Abstract
The primary cilium is an important signaling organelle critical for normal development and tissue homeostasis. Its small dimensions and complexity necessitate advanced imaging approaches to uncover the molecular mechanisms behind its function. Here, we outline how single-molecule fluorescence microscopy can be used for tracking molecular dynamics and interactions and for super-resolution imaging of nanoscale structures in the primary cilium. Specifically, we describe in detail how to capture and quantify the 2D dynamics of individual transmembrane proteins PTCH1 and SMO and how to map the 3D nanoscale distributions of the inversin compartment proteins INVS, ANKS6, and NPHP3. This protocol can, with minor modifications, be adapted for studies of other proteins and cell lines to further elucidate the structure and function of the primary cilium at the molecular level.