fluorogenen

Fluorogens are chemical probes that are nonfluorescent or only faintly emissive until they are activated by a specific trigger, after which they emit strongly in the visible spectrum. This turn-on behavior helps reduce background signal and enables imaging of biological processes with higher contrast than always-on fluorophores.

Activation mechanisms include enzymatic cleavage or chemical reaction that unquenches the fluorophore, binding-induced fluorescence where the fluorogen becomes fluorescent only when bound to a partner protein or RNA aptamer, and environmental activation in which changes in pH, polarity, or viscosity modulate brightness. Based on mechanism, fluorogens are used as enzymatic substrates, binding-activated dyes, or environment-sensitive probes. Notable implementations include fluorogen-activating proteins (FAPs) that induce fluorescence upon dye binding, and fluorogenic RNA aptamers such as Spinach-family sequences that turn on when bound by the corresponding fluorogen.

Applications include live-cell and in vivo imaging, fluorescent reporters of enzyme activity, transcriptional and translational reporters, single-molecule tracking, and multiplexed biosensing. They are valued for improving signal-to-noise ratios and enabling temporal control of fluorescence.

Common examples include the Spinach/DFHBI system, and the FAST system in which fluorogens such as HBR-3,5-DM become bright upon binding to a fluorogen-activating protein. Other fluorogenic substrates are designed for specific enzymes like esterases or proteases. Limitations can include slower on-rates, dependence on expression of an activator partner, variable brightness, and potential off-target activation or photostability concerns.