Over the last decade, single-molecule detection (SMD) gave biologists a tool to turn their dream, to follow a single molecule in live cells, into reality. SMD provides the advantages of identifying subpopulations and of localizing molecules with sub-wavelength precision. The use of nanometre-sized ligand–fluorophore complexes has even made it possible to track targets within confined cellular compartments. In this review, we first describe the main benefits of SMD in cellular imaging. We then show how SMD was used to unravel the membrane diffusion of glutamatergic receptors and how it sheds light on the way neurons can regulate membrane distribution of receptors.

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