Single-molecule spectroscopy of fluorescent proteins

The discovery and use of fluorescent proteins has revolutionized cellular biology. Despite the widespread use of visible fluorescent proteins as reporters and sensors in cellular environments the versatile photophysics of fluorescent proteins is still subject to intense research. Understanding the details of the photophysics of these reporters is essential for accurate interpretation of the biological and biochemical processes illuminated by fluorescent proteins. Some aspects of the complex photophysics of fluorescent proteins can only be observed and understood at the single-molecule level, which removes averaging inherent to ensemble studies. In this paper we review how singlemolecule emission detection has helped understanding of the complex photophysics of fluorescent proteins.