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 µm 2 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.

Table of Contents

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.