Ultrabright and Stable Luminescent Labels for Correlative Cathodoluminescence Electron Microscopy Bioimaging.

The mechanistic understanding of structure-function relationships in biological systems heavily relies on imaging. While fluorescence microscopy allows the study of specific proteins following their labeling with fluorophores, electron microscopy enables holistic ultrastructural analysis based on differences in electron density. To identify specific proteins in electron microscopy, immunogold labeling has become the method of choice. However, the distinction of immunogold-based protein labels from naturally occurring electron dense granules and the identification of several different proteins in the same sample remain challenging. Correlative cathodoluminescence electron microscopy (CCLEM) bioimaging has recently been suggested to provide an attractive alternative based on labels emitting characteristic light. While luminescence excitation by an electron beam enables subdiffraction imaging, structural damage to the sample by high-energy electrons has been identified as a potential obstacle. Here, we investigate the feasibility of various commonly used luminescent labels for CCLEM bioimaging. We demonstrate that organic fluorophores and semiconductor quantum dots suffer from a considerable loss of emission intensity, even when using moderate beam voltages (2 kV) and currents (0.4 nA). Rare-earth element-doped nanocrystals, in particular YO:Tb and YVO:Bi,Eu nanoparticles with green and orange-red emission, respectively, feature remarkably high brightness and stability in the CCLEM bioimaging setting. We further illustrate how these nanocrystals can be readily differentiated from morphologically similar naturally occurring dense granules based on optical emission, making them attractive nanoparticle core materials for molecular labeling and (multi)color CCLEM.