Deciphering cellular function requires visualizing both molecular localization and membrane structure at nanometer resolution. However, current imaging methods force a tradeoff between biomolecule localization and membrane ultrastructure because of the contrast mechanisms involved in light microscopy (LM) and electron microscopy (EM). Here, we introduce a multicolor EM approach that integrates these two types of information. In this approach, the electron beam directly excites small-molecule cathodophores (biomolecule tags) and causes luminescence—a process termed cathodoluminescence (CL)—while simultaneously imaging cellular ultrastructure using heavy metal stains or intrinsic contrast in cryo-EM. By using polystyrene beads and mammalian cells as prototypical systems, we demonstrate CL from a wide range of small-molecule dyes functioning as cathodophores. Furthermore, we extend this method to biological tissues. We performed simultaneous topographic EM and multicolor EM imaging on cells and fungus-infected flies stained with these cathodophores. This work opens a new dimension in biomolecular imaging, enabling nanoscale mapping of molecular localization within cellular and tissue ultrastructure.
Roy et al. (Sun,) studied this question.