Single molecule imaging and spectroscopy are powerful techniques for the study of a wide range of biological processes including protein assembly and trafficking. However, single molecule imaging of biomolecules has been challenging because of difficulties associated with sample preparation and technical challenges associated with isolating single proteins within a biological system. Here we provide a detailed protocol to conduct single molecule imaging where single transmembrane proteins are isolated by rapidly extracting nanovesicles containing receptors of interest from different regions of the brain and subjecting them to single molecule study by using total internal reflection fluorescence (TIRF) microscopy. This protocol discusses the isolation and separation of brain region specific nanovesicles as well as a detailed method to perform TIRF microscopy with those nanovesicles at the single molecule level. This technique can be applied to study trafficking and stoichiometry of various transmembrane proteins from the central nervous system. This approach can be applied to a wide range of animals that are genetically modified to express a membrane protein-fluorescent protein fusion with a wide range of potential applications in many aspects of neurobiology. Graphic abstract: single molecue imaging of membrane receptors.