Fig. 3: Multicolor single-particle CL imaging of spectrally distinct LNPs at the nanoscale.

a Excitation crosstalk between two adjacent NaHoF₄ LNPs as analyzed by Monte Carlo simulations. Nanoparticle 1 (NP1) was excited with a 3 keV electron beam and the percentage of energy absorbed by NP2 (normalized to that of NP1) was calculated at different distances, δ, from NP1. 5000 trajectories were simulated at each distance, δ. Inset: Example of a simulation. b SE image of two LNPs located in direct proximity. c Composite CL image of the nanocrystals in (b), obtained by merging signals in d Ho³⁺ color channel and e Dy³⁺ color channel. f Cross-sectional profiles of the CL image along the dotted line shown in (c). g SE image of a field of view containing NaHoF₄ and NaDyF₄ LNPs. h Dual-color CL image of the field of view in (g), obtained by merging i Dy³⁺ color channel, and j Ho³⁺ color channel. Each LNP was detected in one of the two color channels, even when located close to nanocrystals of a different color; see arrows. k SE image of a field of view containing NaHoF₄, NaDyF₄, and NaTbF₄ LNPs. l Composite CL image of the field of view in (k) obtained by merging CL signals from Ho³⁺, Dy³⁺, and Tb³⁺ color channels. m, n Zoomed-in SE (left) and CL (right) images from regions indicated in (k, l), and CL cross-sectional profiles along the dotted lines in CL images. Scale bars: a 50 nm, b–e 20 nm, g–l 100 nm, m, n 20 nm. Pixel intensity scaling: c–e Dy³⁺ filter 400–700, Ho³⁺ filter: 300–1100 photons s⁻¹; h–j Dy³⁺ filter: 340–1000, Ho³⁺ filter: 700–4050 photons s⁻¹; l Tb³⁺ filter: 300–1100, Dy³⁺ filter: 440–1800, Ho³⁺ filter: 240–2400 photons s⁻¹; m Tb³⁺ filter: 300–600, Dy³⁺ filter: 520–1100, Ho³⁺ filter: 1200–2600 photons s⁻¹; n Tb³⁺ filter: 300–1000, Dy³⁺ filter: 520–1560, Ho³⁺ filter: 300–1600 photons s⁻¹. Source data are provided as a Source Data file.