Tracking receptors using individual fluorescent and nonfluorescent nanolabels

The plasma membrane is a fluid-mosaic structure in which some molecules seem to be randomly distributed and others show a precise compartmentalization that is related to their functional properties. These membrane domains are submicrometer in size and therefore are close to or below the optical diffraction limit. This makes their study difficult by conventional microscopy. Moreover, these compartments are usually dynamic in size and composition as their component molecules can continuously enter and exit by diffusion. Real-time, high-resolution, live-imaging methods rather than static imaging are thus required to reflect the real behavior of membrane molecules. Single-molecule techniques fulfill these requirements, as they provide information about the dynamics of molecules, together with nanometer resolution to study their distribution. Here we describe imaging and tracking techniques using nanometer-sized optical labels for the study of the movement of individual or small assemblies of membrane proteins. These labels include fluorescent dyes, luminescent nanocrystals, and absorbing metallic nanoparticles. Single-molecule tracking (SMT), with the use of organic dyes and semiconductor quantum dots (QDs), and single-particle tracking (SPT), with the use of gold nanoparticles, allow one to study the diffusion of individual molecules, their compartmentalization, and their interactions with other molecules. This protocol describes three methods for imaging and tracking membrane proteins: SMT using an organic dye, quantum dot tracking (QDT), and single-nanoparticle photothermal tracking (SNaPT) using gold nanoparticles. Organic dyes and QDs are tracked by single-molecule epifluorescence microscopy. Gold nanoparticles are detected by photothermal heterodyne imaging (PHI) and tracked using a triangulation scheme.