Clathrin-mediated endocytosis (CME) is a highly dynamic, nanoscale process essential for cellular uptake, signaling, and homeostasis. Traditional fluorescence microscopy has revealed much about CME, but diffraction-limited resolution has restricted our ability to capture the fine details of membrane remodeling and vesicle formation in living cells. Here, we combine total internal reflection fluorescence structured illumination microscopy (TIRF-SIM) and grazing incidence sim (GI-SIM) to achieve real-time, three-dimensional (3D) visualization of CME dynamics at the plasma membrane. This dual-modality approach enables us to follow the emergence, maturation, and internalization of clathrin-coated structures with high spatiotemporal resolution while maintaining live-cell compatibility. Using this approach, we directly track how clathrin assemblies drive local changes in membrane curvature and vesicle budding, providing new insights into the nanoscale choreography of endocytosis. Beyond CME, these imaging strategies open avenues for studying a wide range of membrane-associated and cytoskeletal processes in living systems.
Thompson et al. (2026) studied this question.