Determining the geometry of oligomers of the human epidermal growth factor family on cells with 7Â nm resolution

Dimerisation, oligomerisation, and clustering of receptor molecules are important for control of the signalling process. There has been a lack of suitable methods for the study and quantification of these processes in cells. Here we describe a protocol for a method that we have named “fluorophore localisation imaging with photobleaching” (FLImP), which uses single molecule localisation and single-step photobleaching to determine the separation of two fluorophores with a resolution of 7 nm or better. We describe the procedures required for the collection of FLImP data, and point out some of the pitfalls that must be avoided for the collection of high resolution data. We also present recent data obtained using FLImP, showing that the intracellular domain of the Epidermal Growth Factor Receptor is not required in the basal state for the receptor to form ordered inactive oligomers in the plasma membrane.