Single-Molecule Fluorescence Microscopy in Sensory Cilia of Living Caenorhabditis elegans

Intracellular transport of organelles and biomolecules is vital for several cellular processes. Single-molecule fluorescence microscopy can illuminate molecular aspects of the dynamics of individual biomolecules that remain unresolved in ensemble experiments. For example, studying single-molecule trajectories of moving biomolecules can reveal motility properties such as velocity, diffusivity, location and duration of pauses, etc. We use single-molecule imaging to study the dynamics of microtubule-based motor proteins and their cargo in the primary cilia of living C. elegans. To this end, we employ standard fluorescent proteins, an epi-illuminated, widefield fluorescence microscope, and primarily open-source software. This chapter describes the setup we use, the preparation of samples, a protocol for single-molecule imaging in primary cilia of C. elegans, and data analysis.