000 photons s⁻¹. Error bars in c, e–i show mean and standard deviation. Source data are provided as a Source Data file.