Multicolor cathodoluminescence imaging of single lanthanide nanoparticles

Abstract

Cathodoluminescence (CL) microscopy offers a promising approach to nanoscale analysis, enabling detection of optical emission from a sample while leveraging the high resolution of electron microscopy. However, achieving multicolor single-particle CL imaging remains a significant challenge. Here, using lanthanide nanoparticles as a model system, we identify a critical limitation in CL imaging: nonlocal signal caused by stray electrons. We mitigate these nonlocal excitations and demonstrate multicolor single-particle CL imaging of nanoparticles down to 12 nm in diameter. Using this enhanced sensitivity, we demonstrate that CL brightness increases monotonically with nanoparticle diameter. We propose that multicolor imaging of spectrally distinct nanoparticles in the same field of view, coupled with the scaling of CL brightness with nanoparticle size, is crucial for confirming single-particle CL detection. Finally, we demonstrate the utility of our findings by imaging lanthanide nanoparticles in a biological sample. This work advances our understanding of nanoscale photonic responses to free electrons, establishing CL as a useful contrast mechanism for high-resolution, multicolor electron microscopy.

Fig. 1. Challenges in achieving single-particle CL imaging at the nanoscale.

a Secondary electron (SE) and CL signal collected from single LNPs in samples containing either NaHoF₄ or NaDyF₄ LNPs. Ensemble CL spectra of the LNPs and transmission bands of optical filters (shaded bands) used in CL imaging are also shown. b Observed brightness of NaHoF₄ LNPs as a function of their size. c Simulated dependence of CL signal on NaHoF₄ LNP size at 3 keV electron beam energy. Energy absorbed by the nanoparticles was used as a proxy for CL brightness. The interaction volume of the electron beam inside nanoparticles of different diameters (ø), 15, 20, and 30 nm, is also shown. Colormap shows the normalized electron density. d SE and CL signals collected from a single LNP in a sample containing both NaHoF₄ and NaDyF₄ LNPs. e Illustration showing the origin of nonlocal CL signal due to stray electrons originating from the excited LNP. Scale bars: a, c, d 20 nm. Pixel intensity scaling: a 300–1400 photons s⁻¹, d 300–800 photons s⁻¹. Source data are provided as a Source Data file.

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.

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.

Fig. 4. Characterization of single-particle CL emission.

a CL photon detection rate as a function of diameter (full width at half maximum (FWHM) from SE images) for NaHoF₄, NaDyF₄, NaSmF₄, and NaTbF₄ LNPs across the spectral filters matched to their primary emission peaks. Linear fits to the CL signal as a function of diameter are also shown. CL signal from non-emitting control NaGdF₄ LNPs is shown as black dots. b SNR of CL images for NaHoF₄, NaDyF₄, NaTbF₄, and NaSmF₄ LNPs when imaged through their respective spectral filters. The number of single LNPs imaged was 52, 46, 51, and 27, respectively. SNR from control (NaGdF₄) LNPs is shown as gray dots. 56, 36, 31, and 11 NaGdF₄ LNPs were imaged in Ho³⁺, Dy³⁺, Tb³⁺, and Sm³⁺ filters respectively. All LNPs were 18–23 nm in diameter. P-value was <0.001 (***) for all four LNP types in their respective filters. c Representative CL images with different SNRs. d Comparison of the nanocrystal size calculated using TEM (diameter of the circle fitted to nanocrystals) and SE (FWHM of the nanocrystals) images. A TEM image of nanocrystals is also shown. e Comparison of the CL detection rate from NaHoF₄ LNPs shown in (d) and control (NaGdF₄) LNPs. Representative SE and CL images of a NaHoF₄ LNP are also shown. f CL spectra of single LNPs (SE images on the right) and the corresponding ensemble spectra (gray). Scale bars: c 20 nm, d 10 nm, e 20 nm, f 50 nm. In (a) linear fits are of the form $r=mx+b$, where $r$ is CL brightness in photons s⁻¹, $x$ is the diameter of LNPs in nm, $m$ is the slope in photons s⁻¹ nm⁻¹ and $b$ is the y-intercept in photons s⁻¹. The fits are: Ho³⁺ filter: $r=187x-2607$ (from 165 LNPs), Dy³⁺ filter: $r=117x-1729$ (from 129 LNPs), Sm³⁺ filter: $r=51x-770$ (from 28 LNPs), and Tb³⁺ filter: $r=47x-543$ (from 83 LNPs). Error bars in b show mean and standard deviation. Source data are provided as a Source Data file.

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.