Single-molecule imaging of fluorescent proteins

Abstract

Single molecule imaging techniques overcome the averaging effects inherent in ensemble measurements and enable characterization of the enormous heterogeneity that exists in biomolecular systems. Though long the domain of a few highly specialized labs, optical imaging of single molecules in living cells is becoming a widely accessible technique. The development of commercially available microscopes, robust analysis tools, and sensitive, low-noise detectors has contributed to this dissemination, as has the ever-growing array of fluorescent proteins. The relative ease with which genetically-tagged proteins can be created and introduced into a cell has largely eliminated more cumbersome and less precise means of particle labeling. A number of special considerations apply when using genetically encoded fluorophores for single molecule experiments, however. We discuss the means by which fluorescent proteins can be transfected into living cells to obtain the low particle densities required for single molecule imaging, and consider the limitations that are placed on single molecule analysis by the fluorophore's photophysical properties. We also discuss the types of morphology and subcellular localization that make certain preparations more amenable to single particle imaging than others. Last, we discuss some common pitfalls involved in analyzing single molecule datasets, and consider some of the unique information that can be obtained using these techniques.