Bright sub-20-nm cathodoluminescent nanoprobes for electron microscopy

Abstract

Electron microscopy (EM) has been instrumental in our understanding of complex biological systems. Although EM reveals cellular morphology with nanoscale resolution, it does not provide information on the location of different types of proteins. An EM-based bioimaging technology capable of localizing individual proteins and resolving protein-protein interactions with respect to cellular ultrastructure would provide important insights into the molecular biology of a cell. Here, we synthesized small lanthanide-doped nanoparticles, and measured the absolute photon emission rate of individual nanoparticles resulting from a given electron excitation flux (cathodoluminescence). Our results suggest that the optimization of nanoparticle composition, synthesis protocols and electron imaging conditions can lead to sub-20-nm nano-labels that would enable high signal-to-noise localization of individual biomolecules within a cellular context. In ensemble measurements, these labels exhibit narrow spectra of nine distinct colors, so that the imaging of biomolecules in a multicolor EM modality may be possible.

Correlative Cathodoluminescence Electron Microscopy: Immunolabeling Using Rare‐Earth Element Doped Nanoparticles

“…While the SE and CL images confirm the emission from the overall majority of particles, emission intensities vary for individual particles, in line with previous studies. [ 25,35 ] Slight discrepancies between SE and CL may also be attributed to charging [ 25 ] and carbon deposition [ 18 ] during the imaging process. Additionally, a weak background signal stemming from autofluorescence [ 25 ] was observed.…”

“…[ 25,35 ] Slight discrepancies between SE and CL may also be attributed to charging [ 25 ] and carbon deposition [ 18 ] during the imaging process. Additionally, a weak background signal stemming from autofluorescence [ 25 ] was observed. Again, nanoparticle clusters (containing between 2 and 6 nanoparticles) were observed, indicative of the well‐known receptor clustering and in line with the immunogold findings (Figure 3b).…”

“…[ 22a ] Alternatively, rare‐earth element (REE)‐based nanocrystals have been proposed as prospective protein labels due to their bright emission and high electron beam stability. [ 18,25 ] Small nanocrystals can be doped with different rare‐earth elements to exhibit bright and stable CL emission. [ 18,25 ] Prigozhin et al.…”

“…[ 18,25 ] Small nanocrystals can be doped with different rare‐earth elements to exhibit bright and stable CL emission. [ 18,25 ] Prigozhin et al. [ 25 ] most recently demonstrated sub‐20 nm REE‐based nanocrystals with an impressive nine different emission colors, however, their emission has only been demonstrated on silicon wafers instead of biological samples.…”

“…[ 18,25 ] Prigozhin et al. [ 25 ] most recently demonstrated sub‐20 nm REE‐based nanocrystals with an impressive nine different emission colors, however, their emission has only been demonstrated on silicon wafers instead of biological samples. Appropriate phase transfer and surface functionalization has yet to be established before these labels can be applied for the labeling of molecules in biological samples, provided that the promising CL properties can be preserved.…”