Tracking receptors using individual fluorescent and nonfluorescent nanolabels
Cold Spring Harb Protoc. 2014-02-01; 2014(2): pdb.prot080416
DOI: 10.1101/pdb.prot080416
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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.