Molecular probes for multicolor electron microscopy

Light and electron microscopy provide complementary information on protein localization and nanoscale cellular features. To bridge these techniques, we introduce a new contrast mechanism in bioimaging–cathodoluminescence, which is the emission of light from a material under electron beam excitation. Cathodoluminescence enables simultaneous visualization of proteins and cellular features with perfect spatial overlap in a single electron beam scan. We have developed a versatile library of molecular probes for cathodoluminescence imaging, comprising sub-20 nm multicolor lanthanide nanoparticles and small-molecule dyes commonly used in optical microscopy. By precisely engineering lanthanide nanoparticles, we achieve multicolor imaging of eight distinctly colored particles in a single field of view. In addition, we demonstrate the direct excitation of spectrally distinct small-molecule dyes by an electron beam and their color-based distinction for multicolor CL imaging. This approach allows the visualization of single proteins and cellular organelles in electron microscopy, with applications in connectomics, cellular signaling, and cryo-electron microscopy.