Single molecule measurements of membrane receptor assembly, trafficking, and function are complicated by the inherent properties of the native cellular environment. We utilize a combination of nanoscale plasmonic devices and cell-derived vesicles to isolate single membrane receptors in their native lipid bilayer membrane. This provides a method to extend single molecule studies to proteins that cannot be purified from the cell. This approach also allows us to compare populations of the same protein residing in different organelles such as the endoplasmic reticulum and the plasma membrane.

We applied these methods to determine how nicotinic receptor ligands alter the assembly of neuronal nicotinic acetylcholine receptors. Exposure to nicotine led to increased fractions of the high sensitivity stoichiometry of α4β2 during synthesis in the endoplasmic reticulum showing that nicotine alters the assembly of nascent receptor subunits. The use of nanoscale plasmonic devices and cell derived nanovesicles serves to defeat the concentration barrier associated with single molecule imaging extending these methods to physiological environments.