Fig. 5: Multicolor single-particle CL imaging of LNPs in a biological sample.

a Auto-CL photon detection rates from cells with and without OsO₄ treatment in Ho³⁺ and Dy³⁺ channels. Background CL from the Si substrate is also shown for comparison (dashed line). Five cells were imaged for each condition, and CL brightness was calculated from the pixels within the boundaries of all five cells. Boundaries were manually selected based on CL brightness of cells compared to background CL from Si (see top row). b An illustration showing the sample preparation procedure for imaging LNPs on cells. HEK293T cells were fixed, treated with OsO₄, and dried with hexamethyldisilazane (HMDS) (not shown in the illustration). LNPs were then drop-cast onto the cells. The biological sample with LNPs was sputter coated with 80:20 Pt:Pd mixture for SEM imaging. c CL detection rate in the Ho³⁺ channel from single NaHoF₄ LNPs on Si (with and without sputter coating) and on cells prepared for SEM imaging. Samples were prepared according to (b). The LNPs were 19.4 ± 0.6 nm in diameter, as measured in our CL-SEM. At least 20 LNPs were imaged for each condition. d SE image of a cell prepared according to (b) with NaHoF₄ and NaDyF₄ LNPs on the surface. e, f Zoomed-in SE (e) and CL (f) images of the region marked with a yellow rectangle in (d). g Merged image of the SE and CL channels. Pixel intensity in (g) is scaled differently from (f) to show only LNP signal above the background of cellular features. Scale bars: a 10 µm, d 1 µm, e–g 100 nm. Pixel intensity scaling: f Dy³⁺ filter 300–2000, Ho³⁺ filter: 300–6000 photons s⁻¹, and g Dy³⁺ filter 600–4000, Ho³⁺ filter: 1200–7000 photons s⁻¹. Error bars in a, c show the mean and standard deviation. Source data are provided as a Source Data file.