Background

Single molecule imaging techniques have emerged as powerful methods to study the structure, function, and interactions of biomolecules (Joo et al., 2008; Choi et al., 2019). While ensemble measurements provide important information about the average population, single molecule methods can be used to evaluate the heterogeneity within this population revealing differences in structural and functional states (Lee et al., 2020). Single molecule methods have already been used in a diverse range of applications including measurements of protein and ligand interaction, conformational changes in proteins, and ion channel function (Xia et al., 2013; Martinac, 2017; Fu et al., 2019). These methods have provided powerful ways to characterize individual cellular constituents and single molecules in vitro.

Despite their successful implementations in vitro, single molecule methods for in vivo studies have been particularly challenging. Major challenges for in vivo studies include the heterogenous nature of cellular components, the difficulties associated with light penetration in thick tissues, and the need to isolate a single species from the complex environment within a living system (Fu et al., 2019). In addition, the concentration of protein of interest in some cases is much higher in the native environment than the optimum concentration required for single molecule imaging (Richards et al., 2012). Additionally, membrane proteins are not spatially isolated in a single location within the cellular environment and instead diffuse across a wide region, complicating single molecule studies. This makes isolation and separation of the molecule of interest a key aspect of any ex vivo single molecule imaging protocol. This protocol details an approach to address these challenges, by utilizing nanoscale vesicles generated from primary tissue as a way of isolating and immobilizing membrane proteins. We demonstrate this ex vivo single molecule imaging approach using knock-in mice containing a fluorescent protein labeled nicotinic acetylcholine receptor (nAChRs). Nicotinic acetylcholine receptors (nAChRs) are transmembrane proteins which respond to and can be upregulated during nicotine exposure (Cano et al., 2020). Their trafficking and stoichiometry have also shown to be altered by nicotine (Srinivasan et al., 2011). In this protocol, we used a mouse model where the nAChR α4 subunit is labelled with green fluorescence protein (GFP).

We believe this protocol can be widely applied to virtually any mouse model that focuses on a transmembrane protein that is genetically labeled with a fluorescent protein. This protocol will enable researchers to extend single molecule studies beyond common in vitro approaches by performing ex vivo characterization of physiological changes occurring in live animals at the individual protein level. Specifically, this protocol describes a detailed method to isolate nanovesicles from specific regions of the mouse brain in order to perform single molecule imaging to characterize membrane receptors. This technique can be used to understand brain region-specific structural and functional changes in various animal models. An advantage of this approach is that during the isolation of the vesicles, transmembrane receptors maintain their structural integrity because they remain in their endogenous membrane in the nanovesicles. This allows one to characterize their structural assembly as well as their functional properties as in their native physiological environment (Fu et al., 2019). This single molecule protocol which involves sample preparation and labelling along with Total Internal Reflection Fluorescence (TIRF) Microscopy should be immediately applicable to a wide range of existing mouse models already in use for other neuroscience applications.