Super-Resolution Imaging of the Actin Cytoskeleton in Living Cells Using TIRF-SIM

Super-resolution (SR) imaging techniques have advanced rapidly in recent years, but only a subset of these techniques is gentle enough to be used by cell biologists to study living cells with minimal photodamage. Our research is focused on studies of the dynamic remodeling of the actin cytoskeleton in living pancreatic beta cells during insulin secretion. These studies require super-resolution light microscopic techniques that are gentle enough to record rapid changes of the actin cytoskeleton in real time. In this chapter, we describe an SR technique that breaks the diffraction limit of the conventional light microscope called TIRF-SIM. Using this SR techniques, we have been able to show that (1) microvilli on pancreatic beta cells translocate in the plane of the plasma membrane and (2) the cortical actin network reorganizes when cells are stimulated to secrete insulin. We describe the FIJI plugins that were used to process and analyze the TIRF-SIM images to obtain quantitative data.