Cathodoluminescent and Characteristic X-ray-emissive Rare-Earth-doped Core/Shell Immunolabels for Spectromicroscopic Analysis of Cell Surface Receptors

Understanding the localization and the interactions of biomolecules at the nanoscale and in the cellular context remains challenging. Electron microscopy (EM) as a non-Abbe limited technique gives access to the cellular ultra-structure yet results in grey-scale images and averts unambiguous (co-)localization of biomolecules. Multimodal nanoparticle-based immunolabels for correlative cathodoluminescence electron microscopy (CCLEM) and energy-dispersive X-ray spectromicroscopy (EDX-SM) are presented. The single-particle STEM-cathodoluminescence (CL) and characteristic X-ray emissivity of sub-20 nm lanthanide-doped nanoparticles were exploited as unique spectral fingerprints for precise localization and label identification. To maximize the nanoparticle brightness, lanthanides were incorporated in a low-phonon host lattice and separated from the environment using a passivating shell. The core/shell nanoparticles were then functionalized with either folic (terbium-doped) or caffeic acid (europium-doped). Their potential for immunolabeling was successfully demonstrated using HeLa cells expressing different surface receptors that bind to folic or caffeic acid, respectively. Both particle populations showed single-particle CL emission along with a distinctive energy-dispersive X-ray signal, with the latter enabling colour-based localization of receptors within swift imaging times well below 2 mins per µm2 while offering high resolution with a pixel size of 2.78 nm. Taken together, these results open a route to color immunolabelling based on electron spectromicroscopy. ![Figure][1] Small (sub-20 nm) lanthanide-doped nanoparticles were successfully utilized in electron microscopy to label biological structures and contextualize them in the cell’s ultrastructure. Leveraging unique energy-dispersive X-ray signatures, the nanoparticles’ location and doping-identity was easily and fast retrieved, demonstrating the methods’ potential to (co)-localize labels while supplying a holistic impression of the underlying processes, as entire cells could be mapped. Table of Contents ### Competing Interest Statement The authors declare the following competing financial interest(s): Mathieu Kociak patented and licensed technologies at the basis of the Attolight MÖnch used in this study and is a part time consultant at Attolight. As stated in the affiliation, Christian Monachon works for Attolight. All other authors declare no competing financial interests. [1]: pending:yes