Photoactivatable Azido Push-Pull Fluorophores for Single-Molecule Imaging in and out of Cells

We have designed a series of photoactivatable push-pull fluorophores, single molecules of which can be imaged in living cells. Photoactivatable probes are needed for super-resolution imaging schemes that require active control of single-molecule emission. Dark azido push-pull chromophores have the ability to be photoactivated to produce bright fluorescent labels. Activation of an azide functionality in the fluorogens induces a photochemical conversion to an amine, thus restoring fluorescence. Moreover, photoactivated push-pull dyes can insert into bonds of nearby biomolecules, simultaneously forming a covalent bond and becoming fluorescent (fluorogenic photoaffinity labeling). We demonstrate that the azide-to-amine photoactivation process is generally applicable to a variety of push-pull chromophores, and we characterize the photophysical parameters including photoconversion quantum yield, photostability, and turn-on ratio. Azido push-pull fluorogens provide a new class of photoactivatable single-molecule probes for fluorescent labeling and super-resolution microscopy.View Large Image | View Hi-Res Image | Download PowerPoint Slide(top) Photoconversion of an azido push-pull fluorogen produces a fluorescent amino molecule. (A) Living cells incubated with an azido fluorogen before and after photoactivation. Scalebar approximately 15 um. (B) Image of single molecules in a polymer film immediately after photoactivation. Inset is the frame immediately before activating. Scalebars approximately 2 um.