Light and electron microscopy provide complementary information on protein localization and cellular ultrastructure. To bridge these techniques, we introduce cathodoluminescence, optical emission triggered by electron excitation, as a new contrast mechanism in bioimaging. Cathodoluminescence microscopy enables simultaneous detection of proteins and ultrastructural features with perfect spatial registration in a single electron beam scan. To achieve this, we developed a versatile toolkit of cathodoluminescent protein tags, including multicolor lanthanide nanocrystals and small-molecule dyes. By engineering nanocrystal composition, we demonstrate multicolor imaging of spectrally distinct nanocrystals within the same field of view. We also show direct electron-beam excitation of small-molecule dyes commonly used in fluorescence imaging, and apply them to simultaneously image proteins and ultrastructural features in diverse biological systems, including mammalian cells, fungi-infected fruit fly, and bacteria. Together, these advances establish cathodoluminescence microscopy as a powerful platform for mapping proteins in their ultrastructural context.
Rehman et al. (Sun,) studied this question.