FPs are now indispensable tools for studying dynamic processes in living organisms, tissues and cells [3
]. They have been exploited for diverse purposes, including as reporters of cell-specific gene expression, for visualizing the development and dynamic cellular architecture of complex tissues like brains (for example, Brainbow), and, perhaps most commonly, for determining the subcellular localization and dynamics of proteins. They are also used to observe and quantify trafficking of proteins to the nucleus, the reshaping of the cytoskeleton, delivery of proteins to the plasma membrane, action of molecular motors, motility of membrane proteins, assembly and disassembly of protein complexes and the turnover of proteins. Today, a full palette of color variants is available [3
], which has been key to the development of biosensors (both single fluorophore and Förster Resonance Energy Transfer (FRET) sensors) that, for the first time, have enabled monitoring of small molecules and metabolites with subcellular resolution, as, for example, glutamate release from neurons [8
]. Similarly, they have been used to create sensors for protein activity (reviewed in [8
]). More sophisticated versions of these FPs have even laid the path to breaking Ernst Abbe's Law for optical resolution in PALM super-resolution microscopy [9
Still, the race is on to develop new and improved versions with properties that will help the researcher explore deeper questions. Parameters targeted for improvement include brightness (increased extinction coefficients and quantum yields), novel or narrowed spectral ranges for absorption and emission, improved photostability, increased or decreased oligomerization tendency, faster fluorophore maturation time and reduced sensitivity to environmental fluctuations such as pH changes (Table 1). In addition to engineering efforts designed to modulate the emission spectra, FP mutagenesis has also targeted the separation distance between absorption and emission maxima (Stokes shift) to generate better probes for FRET, fluorescence cross-correlation spectroscopy (FCCS) and multicolor imaging.