Fluorescence imaging has become an indispensable tool in cell and molecular biology. Genetically encoded fluorophores have revolutionized fluorescence microscopy, giving experimenters exquisite control over the localization and specificity of tagged constructs. However, these systems present certain drawbacks and as such, alternative hybrid systems based on the interaction between a small molecule fluorophore and a protein have been developed. A way to avoid unspecific background in cells and achieve high imaging contrast is to use fluorescent probes that display no fluorescence until labeling occurs. Such probes are often called fluorogenic probes to highlight their ability to generate fluorescence upon reaction/interaction with their target. A new fluorogenic hybrid system called the Fluorescence-activation and Absorption Shifting Tag (FAST) was developed, which consists of a small protein tag that reversibly and dynamically binds a small molecule chromophore, activating its fluorescence. Hybrid chemical-genetic systems such as FAST represent unique opportunities to extend the utility of the system as the protein and fluorogen present two opportunities for engineering. I will present work on improvements to the system and the development of biosensors based on such hybrid systems.

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