Red fluorescent proteins: Advanced imaging applications and future design

In the past few years a large series of the advanced red-shifted fluorescent proteins (RFPs) has been developed. These enhanced RFPs provide new possibilities to study biological processes at the levels ranging from single molecules to whole organisms. Herein the relationship between the properties of the RFPs of different phenotypes and their applications to various imaging techniques are described. Existing and emerging imaging approaches are discussed for conventional RFPs, far-red FPs, RFPs with a large Stokes shift, fluorescent timers, irreversibly photoactivatable and reversibly photoswitchable RFPs. Advantages and limitations of specific RFPs for each technique are presented. Recent progress in understanding the chemical transformations of red chromophores allows the future RFP phenotypes and their respective novel imaging applications to be foreseen. Well red: Modern red fluorescent proteins (RFPs) provide new possibilities to study biological processes at the levels from single molecules to whole organisms (see scheme). Conventional and far-red RFPs, RFPs with a large Stokes shift, fluorescent timers, irreversibly photoactivatable, and reversibly photoswitchable RFPs are discussed in relationship to advanced imaging approaches.