Sonic hedgehog is a fascinating protein with great responsibility over the formation and upkeep of our bodies. It is widely studied, not least because dysregulation of the Shh signalling pathway leads to repercussions on human health, such as contraction of cancer. Gaining an understanding of its signalling mechanism is central to inventing preventative measures and treatments against this disease. This thesis focuses on the study of the spatial organisation of Shh multimolecular signalling complexes on the surface of producing cells, and those dispatched in the vicinity of those cells, using high-resolution optical imaging beyond the diffraction limit. With un-precedented resolution, the differences in organisation of Shh pre- and post-release from the surface were characterised, and the influence of the lipid modifications of Shh, namely choles-terol and palmitate, investigated. The main findings were that both lipid adducts are necessary for large-scale multimerisation, but not for the formation of small, sub-diffraction limit oligomers. Together with data I collected about the profile of the clusters’ size distributions, I find that electrostatic interactions between the molecules may be the engine driving the multimerisation process. Furthermore, the role of lipid modifications may, at least in part, be to retain Shh on the surface while multimerisation proceeding according to the law of mass action builds upon the small oligomer nucleation sites prepared presumably by the electrostatic interactions in the first place. Other, more indirect lines of evidence again based on the profile of the multimer size distribution insinuated that Shh complexes may not undergo any proteolytic modifications prior to release – contrary to some reports in the literature. The results presented in this thesis are the fruits of a completely fresh and innovative approach to examining Shh, which for the first time delivers concrete dimensional details about the elusive structure of the Shh multimer.