Cathodoluminescent Probes for Multicolor Electron Microscopy

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 (EM). However, achieving multicolor single-particle CL imaging remains a significant challenge. Here, we establish lanthanide nanoparticles (LNPs) as a model system for multicolor CL imaging. We identify the critical limitation that precluded multicolor CL imaging—nonlocal signal caused by stray electrons—and mitigate these nonlocal excitations to demonstrate multicolor single-particle CL imaging. To be viable for multicolor CL imaging applications, LNPs must be available in multiple emission colors. Therefore, having achieved single-particle CL imaging, we use this method to study the photophysical properties of LNPs and expand their multiplexing capability. We determine the dependence of LNP brightness on lanthanide ion concentration, develop a method to measure CL excited state lifetimes of LNPs, and study energy transfer between lanthanide ions. Next, we combine multiple lanthanide elements to engineer unique LNP colors and use them for seven-color CL imaging. Applying CL probes as bioimaging labels would enable simultaneous visualization of cellular structures (via EM contrast) and specific biomolecules (via CL contrast) at the nanoscale resolution of EM. However, achieving this is challenging because LNP synthesis yields hydrophobic nanoparticles, limiting their utility as bioimaging labels. To address this challenge, we functionalize LNPs with DNA to produce hydrophilic LNPs. We show that their single-particle CL emission is retained after DNA functionalization and after common EM sample preparation steps, and demonstrate nanoscale, multicolor CL imaging of DNA-functionalized LNPs in a biological sample. Finally, we explore the viability of small-molecule fluorescent dyes as CL labels. We show that these dyes can be excited by an electron beam and emit CL signal. We demonstrate three-color CL imaging using dye-loaded polymer beads, and two-color CL imaging of mammalian cells with dye-labeled organelles, illustrating the potential of small-molecule fluorescent dyes for CL bioimaging. Together, this work establishes CL as a useful contrast mechanism for high-resolution, multicolor electron microscopy and represents a significant step toward the application of cathodoluminescent probes for simultaneous imaging of cellular structures and biomolecules.