Methods useful for exploring the formation and functions of primary cilia in living cells are described here. First, multiple protocols for visualizing solitary cilia that extend away from the cell body are described. Primary cilia collect, synthesize, and transmit information about the extracellular space into the cell body to promote critical cellular responses. Problems with cilia formation or function can lead to dramatic changes in cell physiology. These methods can be used to assess cilia formation and length, the location of the cilium relative to other cellular structures, and localization of specific proteins to the cilium. The subsequent protocols describe how to quantify movement of fluorescent molecules within the cilium using kymographs, photobleaching, and photoconversion. The microtubules that form the structural scaffold of the cilium are also critical avenues for kinesin and dynein-mediated movement of proteins within the cilium. Assessing intraflagellar dynamics can provide insight into mechanisms of ciliary-mediated signal perception and transmission.

Fundamental to the success of cell and developmental biology is the ability to tease apart molecular organization in cells and tissues by localizing specific proteins with respect to one another in a native cellular context. However, many key cellular structures (from mitochondrial cristae to nuclear pores) lie below the diffraction limit of visible light, precluding analysis of their organization by conventional approaches. Point-localization superresolution microscopy techniques, such as PALM and STORM, are poised to resolve, with unprecedented clarity, the organizational principles of macromolecular complexes within cells, thus leading to deeper insights into cellular function in both health and disease.