Opening a new window into the cell with super-resolution imaging and in situ cryo-electron tomography

Super-resolution light and electron microscopy have revolutionized our ability to visualize cell machinery. We use live-cell super-resolution imaging including stimulated emission depletion (STED) microscopy together with highly inclined thin illumination (HiLo) and high-speed three-dimensional widefield imaging to visualize organelle dynamics in real time. We have integrated these approaches with in situ cryo- electron tomography (cryo-ET), and cryo-correlative light and electron microscopy (cryo-CLEM) to visualize the endoplasmic reticulum (ER) and its relationships with other intracellular organelles in near-native states. The combination of these methods has revealed a novel ER-derived compartment that is mobile, vesicular, and associated with mammalian 80S cytoplasmic ribosomes. Moreover, with cryo-focused-ion-beam (FIB) milling and cryo-ET, we show that these vesicles exist as discrete structures separate from the intact reticular ER architecture. We call these organelles Ribosome-Associated Vesicles (RAVs). Detailed characterization of the RAVs revealed that these structures are conserved across multiple cell types and species using both conventional transmission electron microscopy (TEM) and cryo-ET.