Cathodoluminescent and Characteristic X‐Ray‐Emissive Rare‐Earth‐Doped Core/Shell Protein Labels for Spectromicroscopic Analysis of Cell Surface Receptors

Abstract Understanding the localization and the interactions of biomolecules at the nanoscale and in the cellular context remains challenging. Electron microscopy (EM), unlike light‐based microscopy, gives access to the cellular ultrastructure yet results in grey‐scale images and averts unambiguous (co‐)localization of biomolecules. Multimodal nanoparticle‐based protein labels 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 are exploited as unique spectral fingerprints for precise label localization and identification. To maximize the nanoparticle brightness, lanthanides are incorporated in a low‐phonon host lattice and separated from the environment using a passivating shell. The core/shell nanoparticles are then functionalized with either folic (terbium‐doped) or caffeic acid (europium‐doped). Their potential for (protein‐)labeling is successfully demonstrated using HeLa cells expressing different surface receptors that bind to folic or caffeic acid, respectively. Both particle populations show single‐particle CL emission along with a distinctive energy‐dispersive X‐ray signal, with the latter enabling color‐based localization of receptors within swift imaging times well below 2 min per 2 while offering high resolution with a pixel size of 2.78 nm. Taken together, these results open a route to multi‐color labeling based on electron spectromicroscopy.